Introduction
A phenol-chloroform extraction is a liquid-liquid extraction. A liquid-liquid extraction is a
method that separates mixtures of molecules based on the differential solubilities of the
individual molecules in two different immiscible liquids (28). Liquid-liquid extractions are
widely used to isolate RNA, DNA, or proteins1.

Brief History
Volkin & Carter reported the first use of guanidinium chloride in the isolation of RNA in 1951
(30). In 1953, Grassmann & Defner described the efficacy of phenol at extracting proteins
from aqueous solution (16). Utilizing this find, Kirby demonstrated the use of phenol to
separate nucleic acids from proteins in 1956 (18). Cox and others renewed interest in the use
of guanidinium chloride in the isolation of RNA from ribonucleoproteins in the 1960s
(11,12,13). From then on, guanidinium extractions were the method of choice for RNA
purification, replacing phenol extraction. The use of guanidinium thiocyanate instead of
guanidinium chloride was first briefly mentioned by Ullrich et al. in 1977 (29), and later
successfully employed by Chirgwin et al. in 1979 (8). Chirgwin et al. used guanidinium
thiocyanate to isolate undegraded RNA from ribonuclease-rich tissues like pancreas. A
combination of guanidinium thiocyanate and hot phenol for RNA isolation was reported by
Feramisco et al. in 1981 (14). In 1987, Chomczynski & Sacchi combined guanidinium
thiocyanate with phenol-chloroform extraction under acidic conditions (9). Since its
inception, the Chomczynski & Sacchi method has been the method of choice to isolate RNA
from cultured cells and most animal tissues (10).

첨부파일 참고

[Guidelines for Nomenclature 3]

Guidelines for Nomenclature of Genes, Genetic Markers, Alleles, and Mutations in Mouse and Rat 

6   Definitions

The following definitions should aid the user in understanding what is being named, and in understanding the principles underlying these guidelines.

6.1   Gene

A gene is a functional unit, usually encoding a protein or RNA, whose inheritance can be followed experimentally. Inheritance is usually assayed in genetic crosses, but identification of the gene in cytogenetic or physical maps are other means of mapping the locus of a gene. The existence of a gene can also be inferred in the absence of any genetic or physical map information, such as from a cDNA sequence.

6.2   Pseudogene

A sequence that closely resembles a known functional gene, at another locus within a genome, that is non-functional as a consequence of (usually several) mutations that prevent either its transcription or translation (or both). In general, pseudogenes result from either reverse transcription of a transcript of their "normal" paralog (in which case the pseudogene typically lacks introns and includes a poly(A) tail; often called processed pseudogenes) or from recombination (in which case the pseudogene is typically a tandem duplication of its "normal" paralog).

6.3   Locus

A locus is a point in the genome, identified by a marker, which can be mapped by some means. It does not necessarily correspond to a gene; it could, for example, be an anonymous non-coding DNA segment or a cytogenetic feature. A single gene may have several loci within it (each defined by different markers) and these markers may be separated in genetic or physical mapping experiments. In such cases, it is useful to define these different loci, but normally the gene name should be used to designate the gene itself, as this usually will convey the most information.

6.4   Marker

A marker is the means by which a gene or a locus is identified. The marker is dependent on an assay, which could, for example, be identification of a mutant phenotype or presence of an enzyme activity, protein band, or DNA fragment. The assay must show genetic variation of the marker to map the locus on a genetic map but not to place it on a physical map.

6.5   Allele

The two copies of an autosomal gene or locus on the maternal and paternal chromosomes are alleles. If the two alleles are identical, the animal is homozygous at that locus. When genetically inherited variants of a gene or locus are detectable by any means, the different alleles enable genetic mapping. A single chromosome can only carry a single allele and, except in cases of duplication, deletion or trisomy, an animal carries two autosomal alleles. In particular, a transgene inserted randomly in the genome is not an allele of the endogenous locus; the condition is termed hemizygous if the transgene is present only in one of the two parental chromosome sets. By contrast, a gene modified by targeting at the endogenous locus is an allele and should be named as such.

6.6   Allelic Variant

Allelic variants are differences between alleles, detectable by any assay. For example, differences in anonymous DNA sequences can be detected as simple sequence length polymorphism (SSLP) or single nucleotide polymorphisms (SNPs). Other types of variants include differences in protein molecular weight or charge, differences in enzyme activity, or differences in single-stranded conformation (SSCP). Many allelic variants, in particular DNA variants, do not confer any external phenotype on the animal. These variants are often termed “polymorphisms” although, strictly speaking, that term applies only to variants with a frequency of more than 1% in the population.

6.7   Splice Variant or Alternative Splice

Alternative splicing of a gene results in different, normally occurring forms of mRNA defined by which exons (or parts of exons) are used. Thus one or more alternative protein products can be produced by a single allele of a gene. Among different alleles, alternative splice forms may or may not differ, depending on whether the sequence difference between the alleles affects the normal splicing mechanism and results in differences in the exon (or partial exon) usage. For example, allele A may produce mRNAs of splice form 1, 2, and 3; while allele B may produce mRNAs of splice form 1, 2, and 4; and Allele C may produce mRNAs of splice form 1, 2, and 3. In this case, each of the alleles A, B, and C by definition must differ in their DNA sequence. However, the difference between allele B versus alleles A and C must include a sequence difference that affects the splicing pattern of the gene.

6.8    Mutation

A mutation is a particular class of variant allele that usually confers a phenotypically identifiable difference to a reference "wild type" phenotype. However, in some cases, such as when homologous recombination is used to target a gene, a readily identified phenotype may not result even though the gene may be rendered non-functional. In such cases, the targeted genes are nevertheless referred to as mutant alleles.

6.9    Dominant and Recessive

Dominant and recessive refer to the nature of inheritance of phenotypes, not to genes, alleles, or mutations. A recessive phenotype is one that is only detected when both alleles have a particular variant or mutation. A dominant phenotype is detectable when only one variant allele is present. If both alleles can be simultaneously detected by an assay, then they are codominant. For example, an assay that detects variation of DNA or protein will almost invariably detect codominant inheritance, as both alleles are detected. If a mutation produces a phenotype in the heterozygote that is intermediate between the homozygous normal and mutant, the phenotype is referred to as semidominant. A single mutation may confer both a dominant and a recessive phenotype. For example, the mouse patch (Ph) mutation has a heterozygous (dominant) pigmentation phenotype but also a homozygous (recessive) lethal phenotype. As the terms are applied to phenotypes not to genes or alleles, then in the case where a gene has multiple mutant alleles, each can confer a phenotype that is dominant to some, but recessive to other, phenotypes due to other alleles.

Penetrance is a quantitative measure of how often the phenotype occurs in a population; and expressivity is a measure of how strongly a phenotype is expressed in an individual. Particularly in segregating crosses, or where there is a threshold effect on phenotypic manifestation, these measures provide additional ways to describe how particular allelic combinations result in a phenotype.

6.10   Genotype

Genotype is the description of the genetic composition of the animals, usually in terms of particular alleles at particular loci. It may refer to single genes or loci or to many. Genotype can only be determined by assaying phenotype, including test mating to reveal carriers of recessive mutations. Strictly speaking, even direct determination of DNA variants is assaying phenotype not genotype as it is dependent on a particular assay, although it is so close to genotype that it serves as a surrogate.

6.11   Phenotype

Phenotype is the result of interaction between genotype and the environment and can be determined by any assay.

6.12   Quantitative Trait Loci (QTLs)

Quantitative Trait Loci (QTL) are polymorphic loci that contain alleles, which differentially affect the expression of continuously distributed phenotypic traits. Usually these are markers described by statistical association to quantitative variation in the particular phenotypic traits that are controlled by the cumulative action of alleles at multiple loci.

6.13   Haplotype

A haplotype is the association of genetically linked alleles, as found in a gamete. They may be a combination of any type of markers, and may extend over large, genetically separable distances, or be within a short distance such as within a gene and not normally separated.

6.14   Homolog

Genes are homologous if they recognizably have evolved from a common ancestor. Note that genes are either homologous or not; there are no degrees of homology! For example, all globin genes, and myoglobin, are homologs, even though some are more closely related to each other than others. When a measure of relatedness between sequences is required, percent identity or similarity should be used.

6.15   Ortholog

Genes in different species are orthologs if they have evolved from a single common ancestral gene. For example, the beta globin genes of mouse, rat and human are orthologs. Note that several genes in the mouse or rat may have a single ortholog in another species and vice versa.

6.16   Paralog

Paralogous genes are genes within the same species that have arisen from a common ancestor by duplication and subsequent divergence. For example, the mouse alpha globin and beta globin genes are paralogs.

7   References

Bestor TH. Transposons reanimated in mice. 2005. Cell 122:322-325.

Committee on Rat Nomenclature, Cochairmen Gill T.J. III, Nomura T. 1992. Definition, nomenclature, and conservation of rat strains. ILAR News 34:S1-S56.

Committee on Standardized Genetic Nomenclature for Mice. 1963. A revision of the standardized genetic nomenclature for mice. J. Hered. 54:159-162.

Committee on Standardized Genetic Nomenclature for Mice. 1973. Guidelines for nomenclature of genetically determined biochemical variants in the house mouse, Mus musculus. Biochem. Genet. 9:369-374.

Committee on Standardized Genetic Nomenclature for Mice, Chair: Lyon, M.F. 1981. Rules and guidelines for gene nomenclature. In: Genetic Variants and Strains of the Laboratory Mouse, Green, M.C. (ed.), First Edition, Gustav Fischer Verlag, Stuttgart, pp. 1-7.

Committee on Standardized Genetic Nomenclature for Mice, Chair: Lyon, M.F. 1989. Rules and guidelines for gene nomenclature. In: Genetic Variants and Strains of the Laboratory Mouse, Lyon, M.F., A.G. Searle (eds.), Second Edition, Oxford University Press, Oxford, pp. 1-11.

Committee on Standardized Genetic Nomenclature for Mice, Chairperson: Davisson, M.T. 1996. Rules and guidelines for gene nomenclature. In: Genetic Variants and Strains of the Laboratory Mouse, Lyon, M.F., Rastan, S., Brown, S.D.M. (eds.), Third Edition, Volume 1, Oxford University Press, Oxford, pp. 1-16.

Ding S, Wu X, Li G, Han M, Zhuang Y, Xu. T. 2005. Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell 122:473-483.

Dunn, L.C., H. Gruneberg, G.D. Snell. 1940. Report of the committee on mouse genetics nomenclature. J. Hered. 31:505-506.

Dupuy AJ, Akagi K, Largaespada DA, Copeland NG, Jenkins NA. 2005. Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature 436:221-226.

Eppig, JT. 2006. Mouse Strain and Genetic Nomenclature: an Abbreviated Guide. In: Fox J, Barthold S, Davvison M, Newcomer C, Quimby F, Smith A (eds) The Mouse in Biomedical Research, Volume 1, Second Edition. Academic Press. pp.79-98.

International Committee on Standardized Genetic Nomenclature for Mice, Chairperson: Davisson, M.T. 1994. Rules and guidelines for genetic nomenclature in mice. Mouse Genome 92 vii-xxxii.

Levan G., H.J. Hedrich, E.F. Remmers, T. Serikawa, M.C. Yoshida. 1995. Standardized rat genetic nomenclature. Mamm. Genome 6:447-448.

[Guidelines for Nomenclature 2]

3   Names and Symbols for Variant and Mutant Alleles

Different alleles of a gene or locus can be distinguished by a number of methods, including DNA fragment length, protein electrophoretic mobility, or variant physiological or morphological phenotype.

All mutant alleles, whether of spontaneous or induced origin, targeted mutations, gene traps, or transgenics should be submitted to MGD (mouse) or RGD (rat) for an allele or gene accession identifier.

3.1   Mutant Phenotypes

3.1.1   Genes Known Only by Mutant Phenotypes

Where a gene is known only by mutant phenotype, the gene is given the name and symbol of the first identified mutant. Symbols of mutations conferring a recessive phenotype begin with a lowercase letter; symbols for dominant or semidominant phenotype genes begin with an uppercase letter.

Examples:

In mouse, recessive spotting, rs; abnormal feet and tail, Aft; circling, cir

In rat, polydactyly-luxate, lx.

Further (allelic) mutations at the same locus, if they have the same phenotype, are given the same name with a Laboratory code preceded by a serial number (if more than one additional allele from the same lab). In the symbol the Laboratory code is added as a superscript.

• For example, agil2J, the second new allele of mouse agitans-like identified at The Jackson Laboratory.

If a new allelic mutation of a gene known only by a mutant phenotype is caused by a transgenic insertion, the symbol of this mutation should use the symbol of the transgene as superscript (see Section 3.4.2 and Section 4).

• awgTg(GBtslenv)832Pkw; mutation of abnormal wobbly gait caused by a transgene, mouse line 832, produced in the laboratory of Paul Wong. (An abbreviated form, awgTg832Pkw can be used if the abbreviated designation is unique).

If the additional allele has a different phenotype, it may be given a different name, but when symbolized the new mutant symbol is superscripted to the original mutant symbol. Also, if a new mutation is described and named but not shown to be an allele of an existing gene until later, the original name of the new mutation can be kept. Even if the phenotype is apparently identical, the original symbol is used, with the new mutation symbol as superscript.

For example

• grey coat is an allele of recessive spotting (rs) in the mouse, and hence is symbolized rsgrc.

3.1.2   Phenotypes Due to Mutations in Structural Genes

When a spontaneous or induced mutant phenotype is subsequently found to be a mutation in a structural gene, or the gene in which the mutation has occurred is cloned, the mutation becomes an allele of that gene and the symbol for the mutant allele is formed by adding the original mutant symbol as a superscript to the new gene symbol. (The mutant symbol should retain its initial upper or lowercase letter).

• The hotfoot (ho) mutation of the mouse glutamate receptor Grid2, Grid2ho.
• The dominant white spotting (W) mutation of mouse Kit, KitW

If the original mutation has multiple alleles, when describing these alleles, their symbols become part of the superscript to the identified structural gene.

• creeper, Grid2ho-cpr.
• viable white spotting, KitW-v; sash, KitW-sh.

Even if the identified gene is novel and unnamed, it is recommended that it is nevertheless given a name and symbol different from the mutant name and symbol. This will more readily allow discrimination between mutant and wild type and between gene and phenotype.

3.1.3   Wild Type Alleles and Revertants

The wild type allele of a gene is indicated by + as superscript to the mutant symbol.

• The wild type allele of the agitans-like mutation, agil+.
• The wild type Kit locus (if necessary to distinguish from mutations), Kit+.

A revertant to wild type of a mutant phenotype locus should be indicated by the symbol + with the mutant symbol as superscript.

• Revertant to wild type at the hairless mutant locus +hr

Additional revertants are given a Laboratory Code and preceded by a serial number if more than one revertant is found in a lab. Serial numbers are independent for mouse and rat revertants and no homology is implied. If the revertant is in a gene that has been cloned, then the mutant symbol is retained as superscript to the gene symbol, and + is appended.

• Revertant to wild type of the dilute mutation of myosin Va; Myo5ad+
• Second such revertant identified at The Jackson Laboratory; Myo5ad+2J.

3.2   Variants

3.2.1   Biochemical Variants

Electrophoretic or other biochemicalvariant alleles of known structural genes are usually given lowercase letters to indicate different alleles, and in the symbol the letter becomes a superscript to the gene symbol.

• For example, glucose phosphate isomerase 1 alleles a and b; Gpi1a, Gpi1b.

  3.2.2   DNA Segment Variants

Variants of DNA segments are indicated by a superscript to the symbol. The symbol is usually an abbreviation for the inbred strain in which the variant is being described. However, a particular allele may be found in several inbred strains, and, furthermore, it may be difficult to establish whether an allele in one strain is identical to one in another. The use of allele symbols for DNA segments is mainly limited to describing inheritance and haplotypes in crosses. As long as the symbols are defined in the description, users are free to use whatever allele symbol best fits their needs. In tables of genotypes, the gene symbol can be omitted and the allele abbreviation used alone.

• D11Mit19a, D11Mit19b, D11Mit19c are variant alleles of D11Mit19 in mouse.

3.2.3   Single Nucleotide Polymorphisms (SNPs)

Polymorphisms defined by SNPs may occur within or outside of a protein coding sequence.

If the SNP occurs within a gene, the SNP allele can be designated based on its dbSNP_id, followed by a hyphen and the specific nucleotide.

Examples:

Park2rs6200232-G	The Park2 rs6200232 SNP allele with the G variant
Park2rs6200232-A	The Park2 rs6200232 SNP allele with the A variant

If the SNP occurs outside of an identified gene, the SNP locus can be designated using the dbSNP_id as the locus symbol and the nucleotide allelic variants are then superscripted as alleles. If a gene is later discovered to include this SNP locus, the same guidelines are applicable as those used when mutant locus symbols become alleles of known genes.

Examples:

rs6200616T	A SNP locus with the T variant
rs6200616C	A SNP locus with the C variant

Note: If a gene Xyz is later discovered to include this SNP locus, rs620061, then the alleles listed above become Xyzrs620061-T and Xyzrs620061-C.

3.3  Variation in Quantitative Trait Loci and in Response and Resistance Genes

Variation in genes that do not give rise to a visible phenotype may be detected by assaying physiological or pathological parameters. Examples of this type of variation include levels of metabolite, immune response to antigen challenge, viral resistance, or response to drugs. Genetic variation may also produce phenotypic variation in morphology, behavior, or other observable traits that interact in a complex manner with other genes and/or with the environment.

These genes can only be identified by virtue of allelic variation. In most cases, there will not be a clear wild type; hence all alleles should be named. In most cases, the alleles should be named according to their strain of origin and symbolized by adding the strain abbreviation as superscript, although for resistance and sensitivity, variants r and s may be used. Bear in mind that resistance alleles deriving from different strains may not be the same and should be given different names and symbols.

Once the gene underlying a quantitative trait has been cloned or identified, the phenotypic name should be replaced by the name of the identified gene. The allele names and symbols should be the same as those used for the phenotype.

Examples:

Slc11a1r	solute carrier family 11, host resistance allele
Slc11a1s	solute carrier family 11, host susceptibility allele  (the QTL originally known as BCG/Lsh resistance has been identified as Slc11a1)
Scc2BALB/cHeA	colon tumor susceptibility 2, BALB/cHeA allele
Scc2STS/A	colon tumor susceptibility 2, STS/A allele (for QTL Scc2, the STS/A allele has increased tumor susceptibility vs. BALB/cHeA)

3.4   Insertional and Induced Mutations

Mutations that are induced, targeted, or selected in structural genes are named as alleles of the structural gene.

3.4.1   Mutations of Structural Genes

Variants of structural genes that are clearly mutations, whether or not they confer a phenotype, are given the superscript m#Labcode, where # is a serial number and is followed by the Laboratory code where the mutation was found or characterized. Serial numbers are independently assigned in mouse and rat and the same assigned serial number does not imply orthology. If the mutation is known to have occurred on a particular allele, that can be specified by preceding the superscript with the allele symbol and a hyphen.

• for example, Mod1a-m1Lws is a mutation of the mouse Mod1a allele, the first found in the laboratory of Susan Lewis.

If the mutation is shown to be a deletion of all or part of the structural gene, the superscript del can be used in place of m. Note that this should be used only for deletions that encompass a single gene; larger deletions should use the chromosomal deletion nomenclature.

3.4.2    Transgenic Insertional Mutations

Mutations produced by random insertion of a transgene (not by gene targeting) are named as a mutant allele of the gene (which should be given a name and symbol if it is a novel gene), with the superscript the symbol for the transgene (see Section 3.1.1 for examples, and Section 4 for naming transgenes).

3.5    Targeted and Trapped Mutations

3.5.1   Knockout, Knockin, Conditional and Other Targeted Mutations

Mutations that are the result of gene targeting by homologous recombination in ES cells are given the symbol of the targeted gene, with a superscript consisting of three parts: the symbol tm to denote a targeted mutation, a serial number from the laboratory of origin and the Laboratory code where the mutation was produced (see Section 2.1).

• For example, Cftrtm1Unc is the first targeted mutation of the cystic fibrosis transmembrane regulator (Cftr) gene produced at the University of North Carolina.

So-called "knock in" mutations,  in which all or part of the coding region of one gene is replaced by another,  should be given a tm symbol and the particular details of the knock-in associated with the name in publications or databases. Where there has been a replacement of the complete coding region, the replacing gene symbol can be used parenthetically as part of the allele symbol of the replaced gene along with a Laboratory code and serial number.

• For example, En1tm1(Otx2)Wrst where the coding region of En1 was replaced by the Otx2 gene, originating from the W. Wurst laboratory.

Knock in alleles expressing a RNAi under the control of the endogenous promoter can be designated using targeted mutation or transgene mutation nomenclature, as appropriate:

Example:

Genetm#(RNAi:Xyz)Labcode

When a targeting vector is used to generate multiple germline transmissible alleles, such as in the Cre-Lox system, the original knock-in of loxP would follow the regular tm designation rules. If a second heritable allele was then generated after mating with a cre transgenic mouse, it would retain the parental designation followed by a decimal point and serial number.

• Tfamtm1Lrsn and Tfamtm1.1Lrsn. In this example, Tfamtm1Lrsn designates a targeted mutation where loxP was inserted into the Tfam gene. Tfamtm1.1Lrsn designates another germline transmissible allele generated after mating with a cre transgenic mouse. Note: somatic events generated in offspring from a Tfamtm1Lrsn bearing mouse and a cre transgenic that cause disruption of Tfam in selective tissues would not be assigned nomenclature.

Other more complex forms of gene replacement, such as partial "knock-in", hit-and-run, double replacements, and loxP mediated integrations are not conveniently abbreviated and should be given a conventional tm#Labcode superscript. Details of the targeted locus should be given in associated publications and database entries.

Note that although subtle alterations made in a gene appear to lend themselves to a simple naming convention whereby the base or amino acid changes are specified, in fact these do not provide unique gene names, as such alterations, which could be made in independent labs, while bearing the same changes, may differ elsewhere in the gene.

Large-scale projects that systematically produce a large number of alleles (>1000) may include a project abbreviation in parentheses as part of the allele designation. These should retain the accepted nomenclature features of other alleles of that class. For example, a targeted allele created by Velocigene (Regeneron) in the KOMP knockout project:

Gstm3tm1(KOMP)Vlcg

Once fully designated in a publication, the allele can be abbreviated by removing the portion of the allele designation in parentheses (in this case, Gstm3tm1Vlcg), providing the symbol remains unique.

3.5.2   Endonuclease-induced Mutations

Endonuclease-induced mutations are targeted mutations generated in pluripotent or totipotent cells by an endonuclease joined to sequence-specific DNA binding domains. The mutation is introduced during homology-directed or non-homologous end-joining repair of the induced DNA break(s). Endonuclease-induced mutations are given the symbol of the mutated gene, with a superscript consisting of three parts: the symbol em to denote an endonuclease-induced mutation, a serial number from the laboratory of origin and the Laboratory code where the mutation was produced.

