Average Mutation Frequency Research Articles

Pol gene quasispecies of human immunodeficiency virus: mutations associated with drug resistance in virus from patients undergoing no drug therapy.

The nucleotide sequences of two pol gene regions (codons 41 to 108 and 181 to 219 of reverse transcriptase) of 60 human immunodeficiency virus type 1 genomes obtained directly from primary lymphocytes from infected individuals are reported. In addition, the mutant spectra of several quasispecies have been sampled by repetitive sequencing of molecular clones representing the same pol genomic regions. Average mutation frequencies ranged from 1.6 x 10(-2) to 3.4 x 10(-2) substitutions per nucleotide for independent samples (relative to their consensus nucleotide sequence) and from 3.6 x 10(-3) to 1.1 x 10(-2) substitutions per nucleotide for individual quasispecies distributions. Several mutations leading to amino acid substitutions related to loss of sensitivity to reverse transcriptase inhibitors have been identified in samples from patients not subjected to antiretroviral therapy. Mutation frequencies in the codons previously identified as involved in resistance to reverse transcriptase inhibitors were very similar to the average mutation frequencies in the pol region analyzed. Thus, the finding of mutations related to drug resistance (even in the absence of positive selection by the corresponding drugs) is the expected consequence of the statistical distribution of mutations along the pol gene. The presence of such critical amino acid replacements in human immunodeficiency virus type 1 populations underscores the importance of viral quasispecies as reservoirs of phenotypic virus variants and has a number of implications for AIDS control.

Efficient recovery of ENU-induced mutations from the zebrafish germline.

We studied the efficiency with which two chemical mutagens, ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) can induce mutations at different stages of spermatogenesis in zebrafish (Brachydanio rerio). Both EMS and ENU induced mutations at high rates in post-meiotic germ cells, as indicated by the incidence of F1 progeny mosaic for the albino mutation. For pre-meiotic germ cells, however, only ENU was found to be an effective mutagen, as indicated by the frequencies of non-mosaic mutant progeny at four different pigmentation loci. Several mutagenic regimens that varied in either the number of treatments or the concentration of ENU were studied to achieve an optimal ratio between the mutagenicity and toxicity. For the two most mutagenic regimens: 4 x 1 hr in 3 mM ENU and 6 x 1 hr in 3 mM ENU, the minimum estimate of frequencies of independent mutations per locus per gamete was 0.9-1.3 X 10(-3). We demonstrate that embryonic lethal mutations induced with ENU were transmitted to offspring and that they could be recovered in an F2 screen. An average frequency of specific-locus mutations of 1.1 X 10(-3) corresponded to approximately 1.7 embryonic lethal mutations per single mutagenized genome. The high rates of mutations achievable with ENU allow for rapid identification of large numbers of genes involved in a variety of aspects of zebrafish development.

A mouse model for the study of in vivo mutational spectra: Sequence specificity of ethylene oxide at the hprt locus

We have developed an approach for determining mutational spectra in exon 3 of the hypoxanthineguanine phosphoribosyl transferase ( hprt) gene in splenic T-lymphocytes of B6C3F1 mice. Hprt − mutants from treated animals were isolated by culturing splenic T-cells in microtiter dishes containing medium supplemented with IL-2, concanavalin A, and 6-thioguanine. DNA was extracted from 6-thioguanine-resistant co colonies and amplified by the polymerase chain reaction (PCR) using primers flanking the exon 3 region of hprt. Identification of samples containing mutant exon 3 sequences and purification of mutant DNA from contaminating wild-type hprt DNA was accomplished using denaturing gradient gel electrophoresis. Purified mutant sequences were then sequenced. This approach is being used to study the sequence specificity of ethylene oxide (ETO). 12-day-old mice were given single i.p. injections of 100 mg ETO/kg every other day or 30, 60, 90 or 120 mg ETO/kg daily for 5 days to achieve different cumulative doses of this compound. In mice exposed every other day, cumulative doses of 200, 600 and 900 mg ETO/kg produced average mutant frequencies of 15 ± 12.8, 45 ± 13.2, and 73 (70, 75) × 10 −6), respectively, 8 weeks after the first treatment. In mice exposed daily, cumulative doses of 150, 300, 450 and 600 mg ETO/kg produced average mutation frequencies of 4.2 ± 10.4, 8.2 ± 10.4, 11.1 ± 1.0 and 15.5 ± 10.7 × 10 −6, respectively, 20 weeks after the first treatment. The mutant fraction in control mice was less than 3 × 10 −6. 123 hprt − mutants from mice exposed to 600 or 900 mg ETO/kg were isolated and analyzed for mutations in exon 3. 18 were located in exon 3 (14.6%). DNA sequencing revealed that 11/18 mutations were base-pair substitutions at 8 different sites in exon 3. Four AT transversions, three AT transitions, two GC transversions, and two GC transitions were observed. Three of the substitutions (2 AT → CG, 1 AT → GC) occurred at one base (203) in a single animal. The remaining 7 mutations, isolated from 4 different animals, were the same + 1 frameshift mutation in a run of 6 consecutive guanine bases (207–212) in exon 3. These results suggest the involvement of both modified guanine and adenine bases in ETO mutagenesis. The mouse T-cell cloning/sequencing assay for hprt described here represents a useful system for studying the molecular mechanism of chemically-induced mutation occurring in vivo at an endogenous gene.

