The mouse Cat4 locus maps to Chromosome 8 and mutants express lens-corneal adhesion

Cat4 is the second largest allelism group in the collection of mouse dominant eye mutations recovered in Neuherberg and carriers express anterior polar cataract, central corneal opacity, and lens-corneal adhesions. We have mapped the Cat4 locus of the mouse to central Chromosome (Chr) 8 at position cM 31. Histological characterization of Cat4 ^a heterozygotes and homozygotes indicates failure of separation of the lens vesicle from the surface ectoderm. Human anterior segment ocular dysgenesis (ASOD) is autosomal dominant, carriers express an eye phenotype similar to that of Cat4 ^a carriers, and it has been mapped to a region of 4q homologous to mouse central Chr 8. Thus, on the basis of phenotype and map position, Cat4 may be a mouse model of human ASOD. The genes Junb, Jund1, Mel, and Zfp42 are discussed as possible candidates for Cat4. Received: 31 October 1996 / Accepted: 20 January 1997     

Further experience of the mouse dominant cataract mutation test from an experiment with ethylnitrosourea

6 mice with inherited cataracts and 1 new allele of microphthalmia were recovered from 923 progeny of untreated, outbred, PT stock females that had been mated to inbred C3H/HeH strain males, whose spermatogonia had been exposed to 250 mg/kg of ethylnitrosourea (ENU). The cataract phenotypes were quite variable in expression and 5/6 showed a similar range of phenotypes. 2 of the 6 mutant mice were daughters of the same ENU-treated C3H/HeH male and probably represent repeats of the same mutation. One mutation, designated lens opacity-4 (Lop-4), has been genetically mapped to the distal region of chromosome 2. The yield of 5 presumably independent cataract mutations from 923 F1 offspring is a little higher than that reported by others in similar but larger scale experiments. Approximately 3-5% of the F1 mice examined had cataracts, yet only 6/49 (12%) of these, in the experimental group, were inherited as simple Mendelian traits. We consider that this high frequency of false positives (88%), and the incomplete penetrance and variable expressivity of the cataract mutations that were found, pose serious problems that could undermine the objective nature of the dominant cataract mutation test. We suggest that further studies be made to evaluate whether the use of inbred strains would reduce the variability in the system and so make the test more objective. However, it seems likely that the high false positive rate will continue to be a serious drawback to this test system.     

A comparison of the dominant cataract and recessive specific-locus mutation rates induced by treatment of male mice with ethylnitrosourea

Mice were derived from parental males treated with 250 mg ethylnitrosourea per kg body weight. The mice were screened simultaneously for induced dominant cataract and recessive specific-locus mutations. In the spermatogonial treatment group, 16 dominant cataract, 1 dominant corneal opacity and 60 recessive specific-locus mutations were recovered and genetically confirmed in 9352 offspring observed. This lower yield of dominant cataract mutations, when compared with the yield of recessive specific-locus mutations, is similar to results observed by Kratochvilova in a series of experiments on dominant cataract mutations induced by radiation treatment. These results taken with reported results from other dominant mutation test systems, suggest a lower per-locus mutation rate to dominant than to recessive alleles. A corollary to the hypothesis that most dominantly expressed alleles code for an alteration in the function of the normal gene product is that a limited subset of mutations could normally lead to a dominantly expressed mutation. This may explain the lower per-locus mutation rate to dominant than to recessive alleles.

Genetic confirmation tests of recovered presumed dominant cataract mutations indicate that a certain category of phenotypic variants (bilateral, severe or unique lens opacity) is likely to be a true mutation but only represents 7 of the 19 mutations recovered. A second category of phenotypic variants (unilateral, neither severe nor unique lens opacity) has an extremely low probability of being a true mutation. Only 1 confirmed mutation in 181 phenotypic variants was obtained. The remaining category of phenotypic variants (either unilateral severe or unique, or bilateral neither severe nor unique lens opacity) represented the majority, 11, of the confirmed mutations obtained. However, 266 presumed mutations in this category were recovered. If a sub-class of phenotypic variants within this category could be identified that could be ignored owing to a very low probability of being a true mutation, the efficiency of recovery of confirmed dominant cataract mutations would be greatly increased with no sacrifice in the accuracy of the observed mutation rate.