Example:

Fgf1em1Mcw I

the first endonuclease-induced mutation of the fibroblast growth factor 1 (Fgf1) gene produced at the Medical College of Wisconsin.

3.5.3    Gene Trap Mutations

Gene trap mutations are symbolized in a similar way to targeted mutations. If the trapped gene is known, the symbol for the trapped allele will be similar to a targeted mutation of the same gene using the format Gt(vector content)#Labcode for the allele designation. Example:

Akap12Gt(ble-lacZ)15Brr

a gene trap allele of the Akap12 gene, where the gene trap vector contains a phleomycin resistance gene (ble) and lacZ, the 15th analyzed in the laboratory of Jacqueline Barra (Brr).

If the trapped gene is novel, it should be given a name and a symbol, which includes the letters Gt for "gene trap," the vector in parentheses, a serial number, and Laboratory code.

• For example, a gene trapped locus (where the gene is unknown) using vector ROSA, the 26th made in P. Soriano's laboratory, is Gt(ROSA)26Sor.

For high throughput systematic gene trap pipelines, the mutant ES cell line's designation can be used in parentheses instead of the vector designation, and the serial number following the parentheses may be omitted.

Examples:

Gt(DTM030)Byg	for a trapped gene (at an undefined locus) in mutant ES cell line DTM030, made by BayGenomics
Osbpl1aGt(OST48536)Lex	gene trap allele of the oxysterol binding protein-like 1A gene, in mutant ES cell line OST48536, made by Lexicon Genetics, Inc.

3.5.4    Enhancer Traps

Enhancer traps are specialized transgenes. One utility of these transgenes is in creating cre driver lines. Enhancer traps of this type that are currently being created may include a minimal promoter, introns, a cre recombinase cassette (sometimes fused with another element such as ERT2), and polyA sites from different sources.

Nomenclature for these enhancer traps consists of 4 parts as follows:

Et		prefix for enhancer trap
cre recombinase cassette		portion in parentheses... for example, cre, icre, or cre/ERT2 (if fused with ERT2)
line number or serial number		to designate lab trap number or serial number
Lab code		ILAR code identifying the creator of this enhancer trap

Examples:

Et(icre)1642Rdav		Enhancer trap 1642, Ron Davis
Et(cre/ERT2)2047Rdav		Enhancer trap 2047, Ron Davis

Note that the minimal promoter, poly A source, etc. are not part of the enhancer trap nomenclature. These are molecular details of the specific construct that will be captured in database records and reported with experimental results.

4   Transgenes

Any DNA that has been stably introduced into the germline of mice or rats is a transgene. Transgenes can be broken down into two categories:

• Those that are produced by homologous recombination as targeted events at particular loci.
• Those that occur by random insertion into the genome (usually by means of microinjection).

Nomenclature for targeted genes is dealt with in Section 3.5. Random insertion of a transgene in or near an endogenous gene may produce a new allele of this gene. This new allele should be named as described in Section 3.4.2. The transgene itself is a new genetic entity for which a name may be required. This section describes the guidelines for naming the inserted transgene.

It is recognized that it is not necessary, or even desirable, to name all transgenes. For example, if a number of transgenic lines are described in a publication but not all are subsequently maintained or archived, then only those that are maintained require standardized names. The following Guidelines were developed by an interspecies committee sponsored by ILAR in 1992 and modified by the Nomenclature Committee in 1999 and 2000. Transgenic symbols should be submitted to MGD or RGD/RatMap through the usual nomenclature submission form for new loci. The transgene symbol is made up of four parts:

• Tg denoting transgene.
• In parentheses, the official gene symbol of the inserted DNA, using nomenclature conventions of the species of origin.
• The laboratory's line or founder designation or a serial number (note that numbering is independent for mouse and rat series).
• The Laboratory code of the originating lab.

Note that, in contrast to gene and allele symbols, transgene symbols are not italicized as they are random insertions of foreign DNA material and are not part of the native mouse genome.

Examples:

Tg(Zfp38)D1Htz	a transgene containing the mouse Zfp38 gene, in line D1 reported by Nathaniel Heintz.
Tg(CD8)1Jwg	a transgene containing the human CD8 gene, the first transgenic line using this construct described by the lab of Jon W. Gordon.
Tg(HLA-B*2705, B2M)33-3Trg	a double transgene in rat containing the human HLA-B*2705 and B2M genes, that were co-injected, giving rise to line 33-3 by Joel D. Taurog.

The *, as used in the last example above, indicates that the included gene is mutant.

Different transgenic constructs containing the same gene should not be differentiated in the symbol; they will use the same gene symbol in parentheses and will be distinguished by the serial number/Laboratory code. Information about the nature of the transgenic entity should be given in associated publications and database entries.

In many cases, a large number of transgenic lines are made from the same gene construct and only differ by tissue specificity of expression. The most common of these are transgenes that use reporter constructs or recombinases (e.g., GFP, lacZ, cre), where the promoter should be specified as the first part of the gene insertion designation, separated by a hyphen from the reporter or recombinase designation. The SV40 large T antigen is another example. The use of promoter designations is helpful in such cases.

Examples:

Tg(Wnt1-LacZ)206Amc	the LacZ transgene with a Wnt1 promoter, from mouse line 206 in the laboratory of Andrew McMahon.
Tg(Zp3-cre)3Mrt	the cre transgene with a Zp3 promoter, the third transgenic mouse line from the laboratory of Gail Martin.

In the case of a fusion gene insert, where roughly equal parts of two genes compose the construct, a forward slash separates the two genes in parentheses.

Example:

Tg(TCF3/HLF)1Mlc

a transgene in which the human transcription factor 3 gene and the hepatic leukemia factor gene were inserted as a fusion chimeric cDNA, the first transgenic mouse line produced by Michael L. Cleary's laboratory (Mlc).

This scheme is to name the transgene entity only. The mouse or rat strain on which the transgene is maintained should be named separately as in the Rules and Guidelines for Nomenclature of Mouse and Rat Strains. In describing a transgenic mouse or rat strain, the strain name should precede the transgene designation.

Examples:

C57BL/6J-Tg(CD8)1Jwg	mouse strain C57BL/6J carrying the Tg(CD8)1Jwg transgene.
F344/CrlBR-Tg(HLA-B*2705, B2M)33-3Trg	rat strain F344/CrlBR carrying the Tg(HLA-B*2705,B2M)33-3Trg double transgene.

For BAC transgenics, the insert designation is the BAC clone and follows the same naming convention as the Clone Registry at NCBI.

Example:

Tg(RP22-412K21)15Som

a BAC transgene where the inserted BAC is from the RP22 BAC library, plate 412, row K, column 21. It is the 15th in the mouse made in the laboratory of Stefan Somlo (Som).

Transgenes containing RNAi constructs can be designated minimally as:

Tg(RNAi:geneX)#Labcode, where
geneX	is the gene that is knocked down
#	is the serial number of the transgene

An expanded version of this designation is:

Tg(Pro-yyRNAi:geneX)#Labcode, where
Pro-	can be used optionally to designate the promoter
yy	can be used optionally for the specific RNAi construct

While there is the option to include significant information on vectors, promoters, etc. within the parentheses of a transgene symbol, this should be minimized for brevity and clarity. The function of a symbol is to provide a unique designation to a gene, locus, or mutation. The fine molecular detail of these loci and mutations should reside in databases such as MGD and RGD.

5    Transposon-induced Mutations and Inserts

Three types of genetic inserts are involved in creating transposon-induced mutations. Two lines, one carrying the transposable-element as a concatamer and the other carrying the transposase are mated. This causes the transposable-element to come in contact with the transposase and to be mobilized from its original site, and, when reintegrated into the genome, can cause a heritable phenotypic mutation. (c.f., Ding, et al.,2005; Bestor, 2005;Dupuy, et al., 2005). Accepted nomenclature for the transposable-element inserts, transposase transgenes, and resulting transposed insertion alleles are given below.

5.1   Transgenic Transposable Element (TE) Concatamers

The transgenic transposable element concatamers are identified with a standard prefix Tg (for transgenic) and Tn (for transposable element). The class of transposable element may be included in parentheses. The general format of the symbol is:

TgTn(transposon_class_abbreviation-vector)#Labcode

Example:  TgTn(sb-T2/GT2/tTA)1Dla

The symbol consists of:

• Tg denoting transgenic
• Tn denoting transposon
• In parentheses, a lowercase abbreviation of the transposon class (in this case sb for Sleeping Beauty), followed by a hyphen and the vector designation
• The laboratory's line or founder designation or a serial number
• The Laboratory Code of the originating lab

5.2  Transposase Inserts

Transposases can be engineered into the genome via transgenesis or specific gene targeting. In these cases the relevant nomenclature for transgenes or targeted mutations is used.

For a transgene, use the standard prefix Tg (for transgene). The contents of the parentheses will usually be the promoter and the symbol for the transposase with which it is associated, separated by a hyphen. The general format of the symbol is:

Tg(promoter-transposase)#Labcode

Example:  Tg(ACTB-sb10)545Abc

The symbol consists of:

• Tg denoting transgene
• In parentheses, the official gene symbol for the promoter, using the nomenclature of the species of origin, followed by a hyphen and a lowercase transposase symbol, in this case sb10 for the Sleeping Beauty 10 transposase
• The laboratory's line or founder designation or a serial number
• The Laboratory code of the originating lab.

For a targeted knock-in of the transposase, use the standard format for a targeted mutation, i.e., the symbol of the targeted gene with a superscripted allele symbol beginning with the prefix tm. The contents of the parentheses will usually be the symbol for the transposase with which it is associated. The general format of the symbol is:

Genetm#(transposase)Labcode

Example:  Gt(ROSA)26Sortm1(sb11)Njen

The symbol consists of:

• The gene into which the transposase was integrated, in this case Gt(ROSA)26Sor
• In the superscript:
  • tm denoting targeted mutation
  • A serial number of the targeted mutation
  • In parentheses, a lowercase transposase symbol, in this case sb11 for the Sleeping Beauty 11 transposase
  • The Laboratory Code of the originating lab

5.3   Transposed Insertion Alleles

These alleles follow the rules for naming all other alleles. In general a transposable element concatamer marker will already be established, as above. The new allele, then, will be a superscripted form of the concatamer symbol. Note that all such alleles that are "derived from" a transposable element concatamer carry the original number with a decimal point and serial number identifying the specific allele. The general format is:

GeneTn(transposon_class_abbreviation-vector)#Labcode

Example: Car12Tn(sb-T2/GT2/tTA)1.1Dla

The symbol consists of:

The gene into which the transposable element was integrated (transposed)

In the superscript:

• Tn denoting transposon
• In parentheses, a lowercase abbreviation of the transposon class (in this case sb for Sleeping Beauty), followed by a hyphen and the vector designation
• A serial number, in which the primary number corresponds to that given to the transposable element concatamer from which it arose, followed by a decimal point and a serial number designating its number within the series of derivative insertion alleles.
• The Laboratory Code of the lab originating the transposable element line.

If a newly transposed insertion occurs in an unknown site or intergenic region, the form:

Tn(transposon_class_abbreviation-vector)#Labcode

is used to symbolize the "genomic mutation" without being superscripted to a gene symbol, similar to the way a random transgene inserted into a non-gene site is designated.

[Guidelines for Nomenclature 1]

Guidelines for Nomenclature of Genes, Genetic Markers, Alleles

1  Principles of Nomenclature

1.1   Key Features

The key component of nomenclature is the gene or locus name and symbol, which identifies a unit of inheritance. Other features, such as alleles, variants and mutations, are secondary to the gene name and become associated with it. Similarly, probes or assays used to detect a gene are not primary features and should not normally be used as names.

The primary purpose of a gene or locus name and symbol is to be a unique identifier so that information about the gene in publications, databases and other forms of communication can be unambiguously associated with the correct gene. These guidelines, therefore, are intended to aid the scientific community as a whole to use genetic information.

Other, secondary, functions of nomenclature for genes are to:

• identify the gene as a member of a family, which may give further information about the gene by reference to other family members
• identify the gene as the ortholog of a gene in another mammal (usually human)

1.2   Definitions

It is important that the user understands what is being named and the principles underlying these guidelines. Section 6 presents definitions that will aid the user in distinguishing, for example, genes, loci, markers, and alleles.

1.3  Stability of Nomenclature

On the whole gene names should be stable; that is, they should not be changed over time. However there are certain circumstances where a change is desirable:

• In cases where a gene has been known only as, and named for, a mutant phenotype: when the mutated gene is identified, then the mutant name becomes the mutant allele name of the identified gene (see Section 3.1.2).
• Where a gene becomes assigned to a gene family (of paralogs), and the nomenclature of the family is established. (see Section 2.6.2).
• Where orthologous gene(s) have been identified between mouse, rat, and human, and a common symbol is adopted for all three species.

1.4   Synonyms

A gene can have several synonyms, which are names or symbols that have been applied to the gene at various times. These synonyms may be associated with the gene in databases and publications, but the established gene name and symbol should always be used as the primary identifier.

1.5    Gene symbols, proteins, and chromosome designations in publications

1.5.1     Gene and allele symbols

Gene symbols are italicized when published, as are allele symbols. Section 2 below specifies naming rules for establishing correct symbols. Help is available for determining correct gene and allele symbol assignment (nomen@jax.org) and symbols can be reserved privately pre-publication.

To distinguish between mRNA, genomic DNA, and cDNA forms within a manuscript, write the relevant prefix in parentheses before the gene symbol, for example, (mRNA) Rbp1.

1.5.2    Protein symbols

Protein designations follow the same rules as gene symbols, with the following two distinctions:

• Protein symbols use all uppercase letters.
• Protein symbols are not italicized.

1.5.3   Chromosome designations

• Use uppercase "C" when referring to a specific mouse chromosome (e.g., Chromosome 15).
• When abbreviating the word Chromosome, do not use a period (".") after the abbreviation (e.g., Chromosome 15 should abbreviated as Chr 15 and not Chr.15).

2   Symbols and Names of Genes and Loci

The prime function of a gene name is to provide a unique identifier.

The Mouse Genome Database (MGD) serves as a central repository of gene names and symbols to avoid use of the same name for different genes or use of multiple names for the same gene (http://www.informatics.jax.org). The MGD Nomenclature Committee (nomen@jax.org) provides advice and assistance in assigning new names and symbols. A web tool for proposing a new mouse locus symbol is located at the MGD site.

For the rat, these functions are carried out by RGD (http://rgd.mcw.edu) assisted by the International Rat Genome and Nomenclature Committee (RGNC). A web tool for proposing a new rat locus symbol is located at the RGD site.

2.1    Laboratory Codes

A key feature of mouse and rat nomenclature is the Laboratory Registration Code or Laboratory code, which is a code of usually three to four letters (first letter uppercase, followed by all lowercase), that identifies a particular institute, laboratory, or investigator that produced, and may hold stocks of, for example, a DNA marker, a mouse or rat strain, or were the creator of a new mutation. Laboratory codes are also used in naming chromosomal aberrations, transgenes, and genetically engineered mutations. Because Laboratory codes are key to identifying original sources, they are not assigned to "projects," but rather to the actual producer/creator individual or site. Laboratory codes can be assigned through MGD or directly by the Institute for Laboratory Animal Research (ILAR) at  http://dels-old.nas.edu/ilar_n/ilarhome/register_lc.php.

Examples:

J	The Jackson Laboratory
Mit	Massachusetts Institute of Technology
Leh	Hans Lehrach
Kyo	Kyoto University
Ztm	Central Animal Laboratory Medical School Hannover

2.2   Identification of New Genes

Identification of new genes in general comes in two ways; identification of a novel protein or DNA sequence or identification of a novel phenotype or trait. In the case of sequences, care should be taken in interpretation of database searches to establish novelty (for example, to distinguish between a new member of a gene family and an allele or alternative transcript of an existing family member). Novel mutant phenotypes or traits should be named according to their primary characteristic, but once the gene responsible for the phenotypic variation is identified, this gives the primary name of the gene and the mutant name becomes the name of the allele (see Section 2.3).

2.3   Gene Symbols and Names

2.3.1   Gene Symbols

Genes are given short symbols as convenient abbreviations for speaking and writing about the genes.

A gene symbol should:

• be unique within the species and should not match a symbol in another species that is not a homolog.
• be short, normally 3-5 characters, and not more than 10 characters.
• use only Roman letters and Arabic numbers.
• begin with an uppercase letter (not a number), followed by all lowercase letters / numbers (see exception below).
• not include tissue specificity or molecular weight designations.
• include punctuation only in specific special cases (see below).
• ideally have the same initial letter as the initial letter of its gene name to aid in indexing. However, letter order in a gene symbol need not follow word order in the name.

Examples:

Plaur	urokinase plasminogen activator receptor
Sta	autosomal striping

• be italicized in published articles. Because they may be difficult to read, depending on the browser, gene symbols are frequently not italicized when posted to a web page.
• use a common stem or root symbol when belonging to a gene family. Family member numbers or subunit designations should be placed at the end of the gene symbol.

Examples:

Glra1	glycine receptor, alpha 1 subunit
Glra2	glycine receptor, alpha 2 subunit
Glra3	glycine receptor, alpha 3 subunit

• use the same symbol whenever possible for orthologs among human, mouse and rat.

Exceptions to the rule of uppercase first letter and lowercase remaining letters in a gene or locus symbol:

• If the gene (locus) is only identified by a recessive mutant phenotype, then the symbol should begin with a lowercase letter. Once the mutant gene product is identified, the gene product is given a name and symbol and the original phenotype-based symbol and name becomes the allele symbol and name. The recessive nature of the allele is still conveyed by an initial lowercase letter.
• Within a gene symbol, Laboratory codes have an initial uppercase letter.
• When describing cross-hybridizing DNA segments, H (human) or other species code is uppercase, for example D2H11S14.
• When no information is available, other than the sequence itself, use the sequence identifier from the Mammalian Gene Collection, RIKEN, or GenBank (e.g., AF171077, 0610008A10Rik). If multiple sequence sources are available for the novel gene, preference is given first to a BC clone id (from Mammalian Gene Collection) followed by a RIKEN clone id, then the GenBank id.

Use of hyphens within the symbol should be kept to a minimum. Situations where hyphens may be used include:

• to separate related sequence and pseudogene symbols from the root

Examples:

Hk1-rs1	hexokinase-1 related sequence 1
Hba-ps3	hemoglobin alpha pseudogene 3

• within superscripted allele symbols (Section 3.1.2).

Example:

Kit W-v

Kit oncogene allele name: viable dominant spotting

2.3.2   Gene Names

Names of genes should be brief, and convey accurate information about the gene. The name should not convey detailed information about the gene or assay used; this can be associated with the gene in publications or databases. While the gene name should ideally be informative as to the function or nature of the gene, care should be taken to avoid putting inaccurate information in the name. For example, a "liver-specific protein" may be shown by subsequent studies to be expressed elsewhere.

A gene name should:

• be specific and brief, conveying the character or function of the gene.
• begin with a lowercase letter, unless it is a person's name or is a typically capitalized word.

Examples:

Blr1	Burkitt lymphoma receptor 1
Acly	ATP citrate lyase

• use American spelling.
• not contain punctuation, except where necessary to separate the main part of the name from modifiers.

Examples:

Acp1	acid phosphatase 1, soluble
Pigq	phosphatidylinositol glycan, class Q

• include the name of the species from which the ortholog/homolog name was derived at the end of the name in parentheses only when that name is not in common usage.

Examples:

Shh	sonic hedgehog  [commonly used, does not include species name]
Fjx1	four jointed box 1 (Drosophila) [name includes species derivative]

• not include the word mouse (for a mouse gene name) or the word rat (for a rat gene name).
• follow the conventions of the established gene family if it is a recognizable member of that family by sequence comparison, structure (motifs/domains), and/or function.
• not contain potentially misleading information that may be experiment or assay specific, such as "kidney-specific" or "59 kDa."

2.4  Structural Genes, Splice Variants, and Promoters

Ultimately, the majority of gene names will be for structural genes that encode protein. The gene should as far as possible be given the same name as the protein, whenever the protein is identified. If the gene is recognizable by sequence comparison as a member of an established gene family, it should be named accordingly (see Section 2.6).

2.4.1   Alternative Transcripts

Alternative transcripts that originate from the same gene are not normally given different gene symbols and names. To refer to specific splice forms of a gene, the following format should be used (gene symbol, followed by underscore, followed by sequence accession ID): Genesymbol_accID

Example:

Gene		Mttp	microsomal triglyceride transfer protein
Splice variant		Mttp_EU553486	microsomal triglyceride transfer protein splice variant defined by transcript sequence EU553486

Using the sequence accession ID provides an unambiguous and precise definition to the splice variant.

2.4.2   Read-through Transcripts

A read-through transcript is a multi-exon transcript that shares one of more exons with non-overlapping shorter transcripts that are considered to represent products of distinct loci. This is usually readily recognized as a distinct pattern, not to be confused with simple alternate splicing for a locus.

Read-through transcript genes should be named with a unique symbol and name. An example is diagrammed below.

2.4.3   Antisense and Opposite Strand Genes

Transcripts from the opposite strand that overlap another gene, or a transcript that is derived principally from the introns of another gene, or one that uses an alternative reading frame to another gene (and does not use the existing frame to a significant extent) should be given a different name.

A gene of unknown function, encoded at the same genomic locus (with overlapping exons) as another gene should have its own symbol. If the new gene regulates the first gene, it may be assigned the symbol of the first gene with the suffix “as” for antisense. The gene symbol should not be written backwards.

Example:

Igf2as

insulin-like growth factor 2, antisense

Genes of unknown function on the opposite strand, which have no proven regulatory function, should be assigned the symbol of the known gene with the suffix “os” for opposite strand.

Example:

Dnm3os

dynamin3, opposite strand

2.4.4   Genes with Homologs in Other Species

To aid interspecific comparison of genetic and other information, a gene that is identifiable as a homolog of an already named gene in another species can be named as "-like" "-homolog" or "-related." (Note: this is not the same as "related sequence" which applies to related sequences within mouse or within rat.) The gene name or symbol should not include the name mouse or the abbreviation "M" for mouse or the name rat or the abbreviation "R" for rat. Where possible, genes that are recognizable orthologs of already-named human genes should be given the same name and symbol as the human gene.

2.5   Phenotype Names and Symbols

Genes named for phenotypes should aim to convey the phenotype briefly and accurately in a few words. It is accepted that the name may not cover all aspects of the phenotype; what is needed is a succinct, memorable and, most importantly, unique, name. Bear in mind that identification of a variant or mutant phenotype is recognition of an allelic form of an as-yet unidentified gene that may already have or will be given a name.