わが国栽培ギクにおける枝変わりの出現と染色体数の変異について

To elucidate the basis of chromosome variations in the Japanese garden chrysanthemum, the present study was carried out using natural sports in flower colour.1. Of 297 chrysanthemum cultivars examined, we found the frequency of sports to be low and varied slightly among the flowering forms. The average mutation frequency within this select group was 10.8%; it ranged from 5.2 to 15.7%.2. Thirty-two sports and 25 parental cultivars of Japanese chrysanthemums were surveyed for chromosome number variations. The chromosome numbers in 5 out of 32 sports (15.6%) differed from the parents by plus or minus one chromosome.3. Several clonal strains of 'Kii-amaga-hara' and their parental cultivar 'Amaga-hara' from which they originated as sports were subjected to karyotype analysis as related to satellite chromosome variations. The chromosome numbers varied little in the sport or parental plants, whereas the karyotype constitutions differed considerably among them. These results indicate that structural changes in chromosomes may have occurred in the sports.4. Four clonal strains of 'Kii-shuho-no-chikara' and five of its parental cultivar 'Shuho-no-chikara' were examined for the variations in chromosome numbers and certain morphological or ecological characteristics. Four parental strains (80% of those examined) had the same chromosome number (2n=54) as the original clone but in the two mutant strains (50% of those examined), the chromosome number was 52. As one might expect, the loss of two chromosomes by the strains resulted in considerable differences in several characteristics as compared with the original clone.5. Our analysis confirms that the appearance of sports is attributable to chromosome variations in the garden chrysanthemums. However, the frequency of these sports depends on the particular cultivar of chrysanthemum.

Assessment of inbreeding by DNA fingerprinting: development of a calibration curve using defined strains of chickens.

By analyzing DNA fingerprints of chickens from seven well-defined genetic groups, a calibration curve was established relating the degree of inbreeding with the average band frequency, allelic frequency and band sharing. The probe used was bacteriophage M13 DNA and digestion of the genomic DNA was carried out with the MspI restriction enzyme. The analysis also provided an estimate of the average allelic frequency at a hypervariable locus and the average mutation frequency per locus and generation. The values of 0.24 and 1.7 X 10(-3), respectively, are similar to the estimates for humans using other probes and hybridization protocols. It is suggested that the calibration curve established can be used for determining inbreeding not only in chickens, but also in other species.

Somaclonal variation in the progeny of plants regenerated from callus cultures of seven inbred lines of maize