Finally, the 17 confirmed dominant cataract mutations obtained included a class of 7 that produced significantly fewer than the Mendelian expectation of offspring exhibiting the mutant phenotype. This class probably represents both mutations with penetrance effects and mutations with viability effects.

The present experiments represent the first systematic comparison of induced genetically confirmed dominant and recessive mutations for a chemical mutagen in mice. Such results contribute to our limited understanding of the mutation process to dominant alleles.     

HSF4 regulates DLAD expression and promotes lens de-nucleation

HSF4 mutations lead to both congenital and age-related cataract. The purpose of this study was to explore the mechanism of cataract formation caused by HSF4 mutations. The degradation of nuclear DNA is essential for the lens fiber differentiation. DNase 2β (DLAD) is highly expressed in lens cells, and mice with deficiencies in the DLAD gene develop nuclear cataracts. In this study, we found that HSF4 promoted the expression and DNase activity of DLAD by directly binding to the DLAD promoter. In contrast, HSF4 cataract causative mutations failed to bind to the DLAD promoter, abrogating the expression and DNase activity of DLAD. These results were confirmed by HSF4 knockdown in zebrafish, which led to incomplete de-nucleation of the lens and decreased expression and activity of DLAD. Together, our results suggest that HSF4 exerts its function on lens differentiation via positive regulation of DLAD expression and activity, thus facilitating de-nucleation of lens fiber cells. Our demonstration that HSF4 cataract causative mutations abrogate the induction of DLAD expression reveals a novel molecular mechanism regarding how HSF4 mutations cause cataractogenesis.     

Characterization of dominant hereditary cataract in DDD/1 mice

We found a female cataractous DDD/1-nu/+ mouse and established a hairy mutant strain (DDD/1-Cti/Cti) with 100% incidence of cataract from it by repeating sibmating. Genetic studies demonstrated that a single autosomal semidominant gene controls cataractogenesis. This gene was named Cti. In homozygotes, DDD/1-Cti/Cti, the lenses began to opacify at 14 days of fetal life and were recognized clinically as cataract at 13-14 days of age when the eyes first open. The opacification became more and more intense with age and looked like mature cataract at 28-42 days of age. However, clarification of the opacified lenses commenced at the periphery after 56 days of age and expanded to the inside with time, and only an opaque spot was left at the center at 140 days of age. In heterozygotes, DDD/1-Cti/+, the lenses were recognizable as cataract after 28 days and became like mature cataract around 35 days of age. The opacity began to be lightened at 42 days and the lenses appeared normal at 56 days of age. Both lenses and eyeballs developed in similar courses in DDD/1(-)+/+, -Cti/+ and -Cti/Cti, although slightly retarded in the last. Microphthalmia was not accompanied even in DDD/1-Cti/Cti. The lens water content remained higher during the time when intense lens opacity continued in DDD/1-Cti/Cti and -Cti/+. Background genes appeared to affect the expression of Cti. DDD/1-Cti(-)+ mice may provide a model for researches into clarification of opaque lenses. A discussion concerning the possible allelism of Cti and Cts with Lop was made based on their phenotypic characteristics.     

Dominant cataract and recessive specific-locus mutations detected in offspring of procarbazine-treated male mice

The induction of dominant cataract mutations by procarbazine was studied concomitantly with the induction of specific-locus mutations in treated male mice. The most effective dose in the specific-locus test, 600 mg/kg of procarbazine, and a fractionated dose of 5 X 200 mg/kg were used. The frequencies of dominant cataract mutations were higher, but not significantly different from the historical control. The ratio between the number of recovered specific-locus and dominant cataract mutations was in accordance with that found in our experiments with gamma-rays (Ehling et al., 1982; Kratochvilova, 1981) or in experiments with ethylnitrosourea (Favor, 1986). A total of 3 dominant cataract mutations were recovered in the offspring of procarbazine-treated spermatogonial stem cells. Two mutations had complete penetrance while the third exhibited a reduced penetrance of approximately 70%. The viability and fertility of the heterozygotes of all 3 mutations were not affected. Only 1 mutation was shown to be viable as a homozygote.     