2.5.1    Lethal Phenotypes

Genes identified solely by a recessive lethal phenotype with no heterozygous effect are named for the chromosomal assignment, a serial number and the name of the laboratory of origin (from the Laboratory code).

Examples:

l5H1	First lethal on Chromosome 5 at Harwell
l4Rn2	Second lethal on Chromosome 4 from laboratory of Gene Rinchik

2.6   Gene Families

Genes that appear to be members of a family should be named as family members. Evidence of gene families comes in a variety of forms, e.g., from a probe detecting multiple bands on a Southern blot, but is principally based on sequence comparisons.

2.6.1    Families Identified by Hybridization

Historically, many gene families have been identified as fragments detected by hybridization to the same probe but which map to different loci. These family members may be functional genes or pseudogenes. The loci can be named "related sequence" of the founder gene with a serial number (symbol -rs1, -rs2, and so on).

Example:

mouse ornithine decarboxylase-related sequences 1 to 21.

Odc-rs1 to Odc-rs21

If the founder or functional gene can not be identified, initially all the fragments are named "related sequence" until it is identified; then that particular "-rs" is dropped, without renumbering. If there is evidence that any loci are pseudogenes, they should be named as such and given serial numbers as in Section 2.6.2.

Once sequence evidence is accumulated on functional family members (which may or may not have been previously identified as members) a systematic naming scheme should be applied to the family as in Section 2.6.2.

2.6.2   Families Identified by Sequence Comparison

Sequencing can identify genes that are clearly members of a family (paralogs). Where possible, members of the family should be named and symbolized using the same stem followed by a serial number. The same family members in different mammalian species (orthologs) should, wherever possible, be given the same name and symbol. Pseudogenes should be suffixed by -ps and a serial number if there are multiple pseudogenes. Note that the numbering of pseudogenes among species is independent and no relationship should be implied among mouse, rat, or human pseudogenes based on their serial numbering.

Examples:

In mouse, phosphoglycerate kinase 1, pseudogenes 1 to 7, Pgk1-ps1 to Pgk1-ps7

In rat, calmodulin pseudogene 1, Calm-ps1

Numerous gene families have been recognized and given systematic nomenclature. Information on these families can be found at family-specific web sites, some of which are linked from MGD and RGD or RatMap. Names and symbols of new members of these families should follow the rules of the particular family and ideally be assigned in consultation with the curator of that family. Nomenclature schemes and curation of new families benefit from examination of existing models.

2.7   ESTs

Expressed Sequence Tags (ESTs) differ from other expressed sequences in that they are short, single pass sequences that are often convenient for PCR amplification from genomic DNA. ESTs that clearly derive from a known gene should be considered simply as an assay (marker) for that known gene. When anonymous ESTs are mapped onto genetic or physical maps, their designations should be symbolized using their sequence database accession number.

2.8   Anonymous DNA Segments

Only anonymous DNA segments that are mapped should be given systematic names and symbols.

2.8.1   Mapped DNA Segments

Anonymous DNA segments are named and symbolized according to the laboratory identifying or mapping the segment as "DNA segment, chromosome N, Lab Name" and a serial number, where N is the chromosomal assignment (1-19, X, Y in the mouse and 1-20, X, Y in the rat) and is symbolized as DNLabcode#.

Examples:

D8Mit17	the 17th locus mapped to mouse Chromosome 8 by M.I.T.
D1Arb27	the 27th locus mapped to rat Chromosome 1 at the Arthritis and Rheumatism Branch, NIAMS.

The same convention is applied to DNA segments that are variant loci within known genes.

Examples:

D4Mit17	an SSLP within the mouse Orm1 gene
D20Wox37	an SSLP within the rat Tnf gene

Mouse or rat DNA segments that are detected by cross-hybridization to human segments are given the human name with "chromosome N, cross-hybridizing to human DNA segment" inserted between DNA segment and the human segment code (see symbols). The same applies for rat DNA segments detected by cross-hybridization to mouse segments (or vice versa).

Examples:

D16H21S56	Mouse DNA segment on Chr 16 that cross-hybridizes with a DNA segment D21S56 from human Chr 21.
D1M7Mit236	Rat DNA segment on Chr 1 that cross-hybridizes with a DNA segment D7Mit236 from mouse Chr 7

2.8.2   STSs Used in Physical Mapping

When physical maps are assembled (YAC or BAC contigs, for example) many markers may be placed on the map in the form of Sequence Tagged Sites (STSs). These might be clone end-fragments, inter-repeat sequence PCR products, or random sequences from within clones. These markers serve to validate the contigs and appear on the maps, but their further utility may be limited. It is not necessary to give them names or symbols other than those assigned by the laboratory that produced and used them. If the STSs are used more widely, they should be assigned anonymous DNA segment names ("D-numbers").

2.9   Gene Trap Loci

Gene trap experiments in embryonic stem (ES) cells produce cell lines in which integration into a putative gene is selected by virtue of its expression in ES cells. The trapped gene is usually (though not necessarily) mutated by the integration. The site of integration can be characterized by a number of means, including cloning or extension of cDNA products. The loci of integration of a series of gene trap lines, once characterized as potentially unique, can be named and symbolized as members of a series, using the prefix Gt (for gene trap), followed by a vector designation in parentheses, a serial number assigned by the laboratory characterizing the locus, and the laboratory ILAR code. For example, the 26th gene "trapped" by the ROSA vector in the laboratory of Phillip Soriano (Sor) is symbolized as:

• Gt(ROSA)26Sor

A gene trap designation becomes an allele of the gene into which it was inserted, once that gene is identified. For example, Gt(ST629)Byg is known to disrupt the netrin 1 (Ntn1) gene; thus the full allele designation for this gene trap mutation is Ntn1Gt(ST629)Byg. See also the examples of gene trap mutations in Section 3.5.2.

2.10    Quantitative Trait Loci, Resistance Genes, and Immune Response Genes

Differences between inbred strains and the phenotype of offspring of crosses between strains provide evidence for the existence of genes affecting disease resistance, immune response, and many other quantitative traits (quantitative trait loci, QTL). Evidence for QTL is generally obtained through extensive genetic crossing and analysis that may uncover many genetic elements contributing to a phenotypic trait. Generally, the number and effects of QTL can only be deduced following experiments to map them. QTL should not be named until such mapping experiments have been performed.

2.10.1    Names and Symbols of QTL

Names and symbols for QTL should be brief and descriptive and reflect the trait or phenotype measured. Those QTL affecting the same trait should be given the same stem and serially numbered. The series is separate for mouse and rat and no homology should be implied by the serial numbers.

Some historically named QTL carry the name of the disease with which they are associated; these names are maintained; but newly identified QTL should be named for the measured trait and not a disease. The suffix "q" may be used optionally as the final letter preceding the serial number in QTL symbols.

Naming and symbolizing QTL follow the same conventions as for naming and symbolizing genes (Section 2.3). Specifically for a QTL, its name should include:

• a root name describing the measured trait
• the designation QTL (recommended)
• a serial number

Examples:

in mouse	Cafq1	caffeine metabolism QTL 1
	Cafq2	caffeine metabolism QTL 2
	Cafq3	caffeine metabolism QTL 3
in rat	Kidm1	kidney mass QTL 1
	Kidm2	kidney mass QTL 2
	Kidm3	kidney mass QTL 3

To obtain the next available serial number for a new QTL with an already established root name, e.g., the next in the series of "liver weight QTL" in mouse (Lwq#) or the next in series of "blood pressure QTL" for rat (Bp#), users should submit their QTL on the "proposing a new locus symbol" form at MGD (for mouse) or RGD (for rat). Note that examining the database content for a QTL is not sufficient, as a laboratory may have a QTL designation reserved and private, pending publication.

2.10.2   Defining uniqueness in QTL

Specific circumstances for naming independent QTL include:

• Independent experiments study the same trait and map that trait to the same chromosomal region

  Because QTL are detected in the context of specific strain combinations in specific crosses and generally in different laboratories using different assays, each experimentally detecting QTL will be given a unique symbol/name even when the trait measured and region defined is superficially the same as that of an existing QTL.

  Example: In mouse, Obq1 (obesity QTL 1) was identified and mapped to Chromosome 7 in a cross between strains 129/Sv and EL/Suz. Another obesity QTL was also mapped to Chromosome 7, but because it involved distinct strains (NZO and SM), it was given a different QTL designation, Obq15.

• A chromosomal region containing many measured "traits"

  If multiple traits are measured in a single experiment and mapped to a single chromosomal region, there may or may not be evidence that different QTL are involved. If the traits are physiologically related, the QTL name should be broad enough to represent all the measured traits or the name should reflect the trait showing the highest LOD score/p-value. Conversely, if there is clear evidence that the traits are independent, each trait will constitute a unique QTL.

  Examples: In mouse, Nidd1 (non-insulin-dependent diabetes mellitus 1) was associated with related measurements of plasma insulin, non-fasted blood glucose, and body weight and given a single QTL designation.

  In rats, Uae5 (urinary albumin excretion QTL 5) and Cm16 (cardiac mass QTL 16) are QTLs derived from the same experiment that map to overlapping regions of Chromosome 1. Because the measured traits are independent, different QTL designations are assigned.

2.11   Chromosomal Regions

Separate documents detail guidelines for nomenclature of chromosomes (for mouse, Rules for Nomenclature of Chromosome Aberrations are online; for rat, see Levan, et al., 1995). However, certain cytological features of normal chromosomes (such as telomeres, centromeres, and nucleolar organizers) and abnormal chromosomes (such as homogeneously-staining regions and end-points of deletions, inversions, and translocations) are genetic loci that are given names and symbols.

2.11.1   Telomeres

The functional telomere should be denoted by the symbol Tel. A DNA segment that includes the telomere repeat sequence (TTAGGG)n and which maps to a telomeric location is symbolized in four parts:

• Tel (for telomere)
• The number of the chromosome
• p or q (for the short or long arm, respectively)
• A serial number, if more than one segment is assigned to the telomere

For example, Tel4q1   telomeric sequence, Chr4, q arm 1

2.11.2   Centromeres and Pericentric Heterochromatin

The functional centromere should be denoted by the symbol Cen. Until the molecular nature of a functional mammalian centromere is defined, DNA segments that map to the centromere should be given anonymous DNA segment symbols as in Section 2.8.1.

Pericentric heterochromatin, that is cytologically visible, is given the symbol Hc#, in which # is the chromosome on which it is located.

• For example, Hc14 is the pericentric heterochromatin on Chromosome 14.

Variation in heterochromatin band size can be denoted by superscripts to the symbol.

• For example, Hc14n is normal heterochromatin; superscripts l and s would be used to denote long and short heterochromatin, respectively.

2.11.3   Nucleolus Organizers

The nucleolus organizer is a cytological structure that contains the ribosomal RNA genes. These genes are given the symbols Rnr and the number of the chromosome on which they are located.

• For example, Rnr12 is the ribosomal RNA locus on Chromosome 12.

If different Rnr loci can be genetically identified on the same chromosome, they are given serial numbers in order of identification.

• For example, Rnr19-1, Rnr19-2.

2.11.4  Homogeneously Staining Regions

Homogeneously staining regions (HSRs) are amplified internal subchromosomal bands that are identified cytologically by their Giemsa staining. A DNA segment that maps within an HSR is given a conventional DNA segment symbol, when its locus is on a normal (unamplified) chromosome. When expanded into an HSR its symbol follows the guidelines for insertions, thus becoming, for example, Is(HSR;1)1Lub.

2.11.5   Chromosomal Rearrangements

Symbols for chromosomal deletions, inversions, and translocations are given in the chromosomal nomenclature section. The end points of each of these rearrangements, however, define a locus. Where there is only a single locus on a chromosome, the chromosome anomaly symbol serves to define it. However, where an anomaly gives two loci on a single chromosome they can bedistinguished by the letters p and d for proximal and distal.

• For example, In(1)1Rk-p, In(1)1Rk-d are the proximal and distal end points of the chromosomal inversion In(1)1Rk in mouse.

2.12   Genes Residing on the Mitochondria

The mitochondria carry essential genes, among them many transfer RNA (tRNA) genes. Genes residing on the mitochondria have a prefix mt- (lowercase mt followed by a hyphen). For transfer RNAs, the symbols consist of three parts, mt-, T (for tRNA), and a single lowercase letter for the amino acid. The chromosomal designation for mitochondrial genes is Chr MT.

Examples:

mt-Tc	tRNA, cysteine, mitochondrial (a tRNA gene residing on the mitochondria)
mt-Atp6	ATP synthase 6, mitochondrial  (a non-tRNA gene residing on the mitochondria)

2.13   RNA Genes Encoded in the Nucleus

There are hundreds of loci encoding transfer RNAs (tRNA) and ribosomal RNAs (rRNA), and many are encoded in the nucleus. The following method symbolizes these nuclear-encoded RNA genes:Naming nuclear encoded transfer-RNAs

Symbols for nuclear encoded transfer-RNAs consist of four parts:

n-		lowercase n followed by a hyphen to indicate nuclear encoding
T		uppercase T to indicate transfer-RNA
aa		the single letter abbreviation for the amino acid
#		serial number for this transfer-RNA
Example:
n-Ta12		nuclear encoded tRNA alanine 12 (anticodon AGC)

Naming nuclear encoded ribosomal-RNAs

Symbols for nuclear encoded ribosomal-RNAs consist of four parts:

n-		lowercase n followed by a hyphen to indicate nuclear encoding
R		uppercase R to indicate ribosomal-RNA
subunit		the subunit designation
#		serial number for this ribosomal-RNA
Example:
n-R5s104		nuclear encoded rRNA 5S 104

2.14   microRNAs and microRNA clusters

MicroRNAs (miRNAs) are abundant, short RNA molecules that are post-transcriptional regulators that bind to complementary sequences on target mRNA transcripts, usually resulting in translational repression or target degradation and gene silencing.

Naming microRNAs Symbols for microRNAs consist of the root symbol Mir followed by the numbering scheme tracked in the miRBase database (www.mirbase.org), a database tracking microRNAs reported for all species.

For example, mouse Mir143 (microRNA 143) is represented as mmu-mir-143 in miRBase, with the mmu signifying mouse.

Naming microRNA clusters A microRNA cluster consists of several microRNAs in immediate genome proximity. These may be given symbols and names to refer unambiguously to the entire cluster.

For a microRNA cluster, the name will consist of the root symbol Mirc (for microRNA cluster) followed by a serial number (1, 2, 3…) for the cluster. MGI (for mouse) or RGD (for rat) should be consulted for the next available cluster number when a new cluster is defined. The list of microRNAs included in each cluster will be recorded in relevant database records for the genes, knockouts, and strains.

(Note that this differs from the definition of miRBase, which simply refers to clustered miRNAs as those less than 10kb from the miRNA of interest. Thus, in miRBase clusters defined based on one miRNA may or may not overlap clusters based on another miRNA.

2.15   Enhancers, Promoters, and Regulatory Regions

Enhancers, promoters, and regulatory regions can influence multiple genes. In addition, they can be localized far away from the gene(s) that they affect. Thus, it is misleading to name them based on the gene for which regulation was first recognized.

Enhancers, promoters, and regulatory regions are to be symbolized as:

Rr#	regulatory region #
where # indicates the next number in the series.

Guidelines for Nomenclature of Genes, Genetic Markers, Alleles, and Mutations in Mouse and Rat 

International Committee on Standardized Genetic Nomenclature for Mice

Chairperson: Dr. Janan T. Eppig 
(e-mail:janan.eppig@jax.org)

Rat Genome and Nomenclature Committee

Chairperson: Dr. Goran Levan
(e-mail: Goran.Levan@gen.gu.se)

Rules for mouse genetic nomenclature were first published by Dunn, Gruneberg, and Snell (1940) and subsequently revised by the International Committee for Standardized Genetic Nomenclature in Mice (1963, 1973, 1981, 1989, 1996). The most recent publication of mouse nomenclature guidelines can be found in Eppig (2006). Users should be advised, however, that this web version represents the current nomenclature policies of the International Committee for Standardized Genetic Nomenclature for Mice and takes precedent over previously published versions.

Rules for rat genetic nomenclature were first published by the Committee on Rat Nomenclature in 1992 and then by Levan et al. in 1995.

In 2003, the International Committee on Standardized Genetic Nomenclature for Mice and the Rat Genome and Nomenclature Committee agreed to unify the rules and guidelines for gene, allele, and mutation nomenclature in mouse and rats. Nomenclature guidelines are now reviewed and updated annually by the two International Committees; current guidelines can be found on the MGD and RGD web sites.

1  Principles of Nomenclature

1.1   Key Features

The key component of nomenclature is the gene or locus name and symbol, which identifies a unit of inheritance. Other features, such as alleles, variants and mutations, are secondary to the gene name and become associated with it. Similarly, probes or assays used to detect a gene are not primary features and should not normally be used as names.

The primary purpose of a gene or locus name and symbol is to be a unique identifier so that information about the gene in publications, databases and other forms of communication can be unambiguously associated with the correct gene. These guidelines, therefore, are intended to aid the scientific community as a whole to use genetic information.

Other, secondary, functions of nomenclature for genes are to:

• identify the gene as a member of a family, which may give further information about the gene by reference to other family members
• identify the gene as the ortholog of a gene in another mammal (usually human)

1.2   Definitions

It is important that the user understands what is being named and the principles underlying these guidelines. Section 6 presents definitions that will aid the user in distinguishing, for example, genes, loci, markers, and alleles.

1.3  Stability of Nomenclature

On the whole gene names should be stable; that is, they should not be changed over time. However there are certain circumstances where a change is desirable:

• In cases where a gene has been known only as, and named for, a mutant phenotype: when the mutated gene is identified, then the mutant name becomes the mutant allele name of the identified gene (see Section 3.1.2).
• Where a gene becomes assigned to a gene family (of paralogs), and the nomenclature of the family is established. (see Section 2.6.2).
• Where orthologous gene(s) have been identified between mouse, rat, and human, and a common symbol is adopted for all three species.

1.4   Synonyms

A gene can have several synonyms, which are names or symbols that have been applied to the gene at various times. These synonyms may be associated with the gene in databases and publications, but the established gene name and symbol should always be used as the primary identifier.

1.5    Gene symbols, proteins, and chromosome designations in publications

1.5.1     Gene and allele symbols

Gene symbols are italicized when published, as are allele symbols. Section 2 below specifies naming rules for establishing correct symbols. Help is available for determining correct gene and allele symbol assignment (nomen@jax.org) and symbols can be reserved privately pre-publication.

To distinguish between mRNA, genomic DNA, and cDNA forms within a manuscript, write the relevant prefix in parentheses before the gene symbol, for example, (mRNA) Rbp1.

1.5.2    Protein symbols

Protein designations follow the same rules as gene symbols, with the following two distinctions:

• Protein symbols use all uppercase letters.
• Protein symbols are not italicized.

1.5.3   Chromosome designations

• Use uppercase "C" when referring to a specific mouse chromosome (e.g., Chromosome 15).
• When abbreviating the word Chromosome, do not use a period (".") after the abbreviation (e.g., Chromosome 15 should abbreviated as Chr 15 and not Chr.15).

2   Symbols and Names of Genes and Loci

The prime function of a gene name is to provide a unique identifier.

The Mouse Genome Database (MGD) serves as a central repository of gene names and symbols to avoid use of the same name for different genes or use of multiple names for the same gene (http://www.informatics.jax.org). The MGD Nomenclature Committee (nomen@jax.org) provides advice and assistance in assigning new names and symbols. A web tool for proposing a new mouse locus symbol is located at the MGD site.

For the rat, these functions are carried out by RGD (http://rgd.mcw.edu) assisted by the International Rat Genome and Nomenclature Committee (RGNC). A web tool for proposing a new rat locus symbol is located at the RGD site.

2.1    Laboratory Codes

A key feature of mouse and rat nomenclature is the Laboratory Registration Code or Laboratory code, which is a code of usually three to four letters (first letter uppercase, followed by all lowercase), that identifies a particular institute, laboratory, or investigator that produced, and may hold stocks of, for example, a DNA marker, a mouse or rat strain, or were the creator of a new mutation. Laboratory codes are also used in naming chromosomal aberrations, transgenes, and genetically engineered mutations. Because Laboratory codes are key to identifying original sources, they are not assigned to "projects," but rather to the actual producer/creator individual or site. Laboratory codes can be assigned through MGD or directly by the Institute for Laboratory Animal Research (ILAR) at  http://dels-old.nas.edu/ilar_n/ilarhome/register_lc.php.

Examples:

J	The Jackson Laboratory
Mit	Massachusetts Institute of Technology
Leh	Hans Lehrach
Kyo	Kyoto University
Ztm	Central Animal Laboratory Medical School Hannover

2.2   Identification of New Genes

Identification of new genes in general comes in two ways; identification of a novel protein or DNA sequence or identification of a novel phenotype or trait. In the case of sequences, care should be taken in interpretation of database searches to establish novelty (for example, to distinguish between a new member of a gene family and an allele or alternative transcript of an existing family member). Novel mutant phenotypes or traits should be named according to their primary characteristic, but once the gene responsible for the phenotypic variation is identified, this gives the primary name of the gene and the mutant name becomes the name of the allele (see Section 2.3).

2.3   Gene Symbols and Names

2.3.1   Gene Symbols

Genes are given short symbols as convenient abbreviations for speaking and writing about the genes.

A gene symbol should:

• be unique within the species and should not match a symbol in another species that is not a homolog.
• be short, normally 3-5 characters, and not more than 10 characters.
• use only Roman letters and Arabic numbers.
• begin with an uppercase letter (not a number), followed by all lowercase letters / numbers (see exception below).
• not include tissue specificity or molecular weight designations.
• include punctuation only in specific special cases (see below).
• ideally have the same initial letter as the initial letter of its gene name to aid in indexing. However, letter order in a gene symbol need not follow word order in the name.

Examples:

Plaur	urokinase plasminogen activator receptor
Sta	autosomal striping

• be italicized in published articles. Because they may be difficult to read, depending on the browser, gene symbols are frequently not italicized when posted to a web page.
• use a common stem or root symbol when belonging to a gene family. Family member numbers or subunit designations should be placed at the end of the gene symbol.

Examples:

Glra1	glycine receptor, alpha 1 subunit
Glra2	glycine receptor, alpha 2 subunit
Glra3	glycine receptor, alpha 3 subunit

• use the same symbol whenever possible for orthologs among human, mouse and rat.