Callus cultures were initiated from immature embryos of three dent corn inbred lines: FR27rhm (B73 derivative), 79-R4443, and EXL1; and four sweet corn (sh-2) inbred lines: f84-267, f84-275, f84-278, and f84-700. Extensive qualitative variation was noted in self-pollinated progeny of plants regenerated from callus of FR27rhm, EXL1, and f84-700; quantitative variation was measured in self-pollinated progeny of plants regenerated from cultures of almost all inbreds. Most qualitative mutations were visible in the first self-pollinated generation; chimerism in regenerated plants was indicated by initial segregation of some mutants after two self-pollinations. All culture-induced mutant phenotypes were similar to known spontaneous mutations in maize; preliminary data indicate most have single-gene recessive inheritance. For qualitative variation, average mutation frequencies per regenerated plant were calculated as 0.18, 0.71, and 0.64 for FR27rhm, EXL1, and f84-700, respectively. Differences in mutation frequency among the four sweet corn lines were determined to have a genetic basis. Significant quantitative variation included alterations in date of pollen shed and silk emergence, silk emergence relative to pollen shed, plant height, ear, and kernel characteristics. These results show genotypic differences in the extent of qualitative somaclonal variation, with some quantitative variation having possible applications for conventional corn breeding programs.

EMS- and relation-induced mutation frequencies at individual loci in Arabidopsis thaliana (L.) Heynh

Average mutation frequencies per locus per dose of mutagen — EMS, X-rays and fast neutrons — were estimated from 15 different loci of Arabidopsis thaliana. These loci related to clearly distinguishable phenotypic mutant groups, each affected by a limited number of loci. There were significant differences in mutation rate between individual loci. The proposition of a linear relationship between DNA content and mutation frequency per locus is rejected.

DEFECTIVE KERNEL MUTANTS OF MAIZE. I. GENETIC AND LETHALITY STUDIES

A planting of 3,919 M(1) kernels from normal ears crossed by EMS-treated pollen produced 3,461 M(1) plants and 3,172 selfed ears. These plants yielded 2,477 (72%) total heritable changes; the selfed ears yielded 2,457 (78%) recessive mutants, including 855 (27%) recessive kernel mutants and 8 (0.23%) viable dominant mutants. The ratio of recessive to dominant mutants was 201:1. The average mutation frequency for four known loci was three per 3,172 genomes analyzed. The estimated total number of loci mutated was 535 and the estimated number of kernel mutant loci mutated was 285. Among the 855 kernel mutants, 432 had a nonviable embryo, and 59 germinated but had a lethal seedling. A sample of 194 of the latter two types was tested for heritability, lethality, chromosome arm location and endosperm-embryo interaction between mutant and nonmutant tissues in special hyper-hypoploid combinations produced by manipulation of B-A translocations. The selected 194 mutants were characterized and catalogued according to endosperm phenotype and investigated to determine their effects on the morphology and development of the associated embryo. The possibility of rescuing some of the lethal mutants by covering the mutant embryo with a normal endosperm was investigated. Ninety of these 194 mutants were located on 17 of the 18 chromosome arms tested. Nineteen of the located mutants were examined to determine the effect of having a normal embryo in the same kernel with a mutant endosperm, and vice versa, as compared to the expression observed in kernels with both embryo and endosperm in a mutant condition. In the first situation, for three of the 19 mutants, the mutant endosperm was less extreme (the embryo helped); for seven cases, the mutant endosperm was more extreme (the embryo hindered); and for nine cases, there was no change. In the reverse situation, for four cases the normal endosperm helped the mutant embryo; for 14 cases there was no change and one case was inconclusive.

Somatic mutation frequencies in the stamen hairs of stable and mutable clones of tradescantia after acute gamma-ray treatments with small doses

Young inflorescences of two different Tradescantia clones heterozygous for flower and stamen-hair color, one stable (KU 9) and the other spontaneously mutable (KU 20), were irradiated acutely with small doses (approx. 3 to 50 R) of 60Co gamma-rays. Somatic mutation frequencies from blue to pink in the stamen hairs scored on post-irradiation days 10 to 16 increased essentially linearly with increasing gamma-ray dose in both clones. Despite about a 5-fold difference in spontaneous mutation frequency per hair found between the two clones, the dose-response curves of pink mutations determined were similar to each other, giving average mutation frequencies of 1.51 and 1.41 pink-mutant events per 1000 hairs per R for KU 9 and KU 20, respectively. These frequencies are comparable to earlier results obtained from acute irradiation treatments of other clones with higher doses. The doubling dose of pink mutation (the radiation dose making the mutation frequency double the spontaneous level) was calculated to be 2.09 R for KU 9, and this low doubling dose must be given full attention. On the other hand, the doubling dose for KU 20 (calculated to be 10.4 R) is of questionable value, being greatly subject to change because of the diversely variable spontaneous mutation frequency of this clone.