Population Genetics of an Expanding Family of Mobile Genetic Elements

A model of an expanding family of dispersed repetitive DNA was studied. Based on the previous result of the model of duplicative transposition, an approximate solution to give allelism and identify coefficients as functions of time was obtained, and theoretical predictions were verified by Monte Carlo experiments. The results show that, even if the copy number per genome increases very rapidly, allelism and identity coefficients may take a long time to reach equilibrium. The changes of allelism and allelic identity are similar to that of homozygosity at an ordinary single locus, whereas that of nonallelic identity can be much slower, particularly when the copy number per genome is large. Thus, many existing families of highly repetitive sequences may represent nonequilibrium states for nonallelic identity. The present model may be extended to include other evolutionary forces such as gene conversion or the recurrent insertion from normal gene copies.     

Copper metabolism in mottled mouse (Mus musculus) mutants. Studies of blotchy (Moblo) mice and a comparison with brindled (Mobr) mice.

1. Copper concentrations were low in many organs of Moblo/Y mice, but very high in the gut. Absorption of 64Cu was seen to be very low when related to the absorption of cyano[57Co]cobalamin. The results in Moblo/+ mice were intermediate. 2. Copper therapy temporarily ameliorated many effects of the mutation in Moblo/Y mice, but did not improve the rate of weight gain as has been achieved previously in Mobr/Y mice. Lower capacity for a 'depot dose' effect at the site of injection may explain the difference. 3. The distribution of 64Cu after administration into the bloodstream of Moblo/Y mice altered from an initially normal state to one that resembled the abnormal distribution of pre-existing copper by 48 h. This indicated that the later mechanisms of copper distribution were at fault. Moblo/+ mice were equally affected. 4. The alteration of copper homoeostasis in blotchy mice was similar to that observed in brindled mice previously and in the present studies, although generally less severe. This is consistent with allelism of the two mutations.     

The frequency of dominant cataract and recessive specific-locus mutations and mutation mosaics in F1 mice derived from post-spermatogonial treatment with ethylnitrosourea

The frequency of dominant cataract and recessive specific-locus mutations and mutation mosaics was determined in F1 mice derived from post-spermatogonial germ-cell stage treatment with 2 X 80, 160 or 250 mg/kg ethylnitrosourea. A total of 5 dominant cataract mutations, 3 dominant cataract mutation mosaics, 1 specific-locus mutation and 9 specific-locus mutation mosaics were recovered in 15,542 screened F1 offspring. Results indicate that ethylnitrosourea treatment increases the mutation rate of dominant cataract and recessive specific-locus alleles in post-spermatogonial germ-cell stages of the mouse and that the mutations occur mainly as mosaics. Genetic confirmation of newly induced mutations occurring as mosaics is more problematical for induced recessive alleles than for induced dominant alleles and should be considered when evaluating such mutagenicity results.     

Induction of mutations in Streptomyces antibioticus

Mutagenic action of UV-light, nitrosoguanidine and nitrosomethylbiuret was studied in Streptomyces antibioticus VNIIA 1607. Nitrosomethylbiuret appeared to be most effective inducer of auxotrophic mutations (mutation frequency reached 15%). By means of hybridological analysis, it was shown that heterokaryons predominated in the progeny of mixed cultures of multiply marked strains. The test for functional allelism using heterokaryons permitted us to divide 93 independently obtained mutations into 28 complementation groups.     