Exceptions to the rule of uppercase first letter and lowercase remaining letters in a gene or locus symbol:

• If the gene (locus) is only identified by a recessive mutant phenotype, then the symbol should begin with a lowercase letter. Once the mutant gene product is identified, the gene product is given a name and symbol and the original phenotype-based symbol and name becomes the allele symbol and name. The recessive nature of the allele is still conveyed by an initial lowercase letter.
• Within a gene symbol, Laboratory codes have an initial uppercase letter.
• When describing cross-hybridizing DNA segments, H (human) or other species code is uppercase, for example D2H11S14.
• When no information is available, other than the sequence itself, use the sequence identifier from the Mammalian Gene Collection, RIKEN, or GenBank (e.g., AF171077, 0610008A10Rik). If multiple sequence sources are available for the novel gene, preference is given first to a BC clone id (from Mammalian Gene Collection) followed by a RIKEN clone id, then the GenBank id.

Use of hyphens within the symbol should be kept to a minimum. Situations where hyphens may be used include:

• to separate related sequence and pseudogene symbols from the root

Examples:

Hk1-rs1	hexokinase-1 related sequence 1
Hba-ps3	hemoglobin alpha pseudogene 3

• within superscripted allele symbols (Section 3.1.2).

Example:

Kit W-v

Kit oncogene allele name: viable dominant spotting

2.3.2   Gene Names

Names of genes should be brief, and convey accurate information about the gene. The name should not convey detailed information about the gene or assay used; this can be associated with the gene in publications or databases. While the gene name should ideally be informative as to the function or nature of the gene, care should be taken to avoid putting inaccurate information in the name. For example, a "liver-specific protein" may be shown by subsequent studies to be expressed elsewhere.

A gene name should:

• be specific and brief, conveying the character or function of the gene.
• begin with a lowercase letter, unless it is a person's name or is a typically capitalized word.

Examples:

Blr1	Burkitt lymphoma receptor 1
Acly	ATP citrate lyase

• use American spelling.
• not contain punctuation, except where necessary to separate the main part of the name from modifiers.

Examples:

Acp1	acid phosphatase 1, soluble
Pigq	phosphatidylinositol glycan, class Q

• include the name of the species from which the ortholog/homolog name was derived at the end of the name in parentheses only when that name is not in common usage.

Examples:

Shh	sonic hedgehog  [commonly used, does not include species name]
Fjx1	four jointed box 1 (Drosophila) [name includes species derivative]

• not include the word mouse (for a mouse gene name) or the word rat (for a rat gene name).
• follow the conventions of the established gene family if it is a recognizable member of that family by sequence comparison, structure (motifs/domains), and/or function.
• not contain potentially misleading information that may be experiment or assay specific, such as "kidney-specific" or "59 kDa."

2.4  Structural Genes, Splice Variants, and Promoters

Ultimately, the majority of gene names will be for structural genes that encode protein. The gene should as far as possible be given the same name as the protein, whenever the protein is identified. If the gene is recognizable by sequence comparison as a member of an established gene family, it should be named accordingly (see Section 2.6).

2.4.1   Alternative Transcripts

Alternative transcripts that originate from the same gene are not normally given different gene symbols and names. To refer to specific splice forms of a gene, the following format should be used (gene symbol, followed by underscore, followed by sequence accession ID): Genesymbol_accID

Example:

Gene		Mttp	microsomal triglyceride transfer protein
Splice variant		Mttp_EU553486	microsomal triglyceride transfer protein splice variant defined by transcript sequence EU553486

Using the sequence accession ID provides an unambiguous and precise definition to the splice variant.

2.4.2   Read-through Transcripts

A read-through transcript is a multi-exon transcript that shares one of more exons with non-overlapping shorter transcripts that are considered to represent products of distinct loci. This is usually readily recognized as a distinct pattern, not to be confused with simple alternate splicing for a locus.

Read-through transcript genes should be named with a unique symbol and name. An example is diagrammed below.

2.4.3   Antisense and Opposite Strand Genes

Transcripts from the opposite strand that overlap another gene, or a transcript that is derived principally from the introns of another gene, or one that uses an alternative reading frame to another gene (and does not use the existing frame to a significant extent) should be given a different name.

A gene of unknown function, encoded at the same genomic locus (with overlapping exons) as another gene should have its own symbol. If the new gene regulates the first gene, it may be assigned the symbol of the first gene with the suffix “as” for antisense. The gene symbol should not be written backwards.

Example:

Igf2as

insulin-like growth factor 2, antisense

Genes of unknown function on the opposite strand, which have no proven regulatory function, should be assigned the symbol of the known gene with the suffix “os” for opposite strand.

Example:

Dnm3os

dynamin3, opposite strand

2.4.4   Genes with Homologs in Other Species

To aid interspecific comparison of genetic and other information, a gene that is identifiable as a homolog of an already named gene in another species can be named as "-like" "-homolog" or "-related." (Note: this is not the same as "related sequence" which applies to related sequences within mouse or within rat.) The gene name or symbol should not include the name mouse or the abbreviation "M" for mouse or the name rat or the abbreviation "R" for rat. Where possible, genes that are recognizable orthologs of already-named human genes should be given the same name and symbol as the human gene.

2.5   Phenotype Names and Symbols

Genes named for phenotypes should aim to convey the phenotype briefly and accurately in a few words. It is accepted that the name may not cover all aspects of the phenotype; what is needed is a succinct, memorable and, most importantly, unique, name. Bear in mind that identification of a variant or mutant phenotype is recognition of an allelic form of an as-yet unidentified gene that may already have or will be given a name.

2.5.1    Lethal Phenotypes

Genes identified solely by a recessive lethal phenotype with no heterozygous effect are named for the chromosomal assignment, a serial number and the name of the laboratory of origin (from the Laboratory code).

Examples:

l5H1	First lethal on Chromosome 5 at Harwell
l4Rn2	Second lethal on Chromosome 4 from laboratory of Gene Rinchik

2.6   Gene Families

Genes that appear to be members of a family should be named as family members. Evidence of gene families comes in a variety of forms, e.g., from a probe detecting multiple bands on a Southern blot, but is principally based on sequence comparisons.

2.6.1    Families Identified by Hybridization

Historically, many gene families have been identified as fragments detected by hybridization to the same probe but which map to different loci. These family members may be functional genes or pseudogenes. The loci can be named "related sequence" of the founder gene with a serial number (symbol -rs1, -rs2, and so on).

Example:

mouse ornithine decarboxylase-related sequences 1 to 21.

Odc-rs1 to Odc-rs21

If the founder or functional gene can not be identified, initially all the fragments are named "related sequence" until it is identified; then that particular "-rs" is dropped, without renumbering. If there is evidence that any loci are pseudogenes, they should be named as such and given serial numbers as in Section 2.6.2.

Once sequence evidence is accumulated on functional family members (which may or may not have been previously identified as members) a systematic naming scheme should be applied to the family as in Section 2.6.2.

2.6.2   Families Identified by Sequence Comparison

Sequencing can identify genes that are clearly members of a family (paralogs). Where possible, members of the family should be named and symbolized using the same stem followed by a serial number. The same family members in different mammalian species (orthologs) should, wherever possible, be given the same name and symbol. Pseudogenes should be suffixed by -ps and a serial number if there are multiple pseudogenes. Note that the numbering of pseudogenes among species is independent and no relationship should be implied among mouse, rat, or human pseudogenes based on their serial numbering.

Examples:

In mouse, phosphoglycerate kinase 1, pseudogenes 1 to 7, Pgk1-ps1 to Pgk1-ps7

In rat, calmodulin pseudogene 1, Calm-ps1

Numerous gene families have been recognized and given systematic nomenclature. Information on these families can be found at family-specific web sites, some of which are linked from MGD and RGD or RatMap. Names and symbols of new members of these families should follow the rules of the particular family and ideally be assigned in consultation with the curator of that family. Nomenclature schemes and curation of new families benefit from examination of existing models.

2.7   ESTs

Expressed Sequence Tags (ESTs) differ from other expressed sequences in that they are short, single pass sequences that are often convenient for PCR amplification from genomic DNA. ESTs that clearly derive from a known gene should be considered simply as an assay (marker) for that known gene. When anonymous ESTs are mapped onto genetic or physical maps, their designations should be symbolized using their sequence database accession number.

2.8   Anonymous DNA Segments

Only anonymous DNA segments that are mapped should be given systematic names and symbols.

2.8.1   Mapped DNA Segments

Anonymous DNA segments are named and symbolized according to the laboratory identifying or mapping the segment as "DNA segment, chromosome N, Lab Name" and a serial number, where N is the chromosomal assignment (1-19, X, Y in the mouse and 1-20, X, Y in the rat) and is symbolized as DNLabcode#.

Examples:

D8Mit17	the 17th locus mapped to mouse Chromosome 8 by M.I.T.
D1Arb27	the 27th locus mapped to rat Chromosome 1 at the Arthritis and Rheumatism Branch, NIAMS.

The same convention is applied to DNA segments that are variant loci within known genes.

Examples:

D4Mit17	an SSLP within the mouse Orm1 gene
D20Wox37	an SSLP within the rat Tnf gene

Mouse or rat DNA segments that are detected by cross-hybridization to human segments are given the human name with "chromosome N, cross-hybridizing to human DNA segment" inserted between DNA segment and the human segment code (see symbols). The same applies for rat DNA segments detected by cross-hybridization to mouse segments (or vice versa).

Examples:

D16H21S56	Mouse DNA segment on Chr 16 that cross-hybridizes with a DNA segment D21S56 from human Chr 21.
D1M7Mit236	Rat DNA segment on Chr 1 that cross-hybridizes with a DNA segment D7Mit236 from mouse Chr 7

2.8.2   STSs Used in Physical Mapping

When physical maps are assembled (YAC or BAC contigs, for example) many markers may be placed on the map in the form of Sequence Tagged Sites (STSs). These might be clone end-fragments, inter-repeat sequence PCR products, or random sequences from within clones. These markers serve to validate the contigs and appear on the maps, but their further utility may be limited. It is not necessary to give them names or symbols other than those assigned by the laboratory that produced and used them. If the STSs are used more widely, they should be assigned anonymous DNA segment names ("D-numbers").

2.9   Gene Trap Loci

Gene trap experiments in embryonic stem (ES) cells produce cell lines in which integration into a putative gene is selected by virtue of its expression in ES cells. The trapped gene is usually (though not necessarily) mutated by the integration. The site of integration can be characterized by a number of means, including cloning or extension of cDNA products. The loci of integration of a series of gene trap lines, once characterized as potentially unique, can be named and symbolized as members of a series, using the prefix Gt (for gene trap), followed by a vector designation in parentheses, a serial number assigned by the laboratory characterizing the locus, and the laboratory ILAR code. For example, the 26th gene "trapped" by the ROSA vector in the laboratory of Phillip Soriano (Sor) is symbolized as:

• Gt(ROSA)26Sor

A gene trap designation becomes an allele of the gene into which it was inserted, once that gene is identified. For example, Gt(ST629)Byg is known to disrupt the netrin 1 (Ntn1) gene; thus the full allele designation for this gene trap mutation is Ntn1Gt(ST629)Byg. See also the examples of gene trap mutations in Section 3.5.2.

2.10    Quantitative Trait Loci, Resistance Genes, and Immune Response Genes

Differences between inbred strains and the phenotype of offspring of crosses between strains provide evidence for the existence of genes affecting disease resistance, immune response, and many other quantitative traits (quantitative trait loci, QTL). Evidence for QTL is generally obtained through extensive genetic crossing and analysis that may uncover many genetic elements contributing to a phenotypic trait. Generally, the number and effects of QTL can only be deduced following experiments to map them. QTL should not be named until such mapping experiments have been performed.

2.10.1    Names and Symbols of QTL

Names and symbols for QTL should be brief and descriptive and reflect the trait or phenotype measured. Those QTL affecting the same trait should be given the same stem and serially numbered. The series is separate for mouse and rat and no homology should be implied by the serial numbers.

Some historically named QTL carry the name of the disease with which they are associated; these names are maintained; but newly identified QTL should be named for the measured trait and not a disease. The suffix "q" may be used optionally as the final letter preceding the serial number in QTL symbols.

Naming and symbolizing QTL follow the same conventions as for naming and symbolizing genes (Section 2.3). Specifically for a QTL, its name should include:

• a root name describing the measured trait
• the designation QTL (recommended)
• a serial number

Examples:

in mouse	Cafq1	caffeine metabolism QTL 1
	Cafq2	caffeine metabolism QTL 2
	Cafq3	caffeine metabolism QTL 3
in rat	Kidm1	kidney mass QTL 1
	Kidm2	kidney mass QTL 2
	Kidm3	kidney mass QTL 3

To obtain the next available serial number for a new QTL with an already established root name, e.g., the next in the series of "liver weight QTL" in mouse (Lwq#) or the next in series of "blood pressure QTL" for rat (Bp#), users should submit their QTL on the "proposing a new locus symbol" form at MGD (for mouse) or RGD (for rat). Note that examining the database content for a QTL is not sufficient, as a laboratory may have a QTL designation reserved and private, pending publication.

2.10.2   Defining uniqueness in QTL

Specific circumstances for naming independent QTL include:

• Independent experiments study the same trait and map that trait to the same chromosomal region

  Because QTL are detected in the context of specific strain combinations in specific crosses and generally in different laboratories using different assays, each experimentally detecting QTL will be given a unique symbol/name even when the trait measured and region defined is superficially the same as that of an existing QTL.

  Example: In mouse, Obq1 (obesity QTL 1) was identified and mapped to Chromosome 7 in a cross between strains 129/Sv and EL/Suz. Another obesity QTL was also mapped to Chromosome 7, but because it involved distinct strains (NZO and SM), it was given a different QTL designation, Obq15.

• A chromosomal region containing many measured "traits"

  If multiple traits are measured in a single experiment and mapped to a single chromosomal region, there may or may not be evidence that different QTL are involved. If the traits are physiologically related, the QTL name should be broad enough to represent all the measured traits or the name should reflect the trait showing the highest LOD score/p-value. Conversely, if there is clear evidence that the traits are independent, each trait will constitute a unique QTL.

  Examples: In mouse, Nidd1 (non-insulin-dependent diabetes mellitus 1) was associated with related measurements of plasma insulin, non-fasted blood glucose, and body weight and given a single QTL designation.

  In rats, Uae5 (urinary albumin excretion QTL 5) and Cm16 (cardiac mass QTL 16) are QTLs derived from the same experiment that map to overlapping regions of Chromosome 1. Because the measured traits are independent, different QTL designations are assigned.

2.11   Chromosomal Regions

Separate documents detail guidelines for nomenclature of chromosomes (for mouse, Rules for Nomenclature of Chromosome Aberrations are online; for rat, see Levan, et al., 1995). However, certain cytological features of normal chromosomes (such as telomeres, centromeres, and nucleolar organizers) and abnormal chromosomes (such as homogeneously-staining regions and end-points of deletions, inversions, and translocations) are genetic loci that are given names and symbols.

2.11.1   Telomeres

The functional telomere should be denoted by the symbol Tel. A DNA segment that includes the telomere repeat sequence (TTAGGG)n and which maps to a telomeric location is symbolized in four parts:

• Tel (for telomere)
• The number of the chromosome
• p or q (for the short or long arm, respectively)
• A serial number, if more than one segment is assigned to the telomere

For example, Tel4q1   telomeric sequence, Chr4, q arm 1

2.11.2   Centromeres and Pericentric Heterochromatin

The functional centromere should be denoted by the symbol Cen. Until the molecular nature of a functional mammalian centromere is defined, DNA segments that map to the centromere should be given anonymous DNA segment symbols as in Section 2.8.1.

Pericentric heterochromatin, that is cytologically visible, is given the symbol Hc#, in which # is the chromosome on which it is located.

• For example, Hc14 is the pericentric heterochromatin on Chromosome 14.

Variation in heterochromatin band size can be denoted by superscripts to the symbol.

• For example, Hc14n is normal heterochromatin; superscripts l and s would be used to denote long and short heterochromatin, respectively.

2.11.3   Nucleolus Organizers

The nucleolus organizer is a cytological structure that contains the ribosomal RNA genes. These genes are given the symbols Rnr and the number of the chromosome on which they are located.

• For example, Rnr12 is the ribosomal RNA locus on Chromosome 12.

If different Rnr loci can be genetically identified on the same chromosome, they are given serial numbers in order of identification.

• For example, Rnr19-1, Rnr19-2.

2.11.4  Homogeneously Staining Regions

Homogeneously staining regions (HSRs) are amplified internal subchromosomal bands that are identified cytologically by their Giemsa staining. A DNA segment that maps within an HSR is given a conventional DNA segment symbol, when its locus is on a normal (unamplified) chromosome. When expanded into an HSR its symbol follows the guidelines for insertions, thus becoming, for example, Is(HSR;1)1Lub.

2.11.5   Chromosomal Rearrangements

Symbols for chromosomal deletions, inversions, and translocations are given in the chromosomal nomenclature section. The end points of each of these rearrangements, however, define a locus. Where there is only a single locus on a chromosome, the chromosome anomaly symbol serves to define it. However, where an anomaly gives two loci on a single chromosome they can bedistinguished by the letters p and d for proximal and distal.

• For example, In(1)1Rk-p, In(1)1Rk-d are the proximal and distal end points of the chromosomal inversion In(1)1Rk in mouse.

2.12   Genes Residing on the Mitochondria

The mitochondria carry essential genes, among them many transfer RNA (tRNA) genes. Genes residing on the mitochondria have a prefix mt- (lowercase mt followed by a hyphen). For transfer RNAs, the symbols consist of three parts, mt-, T (for tRNA), and a single lowercase letter for the amino acid. The chromosomal designation for mitochondrial genes is Chr MT.

Examples:

mt-Tc	tRNA, cysteine, mitochondrial (a tRNA gene residing on the mitochondria)
mt-Atp6	ATP synthase 6, mitochondrial  (a non-tRNA gene residing on the mitochondria)

2.13   RNA Genes Encoded in the Nucleus

There are hundreds of loci encoding transfer RNAs (tRNA) and ribosomal RNAs (rRNA), and many are encoded in the nucleus. The following method symbolizes these nuclear-encoded RNA genes:Naming nuclear encoded transfer-RNAs

Symbols for nuclear encoded transfer-RNAs consist of four parts:

n-		lowercase n followed by a hyphen to indicate nuclear encoding
T		uppercase T to indicate transfer-RNA
aa		the single letter abbreviation for the amino acid
#		serial number for this transfer-RNA
Example:
n-Ta12		nuclear encoded tRNA alanine 12 (anticodon AGC)

Naming nuclear encoded ribosomal-RNAs

Symbols for nuclear encoded ribosomal-RNAs consist of four parts:

n-		lowercase n followed by a hyphen to indicate nuclear encoding
R		uppercase R to indicate ribosomal-RNA
subunit		the subunit designation
#		serial number for this ribosomal-RNA
Example:
n-R5s104		nuclear encoded rRNA 5S 104

2.14   microRNAs and microRNA clusters

MicroRNAs (miRNAs) are abundant, short RNA molecules that are post-transcriptional regulators that bind to complementary sequences on target mRNA transcripts, usually resulting in translational repression or target degradation and gene silencing.

Naming microRNAs Symbols for microRNAs consist of the root symbol Mir followed by the numbering scheme tracked in the miRBase database (www.mirbase.org), a database tracking microRNAs reported for all species.

For example, mouse Mir143 (microRNA 143) is represented as mmu-mir-143 in miRBase, with the mmu signifying mouse.

Naming microRNA clusters A microRNA cluster consists of several microRNAs in immediate genome proximity. These may be given symbols and names to refer unambiguously to the entire cluster.

For a microRNA cluster, the name will consist of the root symbol Mirc (for microRNA cluster) followed by a serial number (1, 2, 3…) for the cluster. MGI (for mouse) or RGD (for rat) should be consulted for the next available cluster number when a new cluster is defined. The list of microRNAs included in each cluster will be recorded in relevant database records for the genes, knockouts, and strains.

(Note that this differs from the definition of miRBase, which simply refers to clustered miRNAs as those less than 10kb from the miRNA of interest. Thus, in miRBase clusters defined based on one miRNA may or may not overlap clusters based on another miRNA.

2.15   Enhancers, Promoters, and Regulatory Regions

Enhancers, promoters, and regulatory regions can influence multiple genes. In addition, they can be localized far away from the gene(s) that they affect. Thus, it is misleading to name them based on the gene for which regulation was first recognized.

Enhancers, promoters, and regulatory regions are to be symbolized as:

Rr#	regulatory region #
where # indicates the next number in the series.

3   Names and Symbols for Variant and Mutant Alleles

Different alleles of a gene or locus can be distinguished by a number of methods, including DNA fragment length, protein electrophoretic mobility, or variant physiological or morphological phenotype.

All mutant alleles, whether of spontaneous or induced origin, targeted mutations, gene traps, or transgenics should be submitted to MGD (mouse) or RGD (rat) for an allele or gene accession identifier.

3.1   Mutant Phenotypes

3.1.1   Genes Known Only by Mutant Phenotypes

Where a gene is known only by mutant phenotype, the gene is given the name and symbol of the first identified mutant. Symbols of mutations conferring a recessive phenotype begin with a lowercase letter; symbols for dominant or semidominant phenotype genes begin with an uppercase letter.

Examples:

In mouse, recessive spotting, rs; abnormal feet and tail, Aft; circling, cir

In rat, polydactyly-luxate, lx.

Further (allelic) mutations at the same locus, if they have the same phenotype, are given the same name with a Laboratory code preceded by a serial number (if more than one additional allele from the same lab). In the symbol the Laboratory code is added as a superscript.

• For example, agil2J, the second new allele of mouse agitans-like identified at The Jackson Laboratory.

If a new allelic mutation of a gene known only by a mutant phenotype is caused by a transgenic insertion, the symbol of this mutation should use the symbol of the transgene as superscript (see Section 3.4.2 and Section 4).

• awgTg(GBtslenv)832Pkw; mutation of abnormal wobbly gait caused by a transgene, mouse line 832, produced in the laboratory of Paul Wong. (An abbreviated form, awgTg832Pkw can be used if the abbreviated designation is unique).

If the additional allele has a different phenotype, it may be given a different name, but when symbolized the new mutant symbol is superscripted to the original mutant symbol. Also, if a new mutation is described and named but not shown to be an allele of an existing gene until later, the original name of the new mutation can be kept. Even if the phenotype is apparently identical, the original symbol is used, with the new mutation symbol as superscript.

For example

• grey coat is an allele of recessive spotting (rs) in the mouse, and hence is symbolized rsgrc.

3.1.2   Phenotypes Due to Mutations in Structural Genes

When a spontaneous or induced mutant phenotype is subsequently found to be a mutation in a structural gene, or the gene in which the mutation has occurred is cloned, the mutation becomes an allele of that gene and the symbol for the mutant allele is formed by adding the original mutant symbol as a superscript to the new gene symbol. (The mutant symbol should retain its initial upper or lowercase letter).