Mutation induction by rodent liver microsomal metabolites of aflatoxins B 1 and G 1 in Neurospora crassa

The mutagenic activities of aflatoxins B 1 and G 1 were studied in the ad-3 test system of Neurospora crassa by treatment of conidia with aflatoxin and liver homogenate for 2 h. No significant increase in the ad-3 mutation frequency over the spontaneous frequency was observed when either aflatoxin or mammalian liver homogenate was omitted from the test system. The ad-3 mutation frequencies increased to between 29 and 87/10 6 survivors, which is a 73- to 217-fold increase over the average spontaneous ad-3 mutation frequency (0.4/10 6 survivors), after conidia of N. crassa were treated with 0.67 mM aflatoxin B 1, hamster liver homogenate, and a NADPH generating system. A 9- to 15-fold increase int eh mutation frequency over the spontaneous mutation frequency was found when 0.67 mM of aflatoxin G 1 instead of aflatoxin B 1 was used in the test system. Treatment of conidia with 0.44 mM aflatoxin B 1 mice liver homogenate and a NADPH generating system caused a small, but significant increase in the ad-3 mutation frequencies. No significant increase in the mutation frequency was found when a single sample of human liver homogenate was used in the test system. These studies show that metabolic activation is necessary for the expression of the mutagenic activity of aflatoxins B 1 and G 1 in N. crassa.

Mutation to resistance for virus N-1 in the blue-green alga Nostoc muscorum.

The spontaneous and nitrosoguanidine induced N-1 virus resistant mutants of the blue-green alga Nostoc muscorum were isolated with the average mutation frequency of 3.55 X 10(-5) and 6.08 X 10(-4) respectively. Of the surviving colonies 8 per cent became virus sensitive,whereas out of the remaining 92 per cent 9.3 per cent showed lysogenic behaviour and 13.3 per cent showed poor or no nitrogen fixing ability. Mutation to virus-resistance was independent of the presence of the virus.Spontaneous host range mutants of virus were not found in repeated experiments.

X-ray-induced specific-locus mutation rates in young male mice

The specific-locus mutation frequency resulting from 300 R of acute X-irradiation has been determined for the germ cells present in male mice at 2, 4, 6, 8, 10, 14, 21, 28, and 35 days of age. The sample size was large enough for each of these nine age groups to ensure that a high mutation rate would be noticed. The testis of the mouse undergoes many developmental changes between birth, when most or all germ cells are gonocytes, and 35 days, when the cell population has come to resemble that of the adult. It was important to know if the germ cells present in these developmental stages of immature male mice yield the same mutation frequency as that found earlier for spermatogonia in the adult by W. L. Russell. None of the nine age groups has a mutation rate statistically significantly higher than that of the adult. Taken together, the nine groups of males have an average mutation frequency quite to that of the adult. This does not rule out the possibility that individual age groups may have a mutation frequency somewhat different from that of the adult. The distribution of mutations among the loci seems to be similar to that found for mutations induced in spermatogonia of the adult. Clusters of specific-locus mutations were found only on day 21. This paper and that presented earlier on the newborn report the first specific-locus mutation-rate studies on male mice irradiated between birth and adulthood. If the results can be carried over to man, it can be concluded that irradiation of the immature testis, from birth to puberty, will not present any greatly increased genetic hazard over that from irradiation of the adult testis. In fact, as the data stand in the mouse, they indicate a mutation rate similar to the adult for all but the earlier stages tested and, for these stages, a probably lower rate, representing a transition from the significantly lower rate reported earlier for newborns.

Effects of radiation pasteurization on Samonella. 3. Radiation lethality and the frequency of mutation to antibiotic resistance.

