// Workers AI · dad joke modeWhat did the specific-locus test say? I'm in a specific spot.
The specific-locus test (SLT) is a genetic test to assess the impact of mutagens such as radiation or chemicals on mutation rates in a selected set of easily measured genes. The SLT measures mutation rates in a laboratory mouse by mating mutagen-exposed wild-type animals with tester-stock mice carrying recessive alleles at a defined set of marker loci, classically seven, with visible traits such as altered coat color or short ears.[1]
Overview
[edit]The specific-locus test is an assay that uses mutagenesis of the mouse germline to detect and quantify heritable mutations; these mutations are those which are transmitted to first-generation offspring, and measured as visible traits.[2] Because the marker genes are a small number of loci (seven, in the most widely used mice), the assay was given the name of the “mouse specific-locus test” or “SLT”.[3] The method was developed for radiation treatment and effects but the same approach was later applied to chemical mutagenesis.[2][4]
The assay is best used for measuring heritable point mutations and small deficiencies affecting the wild-type allele contributed by the treated parent, which are revealed when paired with a recessive marker allele in the offspring.[2]
In the offspring from the cross, a newly induced mutation at one of the marker loci yields the recessive (and visible trait); this allows for rapid and large-scale screening for radiation- and chemical-induced mutations, and it requires no specialized equipment.[4]
Test design and genetic changes detected
[edit]To conduct the test, an individual must be produced that is heterozygous for multiple alleles that each have a distinct visible phenotype in their homozygous recessive state. Therefore, a wild-type parent, either untreated as a control or exposed to the mutagen being tested, is crossed to a multiple-recessive tester stock.[2][3] The standard visible-marker "T stock" has a set of the following seven recessive markers:[2][3]
- a / non-agouti - for coat color variation
- b / brown coat
- c or cch / albino or chinchilla-related locus
- p / pink-eyed dilution
- d / dilute (coat pigment dispersion)
- se / short ear (the BMP5 gene)
- s / piebald or spotted coat
Most markers used in the test are visible through coat color, eye color, or ear morphology.[2][3] The heterozygous offspring of the cross are normally wild type in appearance unless a new mutation has occurred at one of the marked loci.[4] However, a key aspect of the test is that mutant offspring are recognized by the visible recessive phenotype at one of the marker loci.[4] Scoring of the impact of the treatment is possible at about three weeks, although some mutants are recognizable earlier in development. [2] Allelism tests are used to confirm suspected mutations when needed.[2][3]
The types of mutations that yield a visible phenotype are known to be intragenic changes, including base-pair substitutions, frameshifts, and intragenic deletions, plus small deficiencies affecting marked loci, and occasional gross rearrangements.[2] Some dominant visible mutations outside the marked loci are detectable incidentally.[3][2] Double mutants at closely linked loci, especially d and se, sometimes indicate larger deletions.[2][3] The mutational spectrum as measured across the set of marker loci is used to compare qualitative differences among mutagens.[2]
Applications and findings
[edit]The original application of the method was to assess dose responses for radiation mutagenesis in mouse germ cells, as the mutations that are inherited occur in spermatogonial stem cells, post-spermatogonial stages, or oocytes.[2][1][4] This provided a comparison with Drosophila mutation-rate assessments.[4] The specific-locus test was first used to measure the effect of chemical mutagenesis by Bruce Cattanach in his work with triethylenemelamine (TEM).[5] Subsequent larger-scale work analyzed more than 25 chemical agents, and of these, 17 yielded positive or negative classifications.[2][4] A small-scale ENU experiment was sufficient to show strong mutagenicity in mouse spermatogonia; as a result, ENU was later used widely as a mouse mutagen, as it primarily creates mutations in single genes rather than large-scale chromosomal aberrations.[4]