• The hotfoot (ho) mutation of the mouse glutamate receptor Grid2, Grid2ho.
• The dominant white spotting (W) mutation of mouse Kit, KitW

If the original mutation has multiple alleles, when describing these alleles, their symbols become part of the superscript to the identified structural gene.

• creeper, Grid2ho-cpr.
• viable white spotting, KitW-v; sash, KitW-sh.

Even if the identified gene is novel and unnamed, it is recommended that it is nevertheless given a name and symbol different from the mutant name and symbol. This will more readily allow discrimination between mutant and wild type and between gene and phenotype.

3.1.3   Wild Type Alleles and Revertants

The wild type allele of a gene is indicated by + as superscript to the mutant symbol.

• The wild type allele of the agitans-like mutation, agil+.
• The wild type Kit locus (if necessary to distinguish from mutations), Kit+.

A revertant to wild type of a mutant phenotype locus should be indicated by the symbol + with the mutant symbol as superscript.

• Revertant to wild type at the hairless mutant locus +hr

Additional revertants are given a Laboratory Code and preceded by a serial number if more than one revertant is found in a lab. Serial numbers are independent for mouse and rat revertants and no homology is implied. If the revertant is in a gene that has been cloned, then the mutant symbol is retained as superscript to the gene symbol, and + is appended.

• Revertant to wild type of the dilute mutation of myosin Va; Myo5ad+
• Second such revertant identified at The Jackson Laboratory; Myo5ad+2J.

3.2   Variants

3.2.1   Biochemical Variants

Electrophoretic or other biochemicalvariant alleles of known structural genes are usually given lowercase letters to indicate different alleles, and in the symbol the letter becomes a superscript to the gene symbol.

• For example, glucose phosphate isomerase 1 alleles a and b; Gpi1a, Gpi1b.

  3.2.2   DNA Segment Variants

Variants of DNA segments are indicated by a superscript to the symbol. The symbol is usually an abbreviation for the inbred strain in which the variant is being described. However, a particular allele may be found in several inbred strains, and, furthermore, it may be difficult to establish whether an allele in one strain is identical to one in another. The use of allele symbols for DNA segments is mainly limited to describing inheritance and haplotypes in crosses. As long as the symbols are defined in the description, users are free to use whatever allele symbol best fits their needs. In tables of genotypes, the gene symbol can be omitted and the allele abbreviation used alone.

• D11Mit19a, D11Mit19b, D11Mit19c are variant alleles of D11Mit19 in mouse.

3.2.3   Single Nucleotide Polymorphisms (SNPs)

Polymorphisms defined by SNPs may occur within or outside of a protein coding sequence.

If the SNP occurs within a gene, the SNP allele can be designated based on its dbSNP_id, followed by a hyphen and the specific nucleotide.

Examples:

Park2rs6200232-G	The Park2 rs6200232 SNP allele with the G variant
Park2rs6200232-A	The Park2 rs6200232 SNP allele with the A variant

If the SNP occurs outside of an identified gene, the SNP locus can be designated using the dbSNP_id as the locus symbol and the nucleotide allelic variants are then superscripted as alleles. If a gene is later discovered to include this SNP locus, the same guidelines are applicable as those used when mutant locus symbols become alleles of known genes.

Examples:

rs6200616T	A SNP locus with the T variant
rs6200616C	A SNP locus with the C variant

Note: If a gene Xyz is later discovered to include this SNP locus, rs620061, then the alleles listed above become Xyzrs620061-T and Xyzrs620061-C.

3.3  Variation in Quantitative Trait Loci and in Response and Resistance Genes

Variation in genes that do not give rise to a visible phenotype may be detected by assaying physiological or pathological parameters. Examples of this type of variation include levels of metabolite, immune response to antigen challenge, viral resistance, or response to drugs. Genetic variation may also produce phenotypic variation in morphology, behavior, or other observable traits that interact in a complex manner with other genes and/or with the environment.

These genes can only be identified by virtue of allelic variation. In most cases, there will not be a clear wild type; hence all alleles should be named. In most cases, the alleles should be named according to their strain of origin and symbolized by adding the strain abbreviation as superscript, although for resistance and sensitivity, variants r and s may be used. Bear in mind that resistance alleles deriving from different strains may not be the same and should be given different names and symbols.

Once the gene underlying a quantitative trait has been cloned or identified, the phenotypic name should be replaced by the name of the identified gene. The allele names and symbols should be the same as those used for the phenotype.

Examples:

Slc11a1r	solute carrier family 11, host resistance allele
Slc11a1s	solute carrier family 11, host susceptibility allele  (the QTL originally known as BCG/Lsh resistance has been identified as Slc11a1)
Scc2BALB/cHeA	colon tumor susceptibility 2, BALB/cHeA allele
Scc2STS/A	colon tumor susceptibility 2, STS/A allele (for QTL Scc2, the STS/A allele has increased tumor susceptibility vs. BALB/cHeA)

3.4   Insertional and Induced Mutations

Mutations that are induced, targeted, or selected in structural genes are named as alleles of the structural gene.

3.4.1   Mutations of Structural Genes

Variants of structural genes that are clearly mutations, whether or not they confer a phenotype, are given the superscript m#Labcode, where # is a serial number and is followed by the Laboratory code where the mutation was found or characterized. Serial numbers are independently assigned in mouse and rat and the same assigned serial number does not imply orthology. If the mutation is known to have occurred on a particular allele, that can be specified by preceding the superscript with the allele symbol and a hyphen.

• for example, Mod1a-m1Lws is a mutation of the mouse Mod1a allele, the first found in the laboratory of Susan Lewis.

If the mutation is shown to be a deletion of all or part of the structural gene, the superscript del can be used in place of m. Note that this should be used only for deletions that encompass a single gene; larger deletions should use the chromosomal deletion nomenclature.

3.4.2    Transgenic Insertional Mutations

Mutations produced by random insertion of a transgene (not by gene targeting) are named as a mutant allele of the gene (which should be given a name and symbol if it is a novel gene), with the superscript the symbol for the transgene (see Section 3.1.1 for examples, and Section 4 for naming transgenes).

3.5    Targeted and Trapped Mutations

3.5.1   Knockout, Knockin, Conditional and Other Targeted Mutations

Mutations that are the result of gene targeting by homologous recombination in ES cells are given the symbol of the targeted gene, with a superscript consisting of three parts: the symbol tm to denote a targeted mutation, a serial number from the laboratory of origin and the Laboratory code where the mutation was produced (see Section 2.1).

• For example, Cftrtm1Unc is the first targeted mutation of the cystic fibrosis transmembrane regulator (Cftr) gene produced at the University of North Carolina.

So-called "knock in" mutations,  in which all or part of the coding region of one gene is replaced by another,  should be given a tm symbol and the particular details of the knock-in associated with the name in publications or databases. Where there has been a replacement of the complete coding region, the replacing gene symbol can be used parenthetically as part of the allele symbol of the replaced gene along with a Laboratory code and serial number.

• For example, En1tm1(Otx2)Wrst where the coding region of En1 was replaced by the Otx2 gene, originating from the W. Wurst laboratory.

Knock in alleles expressing a RNAi under the control of the endogenous promoter can be designated using targeted mutation or transgene mutation nomenclature, as appropriate:

Example:

Genetm#(RNAi:Xyz)Labcode

When a targeting vector is used to generate multiple germline transmissible alleles, such as in the Cre-Lox system, the original knock-in of loxP would follow the regular tm designation rules. If a second heritable allele was then generated after mating with a cre transgenic mouse, it would retain the parental designation followed by a decimal point and serial number.

• Tfamtm1Lrsn and Tfamtm1.1Lrsn. In this example, Tfamtm1Lrsn designates a targeted mutation where loxP was inserted into the Tfam gene. Tfamtm1.1Lrsn designates another germline transmissible allele generated after mating with a cre transgenic mouse. Note: somatic events generated in offspring from a Tfamtm1Lrsn bearing mouse and a cre transgenic that cause disruption of Tfam in selective tissues would not be assigned nomenclature.

Other more complex forms of gene replacement, such as partial "knock-in", hit-and-run, double replacements, and loxP mediated integrations are not conveniently abbreviated and should be given a conventional tm#Labcode superscript. Details of the targeted locus should be given in associated publications and database entries.

Note that although subtle alterations made in a gene appear to lend themselves to a simple naming convention whereby the base or amino acid changes are specified, in fact these do not provide unique gene names, as such alterations, which could be made in independent labs, while bearing the same changes, may differ elsewhere in the gene.

Large-scale projects that systematically produce a large number of alleles (>1000) may include a project abbreviation in parentheses as part of the allele designation. These should retain the accepted nomenclature features of other alleles of that class. For example, a targeted allele created by Velocigene (Regeneron) in the KOMP knockout project:

Gstm3tm1(KOMP)Vlcg

Once fully designated in a publication, the allele can be abbreviated by removing the portion of the allele designation in parentheses (in this case, Gstm3tm1Vlcg), providing the symbol remains unique.

3.5.2   Endonuclease-induced Mutations

Endonuclease-induced mutations are targeted mutations generated in pluripotent or totipotent cells by an endonuclease joined to sequence-specific DNA binding domains. The mutation is introduced during homology-directed or non-homologous end-joining repair of the induced DNA break(s). Endonuclease-induced mutations are given the symbol of the mutated gene, with a superscript consisting of three parts: the symbol em to denote an endonuclease-induced mutation, a serial number from the laboratory of origin and the Laboratory code where the mutation was produced.

Example:

Fgf1em1Mcw I

the first endonuclease-induced mutation of the fibroblast growth factor 1 (Fgf1) gene produced at the Medical College of Wisconsin.

3.5.3    Gene Trap Mutations

Gene trap mutations are symbolized in a similar way to targeted mutations. If the trapped gene is known, the symbol for the trapped allele will be similar to a targeted mutation of the same gene using the format Gt(vector content)#Labcode for the allele designation. Example:

Akap12Gt(ble-lacZ)15Brr

a gene trap allele of the Akap12 gene, where the gene trap vector contains a phleomycin resistance gene (ble) and lacZ, the 15th analyzed in the laboratory of Jacqueline Barra (Brr).

If the trapped gene is novel, it should be given a name and a symbol, which includes the letters Gt for "gene trap," the vector in parentheses, a serial number, and Laboratory code.

• For example, a gene trapped locus (where the gene is unknown) using vector ROSA, the 26th made in P. Soriano's laboratory, is Gt(ROSA)26Sor.

For high throughput systematic gene trap pipelines, the mutant ES cell line's designation can be used in parentheses instead of the vector designation, and the serial number following the parentheses may be omitted.

Examples:

Gt(DTM030)Byg	for a trapped gene (at an undefined locus) in mutant ES cell line DTM030, made by BayGenomics
Osbpl1aGt(OST48536)Lex	gene trap allele of the oxysterol binding protein-like 1A gene, in mutant ES cell line OST48536, made by Lexicon Genetics, Inc.

3.5.4    Enhancer Traps

Enhancer traps are specialized transgenes. One utility of these transgenes is in creating cre driver lines. Enhancer traps of this type that are currently being created may include a minimal promoter, introns, a cre recombinase cassette (sometimes fused with another element such as ERT2), and polyA sites from different sources.

Nomenclature for these enhancer traps consists of 4 parts as follows:

Et		prefix for enhancer trap
cre recombinase cassette		portion in parentheses... for example, cre, icre, or cre/ERT2 (if fused with ERT2)
line number or serial number		to designate lab trap number or serial number
Lab code		ILAR code identifying the creator of this enhancer trap

Examples:

Et(icre)1642Rdav		Enhancer trap 1642, Ron Davis
Et(cre/ERT2)2047Rdav		Enhancer trap 2047, Ron Davis

Note that the minimal promoter, poly A source, etc. are not part of the enhancer trap nomenclature. These are molecular details of the specific construct that will be captured in database records and reported with experimental results.

4   Transgenes

Any DNA that has been stably introduced into the germline of mice or rats is a transgene. Transgenes can be broken down into two categories:

• Those that are produced by homologous recombination as targeted events at particular loci.
• Those that occur by random insertion into the genome (usually by means of microinjection).

Nomenclature for targeted genes is dealt with in Section 3.5. Random insertion of a transgene in or near an endogenous gene may produce a new allele of this gene. This new allele should be named as described in Section 3.4.2. The transgene itself is a new genetic entity for which a name may be required. This section describes the guidelines for naming the inserted transgene.

It is recognized that it is not necessary, or even desirable, to name all transgenes. For example, if a number of transgenic lines are described in a publication but not all are subsequently maintained or archived, then only those that are maintained require standardized names. The following Guidelines were developed by an interspecies committee sponsored by ILAR in 1992 and modified by the Nomenclature Committee in 1999 and 2000. Transgenic symbols should be submitted to MGD or RGD/RatMap through the usual nomenclature submission form for new loci. The transgene symbol is made up of four parts:

• Tg denoting transgene.
• In parentheses, the official gene symbol of the inserted DNA, using nomenclature conventions of the species of origin.
• The laboratory's line or founder designation or a serial number (note that numbering is independent for mouse and rat series).
• The Laboratory code of the originating lab.

Note that, in contrast to gene and allele symbols, transgene symbols are not italicized as they are random insertions of foreign DNA material and are not part of the native mouse genome.

Examples:

Tg(Zfp38)D1Htz	a transgene containing the mouse Zfp38 gene, in line D1 reported by Nathaniel Heintz.
Tg(CD8)1Jwg	a transgene containing the human CD8 gene, the first transgenic line using this construct described by the lab of Jon W. Gordon.
Tg(HLA-B*2705, B2M)33-3Trg	a double transgene in rat containing the human HLA-B*2705 and B2M genes, that were co-injected, giving rise to line 33-3 by Joel D. Taurog.

The *, as used in the last example above, indicates that the included gene is mutant.

Different transgenic constructs containing the same gene should not be differentiated in the symbol; they will use the same gene symbol in parentheses and will be distinguished by the serial number/Laboratory code. Information about the nature of the transgenic entity should be given in associated publications and database entries.

In many cases, a large number of transgenic lines are made from the same gene construct and only differ by tissue specificity of expression. The most common of these are transgenes that use reporter constructs or recombinases (e.g., GFP, lacZ, cre), where the promoter should be specified as the first part of the gene insertion designation, separated by a hyphen from the reporter or recombinase designation. The SV40 large T antigen is another example. The use of promoter designations is helpful in such cases.

Examples:

Tg(Wnt1-LacZ)206Amc	the LacZ transgene with a Wnt1 promoter, from mouse line 206 in the laboratory of Andrew McMahon.
Tg(Zp3-cre)3Mrt	the cre transgene with a Zp3 promoter, the third transgenic mouse line from the laboratory of Gail Martin.

In the case of a fusion gene insert, where roughly equal parts of two genes compose the construct, a forward slash separates the two genes in parentheses.

Example:

Tg(TCF3/HLF)1Mlc

a transgene in which the human transcription factor 3 gene and the hepatic leukemia factor gene were inserted as a fusion chimeric cDNA, the first transgenic mouse line produced by Michael L. Cleary's laboratory (Mlc).

This scheme is to name the transgene entity only. The mouse or rat strain on which the transgene is maintained should be named separately as in the Rules and Guidelines for Nomenclature of Mouse and Rat Strains. In describing a transgenic mouse or rat strain, the strain name should precede the transgene designation.

Examples:

C57BL/6J-Tg(CD8)1Jwg	mouse strain C57BL/6J carrying the Tg(CD8)1Jwg transgene.
F344/CrlBR-Tg(HLA-B*2705, B2M)33-3Trg	rat strain F344/CrlBR carrying the Tg(HLA-B*2705,B2M)33-3Trg double transgene.

For BAC transgenics, the insert designation is the BAC clone and follows the same naming convention as the Clone Registry at NCBI.

Example:

Tg(RP22-412K21)15Som

a BAC transgene where the inserted BAC is from the RP22 BAC library, plate 412, row K, column 21. It is the 15th in the mouse made in the laboratory of Stefan Somlo (Som).

Transgenes containing RNAi constructs can be designated minimally as:

Tg(RNAi:geneX)#Labcode, where
geneX	is the gene that is knocked down
#	is the serial number of the transgene

An expanded version of this designation is:

Tg(Pro-yyRNAi:geneX)#Labcode, where
Pro-	can be used optionally to designate the promoter
yy	can be used optionally for the specific RNAi construct

While there is the option to include significant information on vectors, promoters, etc. within the parentheses of a transgene symbol, this should be minimized for brevity and clarity. The function of a symbol is to provide a unique designation to a gene, locus, or mutation. The fine molecular detail of these loci and mutations should reside in databases such as MGD and RGD.

5    Transposon-induced Mutations and Inserts

Three types of genetic inserts are involved in creating transposon-induced mutations. Two lines, one carrying the transposable-element as a concatamer and the other carrying the transposase are mated. This causes the transposable-element to come in contact with the transposase and to be mobilized from its original site, and, when reintegrated into the genome, can cause a heritable phenotypic mutation. (c.f., Ding, et al.,2005; Bestor, 2005;Dupuy, et al., 2005). Accepted nomenclature for the transposable-element inserts, transposase transgenes, and resulting transposed insertion alleles are given below.

5.1   Transgenic Transposable Element (TE) Concatamers

The transgenic transposable element concatamers are identified with a standard prefix Tg (for transgenic) and Tn (for transposable element). The class of transposable element may be included in parentheses. The general format of the symbol is:

TgTn(transposon_class_abbreviation-vector)#Labcode

Example:  TgTn(sb-T2/GT2/tTA)1Dla

The symbol consists of:

• Tg denoting transgenic
• Tn denoting transposon
• In parentheses, a lowercase abbreviation of the transposon class (in this case sb for Sleeping Beauty), followed by a hyphen and the vector designation
• The laboratory's line or founder designation or a serial number
• The Laboratory Code of the originating lab

5.2  Transposase Inserts

Transposases can be engineered into the genome via transgenesis or specific gene targeting. In these cases the relevant nomenclature for transgenes or targeted mutations is used.

For a transgene, use the standard prefix Tg (for transgene). The contents of the parentheses will usually be the promoter and the symbol for the transposase with which it is associated, separated by a hyphen. The general format of the symbol is:

Tg(promoter-transposase)#Labcode

Example:  Tg(ACTB-sb10)545Abc

The symbol consists of:

• Tg denoting transgene
• In parentheses, the official gene symbol for the promoter, using the nomenclature of the species of origin, followed by a hyphen and a lowercase transposase symbol, in this case sb10 for the Sleeping Beauty 10 transposase
• The laboratory's line or founder designation or a serial number
• The Laboratory code of the originating lab.

For a targeted knock-in of the transposase, use the standard format for a targeted mutation, i.e., the symbol of the targeted gene with a superscripted allele symbol beginning with the prefix tm. The contents of the parentheses will usually be the symbol for the transposase with which it is associated. The general format of the symbol is:

Genetm#(transposase)Labcode

Example:  Gt(ROSA)26Sortm1(sb11)Njen

The symbol consists of:

• The gene into which the transposase was integrated, in this case Gt(ROSA)26Sor
• In the superscript:
  • tm denoting targeted mutation
  • A serial number of the targeted mutation
  • In parentheses, a lowercase transposase symbol, in this case sb11 for the Sleeping Beauty 11 transposase
  • The Laboratory Code of the originating lab

5.3   Transposed Insertion Alleles

These alleles follow the rules for naming all other alleles. In general a transposable element concatamer marker will already be established, as above. The new allele, then, will be a superscripted form of the concatamer symbol. Note that all such alleles that are "derived from" a transposable element concatamer carry the original number with a decimal point and serial number identifying the specific allele. The general format is:

GeneTn(transposon_class_abbreviation-vector)#Labcode

Example: Car12Tn(sb-T2/GT2/tTA)1.1Dla

The symbol consists of:

The gene into which the transposable element was integrated (transposed)

In the superscript:

• Tn denoting transposon
• In parentheses, a lowercase abbreviation of the transposon class (in this case sb for Sleeping Beauty), followed by a hyphen and the vector designation
• A serial number, in which the primary number corresponds to that given to the transposable element concatamer from which it arose, followed by a decimal point and a serial number designating its number within the series of derivative insertion alleles.
• The Laboratory Code of the lab originating the transposable element line.

If a newly transposed insertion occurs in an unknown site or intergenic region, the form:

Tn(transposon_class_abbreviation-vector)#Labcode

is used to symbolize the "genomic mutation" without being superscripted to a gene symbol, similar to the way a random transgene inserted into a non-gene site is designated.

6   Definitions

The following definitions should aid the user in understanding what is being named, and in understanding the principles underlying these guidelines.

6.1   Gene

A gene is a functional unit, usually encoding a protein or RNA, whose inheritance can be followed experimentally. Inheritance is usually assayed in genetic crosses, but identification of the gene in cytogenetic or physical maps are other means of mapping the locus of a gene. The existence of a gene can also be inferred in the absence of any genetic or physical map information, such as from a cDNA sequence.

6.2   Pseudogene

A sequence that closely resembles a known functional gene, at another locus within a genome, that is non-functional as a consequence of (usually several) mutations that prevent either its transcription or translation (or both). In general, pseudogenes result from either reverse transcription of a transcript of their "normal" paralog (in which case the pseudogene typically lacks introns and includes a poly(A) tail; often called processed pseudogenes) or from recombination (in which case the pseudogene is typically a tandem duplication of its "normal" paralog).

6.3   Locus

A locus is a point in the genome, identified by a marker, which can be mapped by some means. It does not necessarily correspond to a gene; it could, for example, be an anonymous non-coding DNA segment or a cytogenetic feature. A single gene may have several loci within it (each defined by different markers) and these markers may be separated in genetic or physical mapping experiments. In such cases, it is useful to define these different loci, but normally the gene name should be used to designate the gene itself, as this usually will convey the most information.

6.4   Marker

A marker is the means by which a gene or a locus is identified. The marker is dependent on an assay, which could, for example, be identification of a mutant phenotype or presence of an enzyme activity, protein band, or DNA fragment. The assay must show genetic variation of the marker to map the locus on a genetic map but not to place it on a physical map.

6.5   Allele

The two copies of an autosomal gene or locus on the maternal and paternal chromosomes are alleles. If the two alleles are identical, the animal is homozygous at that locus. When genetically inherited variants of a gene or locus are detectable by any means, the different alleles enable genetic mapping. A single chromosome can only carry a single allele and, except in cases of duplication, deletion or trisomy, an animal carries two autosomal alleles. In particular, a transgene inserted randomly in the genome is not an allele of the endogenous locus; the condition is termed hemizygous if the transgene is present only in one of the two parental chromosome sets. By contrast, a gene modified by targeting at the endogenous locus is an allele and should be named as such.