The radiation resistance (D, in megarads) of six strains of Salmonella irradiated at 4C in brain heart infusion suspension ranged from 0.042 for S. enteritidis to 0.084 for S. thompson. The resistance values were 0.048 for S. typhimurium, strain SR-11, 0.060 for S. typhimurium, strain RIA, and S. newport, and 0.078 for S. heidelberg. The mutation frequency to tetracycline resistance of S. typhimurium, strain SR-11, increased between 0 and 0.05 Mrad and declined thereafter with increasing radiation dose. After 0.5-Mrad exposure, the mutation frequency was essentially the same as in control populations. The mutation frequency to streptomycin resistance of S. typhimurium, strain SR-11, decreased at doses greater than 0.05 Mrad and increased only slightly for the more radiation-resistant serotypes, S. typhimurium, strain RIA, S. thompson, and S. heidelberg. The average mutation frequency of the four Salmonella cultures tested was essentially unchanged (less than 1 log difference) between 0.05 and 0.5 Mrad, while the difference in reduction in viable numbers was 5 logs or greater. The evidence presented indicates that with the proper choice of processing parameters, the application of radiation pasteurization could drastically reduce the possibility of transmission of Salmonella by poultry and thereby decrease the public health hazards associated with this microorganism.

Morphological and Cytological Response of Pear Millet, Pennisetum typhoides to Thermal Neutron and Ethyl Methane Sulfonate Seed Treatments1

Three dosages each of thermal neutrons (TN) and ethyl methane sulfonate (EMS) were applied to 10 pearlmillet inbreds evaluated in replicated plots in the field. Both mutagens delayed seedling emergence and days to maturity, and reduced seedling height, plant height, leaves per culm, and selfed and sibbed seed set. EMS reduced percent emergence and plants per plot to reach maturity. This extra space allowed EMS‐treated survivors to produce more culms and yield more. Two‐thirds of the 56 sectorial mutants came from TN‐treated seed. Significant inbred ✕ treatment interactions were obtained for 5 of 13 characters studied. Frequencies of chlorophyll‐deficient seedlings from selfed seed of all plants indicated that the 90‐minute TN treatment and 0.4% EMS soak for 4 hours increased the average mutation frequency most (some 5 fold). Mutation frequency in inbreds varied greatly. The most effective EMS treatment induced over 8 times as many mutants in 1 inbred as in another. The 90‐minute TN treatment produced 4 times as many plants (28%) with chromosome interchanges as the heaviest EMS treatment.

NATURAL AND RADIATION CARCINOGENESIS IN MAN. I. THEORY OF INITIATION PHASE.

A review is given of the evidence and arguments supporting the hypothesis that carcinogenesis is a multi-stage process. In addition to the broad distinction between ‘initiation’and‘promotion’ phases, it is highly probable that initiation itself is multi-stage in character. Previous models of the initiation phase are briefly described; they do not resolve the question as to how many stages are involved. A factor common to all these models, and to the new one described here, is that, other things being equal, cancer incidence should be related to the number of cells at risk. This prediction receives some support from human epidemiological statistics, and from murine experimental evidence. The new model is based on the traditional assumption that each stage of the initiation process is marked by some form of somatic mutation most probably of nuclear genes. It is constructed to permit the analysis of events throughout the life span and not merely during adult life. Equations are derived in which the average somatic mutation frequency appears as a function of the number of somatic mutations needed to initiate any particular stage, at any given age from fertilization. Familial and other evidence shows that certain individuals, comprising a subpopulation, may have a predisposition (usually genetically-based) to a particular malignancy or group of malignancies. The effect of this population heterogeneity on mortality statistics is analysed mathematically.

Influence of seed moisture content on gamma-ray induced mutation rates at a specific locus in oats

Seeds of the Victorgrain variety of oats were equilibrated to five moisture levels, irradiated with Co 60 γ-rays, and the second generation progeny screened for induced mutations at the locus ( Vb) controlling the reaction to toxin of the fungus, Helminthosporium victoriae. A series of radiation doses was given at each of the following moisture levels: 2·5 per cent, 3·5 per cent, 6·8 per cent, 10 per cent and 90 per cent water, by weight. The average mutation frequencies, on a panicle basis, observed at these moisture levels were 0·5, 171·7, 14·8, 4·9 and 7·7− (× 10 −8), respectively. These rates thus reached a peak at the 3·5 per cent moisture level and decreased as the moisture level was changed in either direction. This change in mutation frequency in relation to change in moisture levels is in the same direction as changes in radical signal and seedling heights related to changes in barley seed moisture contents as reported by Conger. Such great range in mutation frequency as brought about by changes in seed moisture content can be attributed to the indirect effects of the γ-rays.