One major finding from the dose-rate analysis performed with radiation mutagenesis is that protracted exposure produced fewer mutations than acute exposure at the same total dose; this provided early evidence for repair of mutational damage.[4] A key 1958 paper tested whether radiation-induced mutation frequency depended on dose rate.[6] The authors found lower specific-locus mutation rates after chronic gamma irradiation of spermatogonia than after acute X-ray exposure.[6] These observations challenged the earlier view, based largely on Drosophila spermatozoa, that mutation frequency was independent of dose rate.[6] Their findings suggested that genetic hazards under some radiation conditions could be lower than estimates based only on acute irradiation experiments.[6]
Strengths and limitations
[edit]The strengths of the SLT are that it provides a direct assay of transmitted genetic damage in a mammal; there is first-generation scoring via simple visible phenotypes that require no specialized instrumentation for classic visible-marker version.[4][2][3] The mutants are recoverable as stocks for additional genetic analysis. [3][4] And the test is useful for comparing mutational spectra among loci.[2]
The limitations of the test are that large numbers of mice are required, and only a small set of loci are monitored.[3] The large numbers of mice takes a lot of space, and breeding the animals takes significant time.[3][2] In addition, mutations without visible effects at the tested loci (like silent mutations) will not be detected, resulting in an under-count of mutational events.[3]
History
[edit]The SLT was independently developed by Toby C. Carter in Edinburgh and William Russell at Oak Ridge National Laboratory.[3][7]
Russell began developing the method in 1947 after joining Oak Ridge National Laboratory, with the early planning stages involving Alexander Hollaender, Sewall Wright, and H. J. Muller.[4] The motivation for the development of the SLT was the need for mammalian data on radiation-induced gene mutations, because at that time, risk estimates depended heavily on Drosophila data and not on a vertebrate animal more closely related to humans.[4]
The first Oak Ridge irradiation experiment was performed in March of 1949, while the first confirmed pilot mutant was at the d locus and the second pilot mutant was the c locus.[4] In a 1951 Cold Spring Harbor Symposium report, the main 600 R X-ray experiment performed by Russell used 48,007 offspring and identified 53 specific-locus mutations, while the control group with 37,868 offspring identified only two mutations, demonstrating that the method has high sensitivity for the detection of radiation-induced mutations.[4][1]
References
[edit]- 1 2 3 Russell, W. L. (1951). "X-ray-induced mutations in mice". Cold Spring Harbor Symposia on Quantitative Biology. 16: 327–336. doi:10.1101/sqb.1951.016.01.024. PMID 14942747.
- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Russell, L. B.; Selby, P. B.; von Halle, E.; Sheridan, W.; Valcovic, L. (1981). "The mouse specific-locus test with agents other than radiations: interpretation of data and recommendations for future work". Mutation Research. 86 (3): 329–354. doi:10.1016/0165-1110(81)90010-5. PMID 7029264.
- 1 2 3 4 5 6 7 8 9 10 11 12 13 Searle, A. G. (1975). "The specific locus test in the mouse". Mutation Research. 31 (5): 277–290. doi:10.1016/0165-1161(75)90093-X. PMID 1196328.
- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Russell, W. L. (1989). "Reminiscences of a mouse specific-locus test addict". Environmental and Molecular Mutagenesis. 14 Suppl 16: 16–22. doi:10.1002/em.2850140606. PMID 2659326.
- ↑ Cattanach, B. M. (1966). "Chemically induced mutations in mice". Mutation Research. 3 (4): 346–353. doi:10.1016/0027-5107(66)90041-8. PMID 5969214.
- 1 2 3 4 Russell, W. L.; Russell, Liane Brauch; Kelly, Elizabeth M. (1958). "Radiation dose rate and mutation frequency". Science. 128 (3338): 1546–1550. doi:10.1126/science.128.3338.1546. PMID 13615306.
- ↑ Carter, T. C.; Lyon, Mary F.; Phillips, Rita J. S. (1956). "Induction of mutations in mice by chronic gamma irradiation; interim report". British Journal of Radiology. 29 (338): 106–108. doi:10.1259/0007-1285-29-338-106. PMID 13284251.