6.6   Allelic Variant

Allelic variants are differences between alleles, detectable by any assay. For example, differences in anonymous DNA sequences can be detected as simple sequence length polymorphism (SSLP) or single nucleotide polymorphisms (SNPs). Other types of variants include differences in protein molecular weight or charge, differences in enzyme activity, or differences in single-stranded conformation (SSCP). Many allelic variants, in particular DNA variants, do not confer any external phenotype on the animal. These variants are often termed “polymorphisms” although, strictly speaking, that term applies only to variants with a frequency of more than 1% in the population.

6.7   Splice Variant or Alternative Splice

Alternative splicing of a gene results in different, normally occurring forms of mRNA defined by which exons (or parts of exons) are used. Thus one or more alternative protein products can be produced by a single allele of a gene. Among different alleles, alternative splice forms may or may not differ, depending on whether the sequence difference between the alleles affects the normal splicing mechanism and results in differences in the exon (or partial exon) usage. For example, allele A may produce mRNAs of splice form 1, 2, and 3; while allele B may produce mRNAs of splice form 1, 2, and 4; and Allele C may produce mRNAs of splice form 1, 2, and 3. In this case, each of the alleles A, B, and C by definition must differ in their DNA sequence. However, the difference between allele B versus alleles A and C must include a sequence difference that affects the splicing pattern of the gene.

6.8    Mutation

A mutation is a particular class of variant allele that usually confers a phenotypically identifiable difference to a reference "wild type" phenotype. However, in some cases, such as when homologous recombination is used to target a gene, a readily identified phenotype may not result even though the gene may be rendered non-functional. In such cases, the targeted genes are nevertheless referred to as mutant alleles.

6.9    Dominant and Recessive

Dominant and recessive refer to the nature of inheritance of phenotypes, not to genes, alleles, or mutations. A recessive phenotype is one that is only detected when both alleles have a particular variant or mutation. A dominant phenotype is detectable when only one variant allele is present. If both alleles can be simultaneously detected by an assay, then they are codominant. For example, an assay that detects variation of DNA or protein will almost invariably detect codominant inheritance, as both alleles are detected. If a mutation produces a phenotype in the heterozygote that is intermediate between the homozygous normal and mutant, the phenotype is referred to as semidominant. A single mutation may confer both a dominant and a recessive phenotype. For example, the mouse patch (Ph) mutation has a heterozygous (dominant) pigmentation phenotype but also a homozygous (recessive) lethal phenotype. As the terms are applied to phenotypes not to genes or alleles, then in the case where a gene has multiple mutant alleles, each can confer a phenotype that is dominant to some, but recessive to other, phenotypes due to other alleles.

Penetrance is a quantitative measure of how often the phenotype occurs in a population; and expressivity is a measure of how strongly a phenotype is expressed in an individual. Particularly in segregating crosses, or where there is a threshold effect on phenotypic manifestation, these measures provide additional ways to describe how particular allelic combinations result in a phenotype.

6.10   Genotype

Genotype is the description of the genetic composition of the animals, usually in terms of particular alleles at particular loci. It may refer to single genes or loci or to many. Genotype can only be determined by assaying phenotype, including test mating to reveal carriers of recessive mutations. Strictly speaking, even direct determination of DNA variants is assaying phenotype not genotype as it is dependent on a particular assay, although it is so close to genotype that it serves as a surrogate.

6.11   Phenotype

Phenotype is the result of interaction between genotype and the environment and can be determined by any assay.

6.12   Quantitative Trait Loci (QTLs)

Quantitative Trait Loci (QTL) are polymorphic loci that contain alleles, which differentially affect the expression of continuously distributed phenotypic traits. Usually these are markers described by statistical association to quantitative variation in the particular phenotypic traits that are controlled by the cumulative action of alleles at multiple loci.

6.13   Haplotype

A haplotype is the association of genetically linked alleles, as found in a gamete. They may be a combination of any type of markers, and may extend over large, genetically separable distances, or be within a short distance such as within a gene and not normally separated.

6.14   Homolog

Genes are homologous if they recognizably have evolved from a common ancestor. Note that genes are either homologous or not; there are no degrees of homology! For example, all globin genes, and myoglobin, are homologs, even though some are more closely related to each other than others. When a measure of relatedness between sequences is required, percent identity or similarity should be used.

6.15   Ortholog

Genes in different species are orthologs if they have evolved from a single common ancestral gene. For example, the beta globin genes of mouse, rat and human are orthologs. Note that several genes in the mouse or rat may have a single ortholog in another species and vice versa.

6.16   Paralog

Paralogous genes are genes within the same species that have arisen from a common ancestor by duplication and subsequent divergence. For example, the mouse alpha globin and beta globin genes are paralogs.

7   References

Bestor TH. Transposons reanimated in mice. 2005. Cell 122:322-325.

Committee on Rat Nomenclature, Cochairmen Gill T.J. III, Nomura T. 1992. Definition, nomenclature, and conservation of rat strains. ILAR News 34:S1-S56.

Committee on Standardized Genetic Nomenclature for Mice. 1963. A revision of the standardized genetic nomenclature for mice. J. Hered. 54:159-162.

Committee on Standardized Genetic Nomenclature for Mice. 1973. Guidelines for nomenclature of genetically determined biochemical variants in the house mouse, Mus musculus. Biochem. Genet. 9:369-374.

Committee on Standardized Genetic Nomenclature for Mice, Chair: Lyon, M.F. 1981. Rules and guidelines for gene nomenclature. In: Genetic Variants and Strains of the Laboratory Mouse, Green, M.C. (ed.), First Edition, Gustav Fischer Verlag, Stuttgart, pp. 1-7.

Committee on Standardized Genetic Nomenclature for Mice, Chair: Lyon, M.F. 1989. Rules and guidelines for gene nomenclature. In: Genetic Variants and Strains of the Laboratory Mouse, Lyon, M.F., A.G. Searle (eds.), Second Edition, Oxford University Press, Oxford, pp. 1-11.

Committee on Standardized Genetic Nomenclature for Mice, Chairperson: Davisson, M.T. 1996. Rules and guidelines for gene nomenclature. In: Genetic Variants and Strains of the Laboratory Mouse, Lyon, M.F., Rastan, S., Brown, S.D.M. (eds.), Third Edition, Volume 1, Oxford University Press, Oxford, pp. 1-16.

Ding S, Wu X, Li G, Han M, Zhuang Y, Xu. T. 2005. Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell 122:473-483.

Dunn, L.C., H. Gruneberg, G.D. Snell. 1940. Report of the committee on mouse genetics nomenclature. J. Hered. 31:505-506.

Dupuy AJ, Akagi K, Largaespada DA, Copeland NG, Jenkins NA. 2005. Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature 436:221-226.

Eppig, JT. 2006. Mouse Strain and Genetic Nomenclature: an Abbreviated Guide. In: Fox J, Barthold S, Davvison M, Newcomer C, Quimby F, Smith A (eds) The Mouse in Biomedical Research, Volume 1, Second Edition. Academic Press. pp.79-98.

International Committee on Standardized Genetic Nomenclature for Mice, Chairperson: Davisson, M.T. 1994. Rules and guidelines for genetic nomenclature in mice. Mouse Genome 92 vii-xxxii.

Levan G., H.J. Hedrich, E.F. Remmers, T. Serikawa, M.C. Yoshida. 1995. Standardized rat genetic nomenclature. Mamm. Genome 6:447-448.

principle of the fluorophore FITC 참고용 자료

FITC and Fluorescein Dyes and Labeling Kits

Reactive derivatives of fluorescein dye, including antibody labeling kits

Amine-reactive Fluorescein Dyes:

Chemical structures of FITC and NHS-Fluorescein. Both of these compounds allow fluorescent labeling of primary amines on proteins and other molecules. See our review ofAmine-Reactive Crosslinker Chemistry.

FITC is the base fluorescein molecule functionalized with an isothiocyanate reactive group (–N=C=S) at one of two hydrogen atoms on the bottom ring of the structure. This derivative is reactive towards primary amine groups on proteins, peptides and other biomolecules. NHS-fluorescein is activated with the N-hydroxy-succinimidyl-ester (NHS ester) functional group. Compared to FITC, the NHS-ester deriviative has greater specificity toward primary amines in the presence of other nucleophiles and results in a more stable linkage following labeling. Pierce Amine-reactive Fluorescein Dyes are mixtures of isomers with reactive groups attached at the 5- and 6-positions of the bottom ring. The properties of these isomers are indistinguishable in terms of excitation and emission spectra, and for protein applications there is no need to isolate a specific isomer.

Sulfhydryl-reactive Fluorescein Dyes:

Chemical structures of Fluorescein-5-maleimide and 5-IAF. Both of these compounds allow fluorescent labeling of sulfhydryl groups on proteins and other molecules. See our review of Sulfhydryl-Reactive Crosslinker Chemistry.

Fluorescein-5-maleimide and 5-IAF are sulfhydryl-reactive derivatives of fluorescein dye. Fluorescein-5-maleimide is the base fluorescein molecule functionalized with a maleimide reactive group by replacing a hydrogen atom on the bottom ring of the structure. 5-IAF is the core fluorescein molecule functionalized with an iodoacetamide group. Both fluorescein derivatives are reactive toward sulfhydryl groups (e.g., reduced cysteine residues) on proteins, peptides and other biomolecules.

Application Data:

A. Fluorescein	B. Hoechst	C. Merged
Detection of α-tubulin in A549 cells demonstrates use of fluorescein-labeled secondary antibody.Cells were grown in 96-well microplates for 18-20 hrs, fixed with 4% paraformaldehyde (Part No. 28906) and permeabilized with 0.1% Surfact-Amps X-100 (Part No. 28314). Cells were then probed with a mouse anti-α-tubulin primary antibody (0.4µg/mL) and Fluorescein-goat anti-mouse secondary antibody (2µg/mL). Nuclei were labeled with Hoechst Dye. Images were acquired by fluorescence microscopy. A.Fluorescence image shows a delicate network of α-tubulin (pseudo-colored green) located exclusively in the cytoplasm. B. Nuclear counterstain with Hoechst Dye (pseudo-colored blue) C. Merged image.

General References:

1. Miki, M. and dos Remedios, C.G. (1988). Fluorescence quenching studies of fluorescein attached to lys-61 or cys-374 in actin: effects of polymerization, myosin sub fragment-1 binding, and tropomyosin-troponin binding. J. Biochem. 104, 232-235.
2. Smith, L.M., et al. (1987). The synthesis and use of fluorescent oligonucleotides in DNA sequence analysis. Meth. Enzymol. 155, 260-301.
3. Vera, J.C., et al. (1988). Purification, amino terminal analysis and peptide mapping of proteins after in situ postelectrophoretic fluorescent labeling. Anal. Biochem. 174, 38-45.
4. Szewczyk, B. and Summers, D.F. (1987). Fluorescent staining of proteins transferred to nitrocellulose allowing for subsequent probing with antisera. Anal. Biochem. 164, 303-306.
5. Der-Balian, G.P., et al. (1988). Fluorescein labeling of Fab while preserving single thiol. Anal. Biochem. 173, 59-63.
6. Vigers, G.P.A., et al. (1988). Fluorescent microtubules break up under illumination. J. Cell Biol. 107, 1011.
7. Goding, J.W. (1976). Conjugation of antibodies with fluorochrome: modifications to the standard methods. J. Immunol. Meth. 13, 215-226.
8. Szewczyk, B., et al. (1987). Use of different fluorochromes for monitoring protein elution and transfer. Electrophoresis 8, 25-28.
9. Smith, L.M., et al. (1986). Fluorescence detection in automated DNA sequence analysis. Nature321, 674-678.
10. Staines, W.A., et al. (1988). Three-color immunofluorescence histochemistry allowing triple labeling within a single section. J. Histochem. Cytochem. 36(2), 145-151.

small_world_bryson.pdf

Small World by Bill Bryson

* 참고로 Bill Bryson은, 작가의 이름만 보고 책을 골라도 절대 후회하지 않을정도로, 재미있고 위트넘치는 글을 잘 쓰기로 유명한 작가래요^^(그래서인지 글들이 서양식 유머나 언어유희적 표현이 너무 많음…..하… 진짜 하다가 죽는줄 알앗음..ㅜㅜ) 

(괄호 안 내용들)은 제가 추가적으로 이해를 돕기위해 덧붙이거나 의역한 글입니다. 의역한 부분중 특이한 부분은 영단어도 써 놓았어요.

네가 미생물 같은것들에 너무 많은 개인적인 흥미를 가지게 되는건 아마도 그렇게 좋은 생각은 아닐거야. 프랑스의 위대한 화학자이자 미생물학자인 루이스 파스테르는 그것들(:미생물들. 교수님 교재에는 his라고 되어있는데 이상해서 본문찾아보니까 them-_-)에 굉장히 심취한 상태여서, 그는 그의 앞에(before him) 놓인 모든 접시들을 돋보기로 아주 자세하게 들여다 보고는 했어- 확언컨대, 그가 저녁 식사에 다시 초대를 받을 수 있을만한 그런 습관은 아니었지.

·       Magnifying glass:돋보기

·       Peer: look closer, examine

사실, 네가 박테리아로부터 피하려고 해도, 그것들은 항상 네 주변에 있고 (심지어) 네 몸 위에도 네가 상상도 하지못할 만큼의 (많은) 수가 존재하기에, 그건 불가능해. 네가 만약 건강하고 위생에 대해 평균적으로 열심히인 편이라면, 너는 아마도 네 피부의 제곱센치미터 네모안에 약 백만마리(a hundred thousand) 정도로, 총 대략 1조마리의(one trillion) 박테리아들이 네 피부위에서 파고들며 갉아먹고 있을거야.

·       Conceive of: 인지하다

·       Herd : Keep or look after (livestock)

·       Graze: Eat grass in a field / 상처나다

·       Fleshy plains : 살

·       Diligent: 성실한

박테리아들은 (우리 신체의) 모든 구멍들과 갈라진 틈(fissure)로부터 새어나오는, 힘이나게 하는 미네랄과 맛있는 모든 기름들 뿐만이 아니라, 네 몸에서 매일 떨어져 나오는 십억또는 그정도의( ten billion) 피부 조각들로 먹고살면서 그곳에 있지. 너는 그것들에게 있어 따뜻함이라는 안락함과, 언제든지 침투해 들어갈수 있는 지속적인 이동성을 가진 궁극적인 뷔페라고 할수 있어. 고마움의 표시로써, 박테리아들은 너에게 체취라는 선물도 준단다.

·       Shed: (피 눈물등을) 쏟다, (허물을)벗다

·       Fortifying :힘을 주는

·       Seep out: 새어나오다

·       Fissure: 갈라진 틈

·       B.O. = body odour체취

그리고 그것들은 그냥 네 피부에 서식하는 박테리아들일 뿐이야. 너의 장과 비강 통로안에 박혀있고, 너의 머리칼이나 속눈썹에 매달려 있으며, 네 눈 표면에서 수영하고, 네 치아의 에나멜을 뚫고 있는 세균들은 수 조(trillions) 마리야. 너의 소화 기관 혼자서만도 최소 400종류의, 100조 마리보다 더 많은 미생물들을 데리고(host) 있어.

·       Tuck away: 게걸스럽게 먹다

·       Gut:장

·       Nasal:코의

·       Cling: hold on tightly

몇몇은 sugars를, 몇몇은 starches를 담당하며, 또 다른 몇몇은 other bacteria를 공격해. 엄청나게 많은 수의 어디에나 존재하는 장(창자) 내 나선형의 박테리아(the ubiquitous intestinal spirochetes) 같은 것들은 탐지가 가능한 어떠한 기능도 전혀 가지고 있지 않아. 그것들은 그저 너랑 함께 하고싶은것 같아 보여. 모든 인간의 몸은 약 1(ten quadrillion)개의 세포로 구성되어 있지만, 약 100억(one hundred quadrillion)개의 박테리아 세포 역시 가지고(host) 있어. 다시말해서, 그것들은 우리 몸의 많은(중요한) 부분을 차지하고 있다는 거지. 물론 박테리아의 관점에서는, 우리는 그들에게 있어 훨씬 작은 부분일 뿐이겠지만.

·       ubiquitous: present everywhere(유비쿼터스, 어디에나 존재하는)

·       intestinal: 장의, 창자의, 장에 존재하

·       spi·ro·chete: A flexible spirally twisted bacterium, esp. one that causes syphilis

·       quadrillion: 1000조

ㅋㅋㅋㅋㅋㅋ내용이 좀 편하게 재밌게 전달하는 내용이라 
        구어체로 적어보려고 했는데ㅋㅋㅋㅋ

더 어려움ㅠㅠ 

문장 깔끔하게 만들기가 그냥 쓰는것보다 배로 더 힘들어서 그냥 ‘–했다’ 체로 갈게요…ㅋㅋ 진짜 번역가들 대단하다고 새삼 느꼈네요..ㅠㅠ 모두들 콩팅팥팅^.^

우리 인간은 항체나 살균제를 생산해서 활용할 만큼 충분히 크고 똑똑하기 때문에, 우리는 스스로에게 우리가 박테리아를 거의 몰아냈다고 믿도록 확신시키기 쉽다.(우리가 박테리아를 존재의 가장자리로까지 추방시켰다고 확신시키기 쉽다.) 스스로를 믿지마라. 박테리아는 도시를 세우거나 사교 생활(소셜라이프)에 대한 흥미를 가지고 있지 않을지는 모르지만, 그것들은 아마 태양이 폭발 할때에도 여기 이곳에 존제 할테니까. 이 곳 (지구)는 그들의 행성이며, 우리는 오직 그들이 우리가 이곳에 있는 것을 허락했기에 이곳에 존재 할 뿐이다.

·       disinfectants: 살균제, 소독제

·       convince: 설득시키다, 확신시키다

·       banish:추방하다

·       fringe: 가장자

절대 잊지말것. 박테리아는 우리가 존재하기 전부터 이곳에 수십억년(billions of years)간 존재해왔다. 우리는 결코 그들 없이 하루도 살아남을 수 없다. 그것들은 우리가 만들어낸 쓰레기들을 처리하고 그것들을 다시 사용가능하게 만든다; 그들의 ‘성실한 씹어먹기’가 없이는 아무것도 썩지 않게 될 것이다. 그것들은 우리의 물을 정화시키고, 우리의 흙을 생산적으로 유지시킨다. 박테리아는 우리 장에서 비타민들을 합성하고, 우리가 먹을 것들을 유용한 sugars나 탄수화물류로 바꾸며, 우리의 식도로 미끄러져 내려온 이방 미생물들과 싸우기도 한다.

·       diligent: 근면한

·       munch: Eat (something) with a continuous and often audible action of the jaws (오독오독, 와삭와삭소리내면서  씹어먹다)

·       gullet: 식도

공기중에서 질소를 뽑아내는 것 부터 이것을 우리에게 유용한 뉴클레오타이드와 아미노산으로 만드는 데에, 우리는 전적으로 박테리아에 의존한다. 이것은 굉장하고 흐뭇한 재능이다. Marguhs 와 Sagan이 언급하듯이, 똑같은 일을 공업적으로 수행 해내기 위해서는,  생산자들은 그 자원물질들을 500도 씨까지 덥펴야 하며, 일반 압력의 300배가 되는 압력으로 짖눌러야 한다. 박테리아는, 고맙게도, 호들갑 떨지 않고 언제나 같이 같은 일들을 한다- 어느 큰 생물체도 박테리아가 생산해낸 질소 없이는 살아나지 못하기 때문이다. 무엇보다도, 미생물들은 우리에게 우리가 숨쉬는 공기를 제공하고, 대기를 안정하게 유지시키는 일을 지속한다. 미생물들( 현대판 시아노박테리아들을 포함해서) 은 이 행성의 숨술만한 산소를 제공하는 아주 중요한 부분이다.

·       Pluck: 풀을/박혀있는 것을 뽑다 (여기서는 뿌리혹박테리아 같은게 질소고정 하는 것을 말하는듯)

·       Prodigious: (격식) (놀라움・감탄을 자아낼 정도로) 엄청난[굉장한]

·       Gratifying: (격식) 흐뭇한, 기쁜

·       Feat: [명사] (호감) 위업, 개가; (뛰어난) 솜씨[재주]

·       Manufacturers: 제조자

·       Fuss: 호들갑

  조류(Algae)와 바다안에서 졸졸 흘러다니는(bubbling away) 다른 작은 생물체들은 1500억 킬로 길이의 물질을 매년 생산해 낸다. 그리고 그것들은 놀랄만큼 자손을 많이 생산한다. 그것들 둥에서 더욱 활발한 것은 새로운 자손세댜룰 10분도 채 안되는 시간내에 만들어 낼 수 있는 것들이다; Clostridium pe$ingens, 조직 괴사를 일으키는 그다지 반갑지 않은 이 작은 유기체는 9분만에 자손을 만들며 바로 다시 spilit을 시작한다.

·       Prolific: 다산하는, (많은 동물들이 살기에) 먹이가 풍부한, 수가 많은

·       Frantic: 정신없이 서두는( 여기서는 활발한..)

·       Disagreeable: 반갑지 않은

·       Gangrene: (조직)괴사 , 괴저

이러한 수치로 보면, 이론적으로 하나의 박테리아는 이틀만에 이 우주에 존재하는 프로톤들 보다 더 많은 자손들을 만들어낼 수 있다. 벨기에의 생화학자이자 노벨상 수상자인Christian  de Duve에 의하면, “일정한 영양의 공급이 주어지면, 하나의 박테리아 세포는280,000 billion 개체를 단 하루만에 만들어 낼 수 있다”고 한다. 같은 기간동안, 하나의 인간 세포는 그저 가까스로 한번의 cell division을 할 수 있을 뿐이다.

·       Adequate: 충분한, 적절한

·       Laureate:수상자

약 백만번의 분열중에 한번씩은, 그것들은 돌연변이 체를 생산한다. 보통 이것은 돌연변이 체에게는 악운이다- 생물체에게있어 변화란 언제든지 위험성을 띠고 있다-그러나 아주 가끔씩은 그 새로운 박테리아가 항체의 공격을 피하거나 무시 할 수 있는 등의 우연한 이점들을 부여받을 수도 있다. 

With this ability to evolve rapidly goes another, even scarier advantage. (해석 잘안됨… 느낌상: 이러한 빠르게 진화하는 능력에 이어, 그것들은 심지어 더 무시무시한 장점을 가지고 있다)
      박테리아는 정보를 공유한다. 어떤 박테리아도 어떤 다른 개체로부터 유전적 암호 정보의 일부를 취할 수 있다. 본질적으로, Margulis 와 Sagan가 말했듯이(put it :어떤 논문같은데 포함시켰나봄), 모든 박테리아는 하나의 유전자 풀에서 수영한다. 박테리아 세상안에서는 하나의 영역 안에서 일어나는 적응 할 수있는 어떠한 변화는 어떤 다른것으로도 퍼질 수 있다. 이것은 마치 인간이 곤충에게로 가서 날개를 돋아내거나 천정을 걸어다니기 위해 필요한 유전적 코드를 받아오는 것과도 같다고 할 수 있다. 이것은 유전적인 관점에서 본다면 박테리아는 작고 분산되어 있지만, 하나의 천하무적 수퍼 생명체가 되었다는 것을 말하는 듯 하다.

·       endow: 기부하다, 부여하다

·       elude : (교묘히) 피하다[빠져나가다]

·       shrug off: ~을 대수롭지 않게 취급하다

·       adaptive: 적응할 수 있는

·       dispersed: 분산된, 멀리 퍼진

·       invincible: 천하무적의, 아무도 꺾을 수 없는

            그것들은 네가 쏟거나 흘린 거의 모든 어떤 것들위에도 잘 자라고 살것이다. 그저 그것들에게 약간의 수분만 제공해 주라-as  when you  run a damp cloth over a counter(해석안됨ㅜㅜ: 마치 네가 젖은 옷을…?)-그러면 그것들은 마치 무에서 창조된 것처럼 꽃피워있을 것이다. 그것들은 나무, 벽지의 풀, 두껍게 페인트된 금속들을 먹는다. 호주의 과학자들은Thiobacillus concretivorans라는 메탈을 녹을정도로 강한 황산에 사는-사실은 없이는 못사는- 미생물을 발견했다.  Micrococcus radiophilus라고 불리는 한 생물은 핵 쳬기물 쓰레기 탱크 안에서 플루토눔, 그리고 그곳에 있는 것이라면 무엇이든지 먹으면서 사는것이 확인 되었다. 몇몇 박테리아들은  어떤 화학물질들을 분해한다- 우리가 말할 수 있는것은, 그것들은 어떠한 이점도 못엇는다는 것이다. 

·       thrive on: 즐기다, 잘 자라다

·       dribble: 질질 흘리다

·       gorge: 게걸스럽게 먹다

그것들은 끓는 진흙 탕과 수산화 나트륨 호수, 바위안 깊은 속, 해저층, 남극 대륙의 the McMurdo Dry Valleys의 숨겨진 얼음물 호수, 그리고 태평양에 위치한 표면보다 약 1000배보다 더한 압력이 있는 또는 초대형 여객기가 짖눌릴 정도에 상응하는 압력의,11킬로미터 깊이 등에서 사는 것이 발견되어왔다. 이중에서 몇몇은 현실적으로 파괴할수 없는 것 처럼 보인다. the Economist 잡지에 따르면, Deinococcus radiodurans는, "방사성에 거의영향을 받지 않는다"고 한다. 방사선으로 인해 DNA가 폭발하고, 그 조각들이 즉시 본모습으로 재형성 되는 것은 마치 호러영화에서 나오는 완전히 죽지 않은 생명체의 황급히 달려가는 팔다리들 같다.

·       caustic soda: 수산화나트륨

·       Antarctica: 남극대륙

·       Pacific: 태평양의

·       equivalent to: -와 같은, 상응하는

·       beneath: 이래에, -보다 못한

·       jumbojet: 점보 제트기 ((초대형 여객기))

·       indestructible: 파괴할 수 없는

·       immune: 면역성이 있는 , 면역이 되어 거의 영향을 받지 않는

·       Blast: 폭발

·       Scuttling: 황급히 달려가는

아마도 지금까지 발견된 가장 이상한 박테리아의 생존은, 달에서 이년동안 서있었던 카메라의 렌즈에 부착되어있다가 발견되었던 Streptococcus bactenum일 것이다. 요약하자면, 극소수의 박테리아가 살 준비가 안된 환경들이 존재한다는 것이다. Victoria Bennett가 내게 말하길,  “과학자들은 탐사선을 바다 열수 통풍구에 넣었을때 너무 뜨거워서 실제로 탐사선이 녹기 시작할 정도인 그곳에 조차도 박테리아들이 있다는 것을 이제야 알아내고 있는 중이다.”

·       ocean vents: 열수 통풍구 (vent 가 통풍구 배출구라는 뜻)

·       probe: 탐사, 탐사

1920년대에, University of Chicago의 두 과학자, Edson Bastin 과 Frank Greer는 그들이 유정에서 600미터의 깊이에서 살아가고 있던 박테리아 균주를 분리해냈다고 발표했다. 그 의견은 본질적으로 상식-600미터아래에서 살수 있는 것은 없다-을 벗어났다고 무시되었다. 그리고 50년동안 그것은 그들의 샘플이 표면에 있는 미생물로 오염되었다고 추측되어졌다. 현재 우리는 수많은 미생물들이 지구 깊숙한 곳에 살고 있으며, 유기적인 세상에서는 아무것도 할 수 없는 많은 미생물들 또한 있다는 것을 알고 있다. 그것들은 바위, 또는 철, 황, 망간 등등의 바위의 안에 있는 물질들을 먹는다. 그리고 그들은 철, chromium, 코발트, 심지어 우라늄과 같은 이상한(odd) 것들도 호흡한다. 특정 프로세스들은 금, 구리, 그리고 다른 소중한 금속들을 모으는, 그리고 아마도 오일이나 천연가스를 퇴적시키는 수단이 될 수도 있다. 그들의 지치지 않는 ‘갉아먹기’들이 지구의 지각을 만들었다고 추정된다.

·       oil wells: 유정(油井)

·       strains: 균주

·       notion:의견 생각

·       dismissed: 해산시키다

·       preposterous: 불합리한, 상식을 벗어난, 터무니 없는

·       instrumental: (격식) 수단[도구]이 되는; (수단으로서) 도움이 되는, […하는 데에] 유익한

·       nibble: 조금씩 갉어 먹다, 물어뜯다

·       crust: 딱딱한 외피, the Earth's crust:지질의 지각

어떤 과학자들은 이제 우리의 발 밑에 100 trillion톤정도의 많은 박테리아들- subsurface lithoautotrophic microbial ecosystems라고 알려진, 줄여서SLiME 라고 하는-이 있을 수 있다고 생각한다. 코넬의 Thomas Gold 은 만약 지구내에 있는 모든 박테리아를 모아서 표면에 쌓으면, 아마도 4층건물 높이정도인 15미터의 폭으로 지구를 덮을 것이라고 어림잡아 추측했다. 만약 그 추축이 맞다면, 지구상에는 표면위에있는 것보다 더 많은 생명이 지표면 안에 존재 할 것 이다.

·       SLiME: subsurface lithoautotrophic microbial ecosystems

깊은 곳에서(At depth 인데 잘 모르겟음ㅜㅜ), 미생물들은 사이즈가 줄어들고 매우 기능이 둔해진다. 그것들 중에서 가장 활기찬 것은 아마 1세기에 한번도 채 분열 하지 않을것이고, 몇몇은 아마 500년중에 한번 정도 일 것이다. the Economist 지가 언급 한 것 처럼, 장수의 비결은, ‘많은것을 하지 않는것’ 인것 처럼 보인다. 삻이 힘들어 질때면 박테리아는 모든 시스템을 종료하고 더 나은 기단을 기다릴 준비를 마친다. 1997년에 과학자들은 노르웨이에 있는Trondheim의 박물관에 디스플레이 되어 80년동안 잠복중이 었던 몇몇 탄저병 포자들을 성공적으로 활성화 시켰다. 또 다른 미생물들은 118년 된 고기 통조림과 166년된 오래된 병맥주로부터 추출된 후, 다시 살아있는 상테로 돌아오기도 했다.  1996년도에는the Russian Academy of Science의 과학자들이 시베리아의 영구 동토층에 3million년 동안 얼어있던 박테리아를 부활시켰다고 부밪하기도 하였다. 그러나 현재 영속성에 대한 타이틀을 차지한 기록은, 2000년도에at West Chester University in Pennsylvania 출신의 Russell Vreeland 와 그의 colleagues들이 만들어낸 기록인, New Mexico주Carlsbad의 지하600미터 아래에있는 소금 결정 퇴적물들 안에 갇혀있던 250million살 먹은     Bacillus permians를 소생시킨 것이다. 만약 그렇다면, 이 미생물은 지구상의 대륙들보다 더 오래된 것이다.

·       sluggish: [기관(器官) 등이] 기능이 둔한

·       lively : 활기찬, 원기[생기] 있는, 활발한

·       anthrax: 탄저, 옴, 악성 부스럼

·       spores: 포낭, 포자

·       dormant: 휴면 중인, 잠복중인

·       leap: 도약하다 (상태에)도달하다

·       permafrost:영구 동토층( 언 땅)

·       durability: 내구성, 영속성

·       claim: (타이틀을) 차지하다, 얻다

·       resuscitated :소생시키다

            그 레포트는 몇몇의 이해하기 쉬운 불확실함들과 맞닥뜨린다. 많은 생화학자들은 주장했다,that over such a span the microbe's components would have become uselessly degraded unless the bacterium roused itself from time to time(잘 해석안되지만ㅜㅜ >그런 긴 기간동안 때때로 깨어나 활동한 것을 제외하고, 미생물의 구성물질들이 무의미하게 퇴화했다고.) 그러나, 만약 박테리아가 때때로 정말로 그렇게 활발히 움직이면, 아마 그렇게 오랫동안 남아있을 법한 체내 에너지가 전혀 없을 것이다. 의심많은 과학자들은 만약 회생되는 기간동안이 아니라면 아마도 파묻혀 있을 동안 샘플이 오염되었을지도 모른다는 의견을 제시한다. 2001년도에, Tel Aviv Universit의 한 팀은B. permians는 현대의 박테리아Bacillus marisrnortui 균주와 거의 동일했다고 주장했다. 오직 단 두개의 제네틱 시퀀스만이 아주 살짝 다를 뿐이었다.

·       dubiousness: 의심함, 미심쩍어 함; 수상쩍음; 불확실함.

·       Maintain: 주장을 유지하다

·       Span: 기간

·       Uselessly: 무의미하게

·       Degraded: 퇴화한

·       from time to time: 때때로

·       stir: 활발히 움직이다

·       plausible: [논의·진술·이야기 등이] 그럴듯한, 정말 같은

·       retrieval: 회복, 만회, 수복, 복구; 구조; 보상.

            이스라엘의 연구자들은 "우리가 믿을 수 있는가, 250 million살 된 B. permians가 축적해온 유전적 변이가 실험실에서 단지 3-7일정도면 얻을 수 있을 정도의 유전적 차이와 같은 양이라는것을?” 이라고 질문했다. 그에 대한 답변으로, Vreeland는 “ 박테리아는 야생에서 보다 랩안에서 더 빠르게 진화한다”고 제시했다. 아마도, 그럴지도 모른다.

·       accumulate: 축적하다 모으다

 이러한 최신 시대에도, 대부분의 학교 교과서는 이 세상을 그저 –식물과 동물-이라는 단지 두개의 카테고리로 생명체를  분류해 놓았다는 것은 놀라운 사실이다. 미생물들은 거의 다루어지지 않는다. 아메바나 그와 비슷한 단세포 유기체들은 원시 동물로, 조류(algae)는 원시식물로써 취급된다. 박테리아는 비록 모든사람들이 그렇지 않다는 것을 알고 있음에도 불구하고,  식물들과도 주로 같은 묶음으로 여겨지기도 한다. 19세기 말로 돌아가면, 독일의 박물학자인Ernst Haeckel는, 박테리아는Moner라고 불리는 또 다른 별개의 Kingdom으로 나뉘어 분류해야 한다고 주장했다. 그러나 이 아이디어는 1960년대까지 오직 일부 과학자들만을 관심을 가졌을 뿐, 대부분의 생물학자들의 관심을 끌지 못했다.

·       the space age: 최신 시대

·       feature: 다루다

·       Amoeba: 아메바

·       proto-animals: 원시동물

·       proto-plants: 원시 식물

·       lump: …을 […과] 묶음[뭉치]으로 하다, 일률[총괄]적으로 취급[생각]하다[together／with ‥]

·       naturalist: 박물학자, 자연주의자

·       deserve: 할 가치가 있다.

가시적인 세상에 있는 많은 유기체들 역시 이런 전통적인 분류에서는 제대로 다루어지지 않았다. 버섯, 세포점균류(molds), 흰곰팡이(mildews), 효모, 그리고 말불버섯류(puffballs)들을 포함한 집단인, Fungi는 거의 항상 식물류로 여겨졌다, 비록 거의 모든것들- 그것들이 생식과 호흡을하고, 자신을 구성하는 방법들이- 식물 나라의 어떤 것과도 일치하지 않는데도 말이다. 구조적으로 보면, 그것들은  자신의 세포를 그들만의 구별되는 텍스쳐를 가질수 있게 해주는 물질인 키틴(chitin)을 이용해  만드는 방법에 있어 오히려 동물과 더 많은 공통점을 가지고 있다. 동일한 물질이 곤충의 껍질과 포유류의 발톱을 만드는데 사용된다, 비록 사슴벌레는 포타벨로 버섯만큼 맛있지는 않지만.(< 개그임-_-하하하……). 식물과는 다르게, 위에 언급된 fungi들은 광합성을 하지 않고, 그래서 그들은 엽록소들을가지고 있지않으며 따라서 초록색깔을 띄지 않는다. 대신에, 그것들은 그들의 음식원료위에 바로 성장하며, 그 자원은 거의 모든것들이 될 수 있다. Fungi는 어떤 식물들도 하지않는 두가지 일:콘크리트 벽의 황을 먹어치우고, 네 발가락 사이에있는 물질들을 분해시키는- 일을 한다. 거의 식물만이 할 수 있는 특성중에서 그들이 가진것은, fungi도 뿌리를 내린다는 것이다.

·       botanical: 식물학적인

·       respire: 호흡하다

·       stag beetle: 사슴벌레

·       Portobello mushroom:포타벨로 버섯. 맛이 진하고 고기씹는듯한 감촉: 채식주의자들이 좋아함.

공식적으로 Myxomycetes(변형균)이라고 불리지만slime molds(점균류)라고 더 잘 알려진 이 특징적인 유기 생물체들의 그룹은, 그 분류된 범주화를 훨씬 받아들이기가 어렵다.

Anappellation that sounded a-little more dynamic-"ambulant self- activating protoplasm,"say-and less like the stuff you find when you reach deep into a clogged drain would almost certainly have earned these extraordinary entities a more immediate share of the attention they deserve, for slime molds are, make no mistake, among the most interesting organisms in nature.( 한문장…ㅋ 완벽한 해석불가 ㅜㅜ )

그 이름은 의심의 여지가 없이 애매한 상태이다. ‘순회하는 자가활성 원형질’이라는 살짝은 역동적인 소리로 발음되며, 막힝 하수의 깊숙한 곳에 네가 도달했을때 찾은 물건과는 다른 느낌인 그 명명은(해석불가…ㅠㅠ), 아마 거의 확실히 그들이 원할만한 집중을 끌 수 있는 더 많은 즉각적인 공감이 아무 실수를 저지르지 않는 독특한 실체를얻었을 것이다.?????????

When times are good, they exist as one-celled individuals, much like amoebas. But when conditions grow tough, they crawl to a cen tral gathering place and become, almost miraculously, a slug.

좋은 시기에는, 그것들은 각개 개체로서 존재한다, 아메바와 배우 비슷한 상태로. 그러나 주위상황이 좋지 않아지며느 그들은  하나의 모임 중심으로 기어가서, 굉장히 놀랍게도, 민달팽이(Slug, 민달팽이라고 해석은 했지만, 그냥 민달팽이 형태의 큰 복합체가 되서 하나의 유기체처럼 움직인다는 말인것 같음.)가 된다.

The slug is not a thing of beauty and it doesn't go terribly far-usually just from the bottom of a pile of leaf litter to the top, where it is in a slightly more ex- posed position-but for millions of years this may well have been the niftiest trick in the universe.

그 슬러그는 아름다운 것은 아니지만,  (여기부터 해석 어색ㅜㅜ) 그렇다고 보통의 그 낙옆더미의 바닥으로부터 약간 더 노출된 장소인 꼭대기까지, 이것은 아름다움으로 부터 완전히 거리가 먼 것은 아니다, 그래서 아마 수백만년동안의 우주의 멋진 재치로 잘 남아있는 것은 아닐까.

·       Susceptible; (…의) 영향[작용 (등)]을 받기 쉬운, […에] 흔들리기 쉬운, 감염되기 쉬운[to ‥]

·       Peculiar: [사람·시대·지역 등에] 특유한, 독특한, 특징적인[to ‥]

·       Myxomycetes: 변형균, 잡균

·       slime molds: 세포 점균류

·       obscurity: [불] 애매한 상태; 불명료, 몽롱; (문어) 어둠, 어두컴컴함, 알려지지 않은 부분

·       Anappellation: 명칭, 호칭, 명명

·       Ambulant: 이동, 순회하는

·       Protoplasm:원형질, 세포질

·       Entities: 독립체, 본질, 실체

·       Miraculously: 기적적으로, 놀랄만하게

·       Slug:민달팽이

·       Nifty:멋진, 재치있는, 재치있는것

            그리고 이것은 여기에서 끝나지 않는다. 그 스스로를 좀 더 선호하는 장소로 가면서, the slime mold는 다시한번 식물의 형태로 자기자신의 형태를 바꾼다. 몇몇 호기심을 유발시키는 질서정연한 과정에 의해 셀들은, ‘자실체(fruiting body)’라고 알려진 동그란 형태의 꼭대기를 가진 가느다란 자루 형태로 만들기 위해, 작고 가느다란 튀어나온 밴드의 구성원같이 형태를 재조정한다. 자실체 안에는 수백만개의 포자들이 있으며, 그 포자들은 아주 적절한 시기에 이 전체 프로세스를 다시 시작 할 수 있는 단세포 유기체를 만들기 위해, 바람을 통해 흩날리며 밖으로 방출된다.

·       Haul: 잡아끌다, 수송하다, 체포하다

·       locale : 장소, 현장

·       reconfigure: 형/부품을 바꾸다

·       march: 행진하다, 앞으로 나아가다

·       stalk: 식물의 줄기, 자루, 가느다란 버팀대

·       atop: -의 꼭대기

·       bulb: [식물] (양파의) 구근(球根)/  원통형 물건 끝의 둥그스름한 부분(스포이트의 고무 등)

·       fruiting body: [식물] 자실체(子實體): 균류의 포자를 만드는 조직.

수년동안, slime molds는  동물학자들에 의해단세포 동물로, 균류학자들에 의서는fungi라고 주장되어 왔다, 비록 대부분의 사람들은 그것들이 어느곳에도 속하는 것 같지 않다고 생각했지만 말이다. 유전적 실험정보가 도착했을때(밝혀졌을때), 실험복을 입고있던 사람들은(과학자들은) slime molds가 굉장히 뚜렷히 구별되고 특징적이어서 그것들이 자연에있는 어떤 것들과도 실질적으로 바로 연관성이 없다는 것 –심지어 때때로는 그들 사이에서 조차도- 라는 것을 알고 깜짝 놀랐다.

·       Protozoa: 원생 동물; 단세포 동물.

·       Mycologists: 균류학자

·       distinctive : 구별되는

·       peculiar: 고유의 특징적인

1969년에, 분류 체계의 부족함을 채우려는 시도로써, Cornell University의 한 생태학자, R H. Whittaker는  Science지에서, 생명체를 다섯개의 주된 가지들을 ‘kingdom’ 으로, Animalia, Plan- tae, Fungi, Protista,  Monera으로 나누기 위한 자신의 목적을 밝혔다. Protista는, 약 한 세기 전에 제시되어 사용되었던  스코트탠드 생물학자인John Hogg가 식물과 동물 어느곳에도 속하지 않는 모든 유기체들을 설명하기 위해 만든  Protoctista 라는 구시대의 용어를 변경한 것이다.

·       inadequacies: 부적당 불충분 부족

·       classification: 분류, 등급가르기

·       Protista: 원생생물

·       Modification:번경

            Whittaker의 이러한 새로운 체계는 엄청난 진보였음에도 불구하고, Protista는 정의내리기 힘든채로 남아있었다. 몇몇 분류학자들은 그 용어를 큰 단세포 유기체- eukaryotes-를 위해 따로 놔두었지만, 그러나 그외 다른 사람들은 그것을 어느곳에도 맞지 않으면 무엇이든지 다 같이 묶어서 처리해 버림으로써, 마치 생물학버전의 한짝만 남은 양말을 처박아두는 서랍장같이 다루었다. 이 것은 slime molds와 아메바, 그리고 심지어 미역(seaweed)과 기타 등등역시 포함하고 있었다. 대충 계산해 보았을때, 이것은 200000종의 수많은 다양한 유기체들이 전부 포함되어있었다. 그건 너무 많은 한짝 남은 양말들이지.ㅋ(<계속 개그임)

·       Scheme: 계획, 기획, 체계

·       Taxonomists: 분류학자, 계통주의자

아이러니 하게도, Whittaker의 5 kingdom 분류 쳬계과 비슷한 류의 방법들이 교과서에서 발견되기 시작했다. the University of Illinois의 어떤 평범한 학자는 모든것을 고무시키는 발견을 통해 그의 방법을 그룹화했다. 그의 이름은Carl Woese로, 1960년대  중반부터 –또는 아마 그렇게 하기가 가능 했을 일찍 부터- 그는 조용히 박테리아의 유전자 서열들을 연구하고 있었다. 옛날 시절에는 이것은 몹시 힘들고 많은 수고를 요하는 과정이었다. 하나의 박테리아를 연구하는데에 일년을 소비하기 일 수 였다. Woese에 의하면, 그 당시에는  오직 약 500종의 박테리아만 알려져 있었으며 그것은 우리 입에 있는 종들의 수보다도 적은 수치인 것이었다. 오늘날, 그 숫자는 그 당시의 것보다 약 열바정도 많은 숫자이지만, 여전히26,900 종의 algae나 70,000종의 fungi, 30,800종의  amoebas들, 그리고 생물학의 연대기를 채울만한 일대기들과 관련된 유기생명체들에 비하면 매우 적은 수치이다.

·       Unassuming: 참견하지 않는, 젠체하지 않는, 허세 부리지 않는

·       Exceedingly: 대단히, 몹시

·       Painstaking: 수고를 아끼지 않은 힘든 부지런한 애쓰는

·       Biographies: 일대기, 전기

·       Annals: 연대기 , 연보

이것은 전체량을 낮게 유지할만큼 단순히 대수롭지 않은것이 아니다. 박테리아는 짜증스러울 정도로 분리 해내고 공부하기 어려울 수 있다. 오직 약 1%만이 배양 된다. 그것들이 자연에 얼마나 야생적으로 순응하는지를 생각해 보면, 그들은 살고 싶지 않아 보이는 그 하나의 장소가 페트리 접시라는 것은 기묘한 사실이다. 박테리아들을 agar침대에 퐁당 떨어뜨려서 네가 잘 할테지만 애지중지 하라,그리고 대부분은  그저 거기에 자리잡을 것이다, 박테리아가 꽃피우려는 시도들은 점점 쇠퇴하면서. 실험실에서 번성한 박테리아들은 예외적으로 정의되고, 그들은  거의 독보적으로 미생물학자들에 의해 연구되는 유기체들이다. Woese가 말하기를, “이것은 마치 동물원들을 방문함으로써 동물들에 대해 비우는 것과 같은 것이다.”

·       indifference: 무관심, 냉담

·       exasperatingly: 정말 짜증스럽게[화나게].

·       Plop: …을 퐁당[풍덩, 털썩] 떨어뜨리다.

·       Pamper: [사람·동물을] 지나치게 소중히 하다, 애지중지하다

·       Decline: 거절하다, 내리막길처럼 기울이다

·       Inducement: ① 유인 ② 권유 ③ 동기

·       Thrive: 번성, 번영하

·       Exclusively: 배타적으로, 독점적으로 , 오로지

            그러나 genes은, Woese가 미생물들에 대해 또다른 각도로 접근할 수 있게끔 했다. 그가 일했던 것처럼, Woese는 미생물 세상에는 어느 누가 예상했던 것 보다도 더 근본적인 분류가 존재한다는 것을 깨달았다. 박테리아처럼 생겼으며, 박테리아처럼 행동하는 수많은 작은 유기체들이 전적으로 사실은 완전히 다른 -오랜 시간전에 박테리아 종우로 부터 분리되어 나온- 무언가였다. Woese는 이러한  유기체들을archaebacteria라고 불렀으며, 이는 나중에archaea로 짧아졌다.

·       Altogether: 전적으로, 아주, 완전히

Archaea를 박테리아로부터 분리 구별 해낸  그 기여는 생물학자들이 흥분할만큼 가슴을 뛰게하는 그런 일이었다. 그것들은 그들의 지질과peptidoglycan이라고 불리는 물질면에서 큰 차이를 보였다. 그러나 실질적으로 그것들은 다른 세상을 만들었다. 나랑 당신을 게와 거미와 비교했을 때보다, Archaea와 박테리아의 차이가 훨씬 더 크다. Woese는 단독으로 생명체의 예상되지 않았던 새로운 분류 그룹을 발견해 내었고, 그래서 이는 the level of kingdom위에 기초하여 보편적인Tree of Life라는 정점을 찍을 수 있게 되었다.

·       Single handedly: 단독으로

·       Apogee: 정점

·       Reverentially: 경건하게, 공경하는 마음 가득하게

1976년도에 그는 세계를 깜짝 놀라게 했다- 또는 최소한 그런 약간의 관심을 끌게 되었다- 그건  Tree of Life를 다섯개의  큰 분류가 아닌 23개로 구체화 해서 재 구성함을 통해서 였다. 그가 그룹지은 것들은 크게 세개의 새로운 주 분류-Bacteria, Archaea, 그리고Eukarya (가끔 Eucarya이라고 쓰이기도 함) 아래에 존재했으며, 이들은 도메인이라고 불렸다.

·       startle: 깜짝 놀라게 하다

·       incorporate: 구체화 하다

*<<이곳에  도메인 체계가 들어감. 해석은 필요없는 부분이라 생략^.^>>

Woese의 새로운 분류체계는 생물학 세계를 폭풍으로 몰고가지는 않았다. 몇몇은 미생물들 분야가 너무 큰 비중을 차지하고 있는 점을 들어 받아들이기를 거절했다.많은 이가 그저 무시했다. 프랑스의Ashcrok에 따르면Woese는 ‘매우 쓴 실망을 맛보았다’고 한다. 그렇지만 그의 새로운 제안은 아주 천천히 미생물학자들의 관심을 사로잡기 시작했다. 식물학자와 동물학자들은 그것의 가치에 대한 평가를 훨씬 더 느리게 받아들였다. 왜 그랬는지를 알아보는건 어렵지 않다. Woese의 모델에서, 식물학과 동물학의 세계는 작은 가지 체계로 강등되어, Eykaryan도메인의 가장 바깥쪽에 위치하고 있었기 떄문이다. 모든 것들 역시 단세포 개체로써 포함되었다.

·       Botany:식물학

·       Zoology:동물학

·       Relegated: [A(사람·사물)를 B(보다 낮은 지위·상태·장소)로] 쫓아보내다, 물리치다, 좌천시키다,  추방하다(banish); (축구에서) [팀을] 보다 하위 리그로 강등시키다

·       Twigs : (보통 잎이 없는 가느다란) 어린[작은] 가지. cf. BRANCH

·       Outermost: 『부사』 가장 바깥쪽의[에], 가장 먼[멀게]

이러한 사람들은 총체적인 형태학적 유사성과 차이점들에 관하여 분류하기를 원하는 쪽이었다. Woese는 1996년 인터뷰중에, ‘분자적 시퀀스에 관한 것들을 한다는 생각은 많은 사람들에게 있어 그들이 받아들이기 약간은 힘들 수도 있습니다’라고 했다. 짧게 요약 말하면, 만약 그들이 그들의 눈으로 차이점들을 볼 수 없다면 그들은 그것을 달가워 하지 않는다는 것이다. 그리고 그래서 그들은 더 널리 알려진 다섯개의 왕국 분류 체계- Woese가 말하기를 ‘매우 유용하지 못한’ 그리고 ‘ 잘못된 방향으로 이끌어나가는’ 정렬방식- 에 계속 잔류해있는 것이다. Woese 는 이렇게 적었다; ‘생물학은, 흥미로운 물체나 그것의 상호작용들이 때때로는 직관적인 관찰을 통해 받아들여 지지 않기도 하는 레벨로 올라가기 바로 전의 물리학과 같은 것.’

·       Folks: 사람들, 민속의

·       Gross: 총체[총계]의, 전부[전체]의

·       Morphological: 형태학적인

·       in terms of: …에 말로, …의 항으로, …에 관하여

·       persisted: 지속하다 잔존하다 영향이 남다

1998년도, 위대한 학자였던Harvard의Ernst Mayr는 더 나아가 생명체의 분류에 대해 크게, 중요한  두개의empires로 나눌 것을 제안하면서 상황을 복잡하게 만들었다. the Proceedings of the National Academy of Sciences에 게시된 Mayr의 논문에서는,  Woese의 발견은 흥미롭지만 궁극적으로는 잘못 되었으며, Woese는 생물학자로써 교육받지 않았고 그래서 자연스럽게 분류에 대한 원리 비슷하게 확장된 비슷한 점 조자도 가지고있지 않을만큼 아무것도 없다고 말했으며, 이것은 거의 ‘그는 그 자신이 말하고 있는 것에 대해서 아무것도 모른다’고 말하는 것과 비슷한 것이었다.

·       Declaring: 신고하다 선언하다 단언하다

·       Prime: 가장 중요한

·       stirred the pot: 상황을 복잡하게 만들다

Mayr의 비판중 특정한 부분들은 굉장히 전문적이었다- 그것들은meiotic sexuality에 대한 부분과, Hennigian에 대한 해명,  Methanobmeriurn thermoautrophicum의 게놈에 대해 논쟁이 되고있는 해석, 그리고 또다른 많은 부분들 역시 포함시키고 있었다- 그러나 그가 가장 중요하게 주장한것은, Woese의 정리가the tree of life의 균형을 꺠뜨렸다는 것이었다.

·       Technical: 전문적인

·       Extensive: 광활한

·       Cladification: 정화, 해명, 설명

·       Controversial: 논쟁상의

·       Interpretations: 해석

 Mayr는, 박테리아 영역은 몇쳔개가 안되는 종으로 구성되어있는 반면, archaean은 175개의 이름지어진 종에 불과 하다고 말했다- 아마도 나중에 발견될 약 몇 천개가 존재 하지만 그것보다 더 많지는 않을 것이라고 말했다. 대조적으로the eukaryotic영역에서는 – 액이 있는 더 복잡한 생물체, 우리 같은- 이미 수백만을 넘어가는 숫자가 존재 한다. ‘균형 이론’에 따르면Mayr는 양쪽의 균형을 맞추기 위해서, 단순 박테리아 생물페들을 하나의 카테고리인  Prokaryota에 합치는 반면,  훨씬 더 복잡하고 고등진화된 나머지들은Eukaryota 라는 empire에 위치시키자고 주장했다. 다른 방법에 적용 시키면, 그는 그것들이 예전에 있었던 대로 그대로 유지시키기 위해 주장했다. 이런 단순한 세포와 복잡한 세포들 사이의 분류는 생명체 세상에서의 가장 큰 차이가 일어나는 곳이었다.

·       Realm: 영역

·       Mere: 단지-에 불과한

·       Nucleated: 핵이 있는, 핵에서 생기는, 응집시키다

·       Remainder:나머지, 잔여품

·       Alongside:- 옆에

만약Woese의 새로운 정렬방식이 우리에게 무엇인가를 가르쳐 주었다면, 그것은 생명은 매우 다양하고 그 다양함은 작고, 단세포이며 친숙하지 않은 것에 대부분이라는 것이다. 발전의 긴 사슬, 끝없는 커짐과 복잡함을 향한 진보같은 것들과 관련 된-한마디로 말하면, 우리들에 관한,  ‘진화’에 대해 생각하는 것은 자연스러운 인간의 충동이다. 진화와 관련된 진정한 다양성의 대부분은 작은 스케일이어왔다. 우리같은 큰 생물체들은 그저 흥미로운 옆가지중 하나가 되는 뜻밖의 행운이 있었을 뿐이다. 생명체에 대한 23개의 메인 분류, 오직 식물, 동물, fungi로 나눈 세개의 분류들은, 인간의 눈으로 볼 수 있을만큼 충분히 크나 현미경을 통해 서만 보이는 종들도 포함하고 있다. 그리고 실제로 Woese에 따르면,  만약 식물을 포함한 살아있는 모든것들을 포함시킨 이 행성의 모든 바이오매스를 합치면, 그중 미생물이 최소 80퍼센트 또는 그 이상이 될 것이라고 한다. 이 세상은 그 매우 작은 것들의 것이다- 그리고 이것은 아주 오랫동안 그래왔다.

·       impulse : 충동, 자극

·       in a word: 한마디로 말해서, 요컨대

·       flukes: 요행, 뜻밖의 행운

·       microscopic: 미시적인

교재2..... (오ㅅㅈ 교수님이 계속 가르친다면 이걸로 계속 하실것 같은데.ㅋㅋ)

…… 저는 생물책이아니라 무슨 소설책이나 시집 읽는 줄 알았어요…

한문장이 네줄 넘어가는 것도 있고ㅜㅜ 내용이 아주 흥미 진진하게 전개되네요…ㅋㅋㅋㅋㅋㅋㅋㅋ ㅜㅜ  

우선 이부분은 19페이지(오 교수님 교재 기준) 별표까지입니다*

나중에 시간나면 업뎃 할 수도 있긴한데.. 아마 안할거에요ㅋㅋㅋ 그러니 요청은 안받을게요ㅋㅋ 제가 업뎃하면 이건 재수강한다는 뜻........ ㅠㅠ

이 글을 보는 사람들은 아마도 생명과학기술영어 수업을 듣는 제 후배들이겠죠?^.^
열공해서 좋은 학점 받길 바랄게요!ㅋㅋ

제가 직접 해석한 것들이라서 아마 조금...(많이..ㅋㅋ) 어색한 부분들이 있을텐데, 친구들하고 스터디 하고 교수님께 질문하면서 잘 해결해 나가길 빌게요!

제 머리로는 해석이 불가능 한것들이 좀 많이 있어서 최대한 해석해보려고 노력은 했는데 많이 부족하네요. 피드백해주세요^.^ 

형질전환(Transformation)

1. 실험배경과 목적

한 세균으로부터 나온 유래한 DNA를 다른 세균이 받아들임으로써 유전형질이 전환되는 유전자 전달방식인데 Streptococcus pneumoniae, Haemophilus influenza, Bacillus subtilis, Streptococcus sanguis, Neisseria gonorrhoeae, Acinetobacter calcoaceticus에서 잘 연구된 바 있으나, 요즈음은 유전자조작 기술의 발달과 더불어 여러 종류의 세균에서 적용되고 있다. 그런데 어떤 세포에서의 형질전환 과정이 자연적인 과정의 하나로 이루어지기 위해서는 유전자 수용세균이 배지내에 있는 DNA를 세포내로 수용할 수 있는 상태(competent)에 있어야 한다. 또한 접합실험과 마찬가지로 유전자 공여체와 수용세균이 서로 다른 유전형질을 가지고 있어야만 형질전환체(transformant)의 선별이 가능하다.

유전자 공여체로 바이러스 DNA를 사용하는 경우를 특별히 transfection이라고 한다. 여기서는 이미 추출한 플라스미드 DNA로 형질전환시키는 방법 중 가장 많이 이용되는 Mandel 과 Higa(1970)의 CaCl2처리법을 살펴보자.

2. 실험 재료 및 방법

▣ 재 료

․Escherichia coli HB101, 플라스미드 pBR322 DNA, LB broth 배지

․Ampicillin(Am)이 포함된 LB-한천평판배지, 10mM Tris-HCl(pH8.0)에 녹인 50mM CaCl2 용액 TE 완충용액(pH8.0)

▣ 과 정

① 500 ml짜리 플라스크에 들어 있는 100 ml의 L-액체배지에 하룻밤 배양한 E.coli HB101 배양액 1ml을 가하고 격렬하게 진탕하면서 37℃에서 2~4시간 배양하여 균의 밀도가 약 5× 107 세균/ml (O.D. 600≒0.5에 해당됨) 되게 한다.

② 배양체를 얼음에 약 10분 담그어 식힌 후 20ml을 취하여 4℃, 4000g에서 5분간 원심분리한다.

③ 상등액을 버린 다음, ice-cold한 멸균된 50 mM CaCl2 용액을 원래의 ½부피(15ml)만큼 가하여 현탁시킨다.

④ 얼음에 15분 방치한 후, 다시 원심분리한다.

⑤ 이를 다시 원래의 1/15부피만큼(2ml)의 50mM CaCl2 용액에 다시 현탁시킨 후, 미리 차게 식혀 둔 미세원심분리관에 0.2ml씩 나누어 담고 4℃에서 12 - 24시간 보관한다.

⑥ TE 완충용액에 0.5㎍/ml 되게 녹인 pBR322 DNA용액 (또는 ligation된 재조합 DNA)을 CaCl2 처리한 E. coli가 들어 있는 미세원심분리관에 2㎕씩 가한다.

⑦ 42℃항온수조에서 1분 방치한다.

⑧ L-액체배지 1㎖을 가하고 37℃에서 1시간 배양한다.

⑨ 적당량(0.1 - 0.2 ml)의 배양액을 항생제가 첨가된 LB 한천평판에 삼각유리봉으로 도말 한다.

⑩ 평판을 뒤집어 37℃에서 배양한다. 대개 12 - 16시간이면 콜로니가 형성된다.

3. 실험수행상 주의 할 점

․형질전환률을 최대로 하기 위해서는 대수기(exponential phase)의 세균을 사용하고,

CaCl2 처리시에는 세균 밀도가 낮은 것이 좋다.

또한 ⑤과정에서와 같이 4℃에서 12 - 24시간보존한 후 DNA와 반응시키면 형질전환 률이 4 - 6배 증가하나, 24시간을 경과하면 감소함에 유의할 것.

․Amp 내성 형질전환체를 선별하고자 할 때는 평판배지 당 세균의 밀도가 낮게 접종하고 배양 16 - 24시간 후에는 4℃에 보관하도록 한다. 왜냐하면 내성균이 분비하는 배지내의 β-lactramaserk 확산되어 콜로니주변 배지의 Amp이 분해되어 접종 밀도가 높거나, 배양 시간이 길면 Amp 감수성 균도 자랄 수 있게 되게 때문이다.

실험보고서

[결과]

형질전환률을 다음에 의거하여 구하라.

형질전환률 = 형질전환체의 수/플라스미드 DNA랑(㎍)

[물음]

1. 형질전환시 플라스미드 DNA의 농도가 높고, 가하는 DNA용액의 부피가 크면 형질전환률이 떨어지는 이유를 생각해 보라.

2. CaCl2 처리의 목적을 살펴보고, 이와 동일한 효과를 내는 처리법들이 (즉 DNA가 세균 세포내로 도입되도록 도와주는 방법)어떠한 것이 있는가 살펴보자.

DNA 전기영동

1. 실험 제목: DNA 전기영동 (DNA Electrophoresis)

2. 실험 목적 등 배경

전기영동(Electrophoresis)이란 특이한 매질 (agarose나 acrylamide)을 이용하여 DNA나 단백질 등의 고분자 물질을 분리하는 방법이다. Agarose에 DNA를 넣고 전기를 걸어주면 DNA는 인산기 때문에 음전하를 띠므로 매질을 타고 양전하 방향으로 움직이게 된다. 이 때 DNA의 움직이는 속도는 DNA의 크기, 형태 (선형, 원형 등)에 따라 다르므로 매질 내에서 그 특성에 따라 분리되어 진다.

본 실험에서는 제한 효소로 자른 plasmid DNA (특별한 구조를 지니지 않은 선형 DNA)와 제한효소를 처리하지 않은 원형 DNA (superhelical 구조를 지님)를 agarose gel 상에서 직접 전기영동하고 이를 ethidium bromide로 염색 후에 자외선을 이용해 눈으로 직접 확인해 봄으로서 DNA 전기영동의 원리와 과정들에 대해 이해해 보고자 한다.

3. 재료 및 실험 기구 및 기기

agarose powder 2 g

1X TBE (Tris-borate buffer) 용액 200 ml X 8조

5 X Gel loading dye 용액 100 ul

10 mg/ml ethdium bromide 용액 100 ul

agarose gel caster 8 대

전기영동 장치 (chamber 및 power supply) 4 대

DNA sample (1 kb ladder, pBlueScript plamsid DNA) 각 20 ug

lambda pippetman and yellow tip8 개

삼각 플라스크8 개

전자렌지 또는 전열기1 대

UV transilluminator, 사진기, balance1 대

Mass cycliner1 개

* 4명 1조, 8조를 기준으로 작성

4. 실험 방법

4-1. agarose gel 만들기

1) 2 liter의 1X TAE buffer 용액 (90 mM Tris-borate, 2 mM EDTA)을 먼저 만든다. 5X TAE buffer 용액 400 ml에 증류수를 첨가하여 2 liter를 만든다

* 5X TAE stock 용액 제조: 54 g의 Tris base, 27.5 g의 boric acid,

20 ml의 0.5 M ETDA 용액에 증류수를 첨가하여 최종 1 liter를 만든다.

2) 삼각 플라스크에 1X TAE 용액 25 ml과 0.25 g의 agarose powder를 넣어 전자렌지에서 agarose 입자가 완전히 녹을 때까지 끓여준다.

3) 손으로 댈 수 있을 때 까지 식힌 후에 10 mg/ml의 ethidium bromide 용액을 2 ul 첨가하여 잘 섞은 후에 gel caster에 부어 완전히 굳을 때 까지 기다린다.

4-2. DNA 시료의 준비

Gel이 식는 동안 분석하려는 DNA 샘플 5 ul (300 - 500 ng)당 1 ul의 loading dye를 첨가하여 준비한다. 또한 5 ul의 1kb ladder 마커 (1 ug)에 1 ul loading dye를 섞어 분자의 크기를 알 수 있는 표준 샘플도 준비한다.

* loading dye는 50% glycerol에 bromophenol blue를 섞은 것으로 DNA가 gel에 loading될 수 있게 하며 전기영동 동안에 시료의 이동을 확인할 수 있게 한다.

4-3. 시료 주입(sample loading)

1) 완전히 굳은 agarose gel에서 조심히 comb을 제거한 후 gel casting tray를 전기영동 탱크에 넣은 후 TBE buffer 용액를 탱크에 부어 gel이 수면으로부터 1-2 mm정도 잠기게 한다.

2) Well의 가장 왼편에 DNA 마커 샘플을 주입하고, 다음 well부터 샘플 (6ul)을 순서대로 주입한다.

3) 샘플을 다 주입하면, gel tank에 전극을 맞추어 연결한다. [gel의 well쪽이 (-)전극 (검정색) 쪽에 위치, DNA는 (+)전극쪽 (빨강색)으로 이동하게 됨]

4) Loading dye의 이동이 gel의 거의 3/4지점까지 내려갈 때까지 전기영동한다.

4-5. 사진찍기

1) Gel 탱크의 전원을 끄고 gel을 UV transilluminator로 옮긴 후 보호 장치를 하고 UV light를 켠다.

*주의 : UV transilluminator는 노출 시 피부와 눈에 손상을 입히는 단파장의 UV light를 발산하기 때문에, 반드시 비닐장갑이나 보호장구를 착용해야함.

2) Transilluminator 전원을 켜고 gel을 관찰한다. DNA 밴드(orange band)를 확인 후 사진을 찍고 전원을 끈다.

실험보고서

1. 1kb ladder DNA 마커들이 움직인 거리와 그 크기 사이에는 어떤 상관 관계가 있는지 실측하여 공식으로 나타내 보자.

2. Supercoil plasmid DNA 시료에서 보이는 다양한 종류의 DNA들은 각각 어떤 형태이며 이들의 움직임에 차이가 나는 이유는 무엇인가?

3. UV를 조사하였을 때 DNA가 오렌지색으로 보이게 되는 이유와 원리는 무엇인가?

제한효소 (restiction enzyme) 반응

1. 실험목적 : DNA를 제한효소로 잘라서 정기영동하여 분석함으로써 제한효소의 특성과 반응을 이해하고 DNA상의 제한효소 지도 (map)를 작성할 수 있는 능력을 기른다.

2. 재료, 실험기구 및 기기 :

Plasmid DNA, DNA size marker,

제한효소와 reaction buffer, 3차 증류수,

eppendorf tubes, micropipetter (20 ㎕, 200 ㎕),

piptet tips, 37℃ water bath,

ice bucket, ice,

microcentrifuge, microwave,

agarose, flask,

미세 저울, ethidium bromide (10 ㎍/ml stock),

6X DNA loading dye, 50X TAE buffer,

agarose 전기영동 시스템과 power supply,

UV box와 사진촬영 시스템

3. 실험 방법 :

DNA를 자르기 위해서는 eppendorf tube에 reaction buffer, DNA 그리고 효소를 같이 넣고 특정온도에서 일정시간동안 반응을 시켜야한다. 먼저 반응을 시키기 전에 자르려고하는 DNA의 양과 reaction volume을 결정해야 한다 (대체적으로 1 ㎕ reaction volume 당 최대 약 100 ng DNA 까지 사용한다). 완전하게 DNA를 자르기 위해서는 효소의 충분한 (3-5배) 활성 단위(unit)로 잘라야한다. 효소 단위