The frequency of dominant cataract and recessive specific-locus mutations in mice derived from 80 or 160 mg ethylnitrosourea per kg body weight treated spermatogonia

A systematic comparison of the frequency of dominant cataract and recessive specific-locus mutations in mice has been extended to include results for 80 and 160 mg ethylnitrosourea per kg body weight spermatogonial treatment. The frequency of confirmed dominant cataract mutations in the historical control, 80 and 160 mg/kg ethylnitrosourea treatment groups was 1/22594, 8/5090 and 14/6435, respectively. The frequency of recessive specific-locus mutations in the same dose groups was, respectively, 19/227805, 20/13274 and 35/8658. These present results confirm previous results, which indicate that ethylnitrosourea is effective in inducing both recessive specific-locus and dominant cataract mutations although the per locus mutation rate to recessive alleles was observed to be approximately 6 times greater than the per locus mutation rate to dominant alleles. The exclusion of certain classes of lens opacity variant phenotypes, previously demonstrated not to be due to a dominant mutation, from the group of suspected dominant cataract mutations subjected to a genetic confirmation test has greatly improved the efficiency of the test. A total of 23 dominant cataract mutations were confirmed from a group of 67 phenotypic variants. Of the 23 confirmed dominant cataract mutations, 8 were shown to have reduced transmission to the following generation of offspring expressing the mutant phenotype. These results are also consistent with previous results for ethylnitrosourea or radiation treatment in which it was shown that approximately one-third of the recovered mutations have reduced penetrance. One group of dominant cataract mutations, with phenotypic effects on the polar, sub-capsular or corneal regions, is overly represented in the group of recovered mutations with a reduced transmission of offspring expressing the mutant phenotype. Two hypotheses are suggested for this observation, both dependent on the fact that the regions affected indicate that the mutations are expressed later in the development of the eye. Either all carrier individuals have not expressed the phenotype at the time of examination and classification, or later acting mutations are more subject to environmental interactions resulting in more variable expression. Finally, it is argued that a dominant cataract mutation test represents a most practicable protocol to screen for induced dominant mutations in germ cells of the mouse. The imposition of the criterion that suspected variants be subjected to a genetic confirmation test has at least two advantages beside the fact that results represent unambiguous mutational events.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic analyses of plumage color mutations on the Z chromosome of Japanese quail

Genetic analyses were performed with four sex-linked plumage color mutations (roux, brown, imperfect albino, and cinnamon) in Japanese quail (Coturnix japonica). Roux and brown quail have similar plumage color, but plumage of roux quail is paler. Pure, F1 and F2 matings were carried out with roux and brown stocks, and 357, 338, and 273 progeny with either roux or brown plumage color were obtained from each mating type, respectively. These allelism tests showed that mutations for roux and brown colors were alleles (*R and *B) from the same locus BR, and that BR*B was dominant over BR*R. Two alleles at the AL locus, AL*A (imperfect albino) and AL*C (cinnamon) were used to estimate the recombination frequency between the BR and AL loci on the Z chromosome. It was estimated to be 38.1+/-1.0% based on 4615 chicks from the test crosses.     

Allelic relationships of pea nodulation mutants

Thirteen stable nonnodulating mutant lines of pea (Pisum sativum L.) originating from cv. Finale were tested for allelism in pairwise crosses. The F(1) plants were evaluated for the symbiotic phenotype under controlled growth conditions against the nodule bacterium Rhizobium leguminosarum bv. viciae strain 248. All mutations were found to be recessive and the lines were classified into eight complementation groups comprising Risnod1-Risnod23, Risnod8, Risnod9-Risnod22, Risnod14, Risnod19-Risnod25, Risnod20, Risnod24-Risnod26, and Risnod32. Position of Risnod21 was not firmly established, leaving the possibility of allelism both with Risnod19-Risnod25 and Risnod20. The results were partially consistent with the previous reports on the allelism of these lines. Additional crosses confirmed the correspondence of Risnod14 with the locus sym7 and of Risnod19-Risnod25 with sym8. The high number of eight complementation groups formed by 13 mutants provides an indication of additional nodulation loci in pea to those already reported and confirms the complexity of the genetic control of the early stages of nodulation.     

HSF4 is involved in DNA damage repair through regulation of Rad51

Heat shock factor protein 4 (HSF4) is expressed exclusively in the ocular lens and plays a critical role in the lens formation and differentiation. Mutations in the HSF4 gene lead to congenital and senile cataract. However, the molecular mechanisms causing this disease have not been well characterized. DNA damage in lens is a crucial risk factor in senile cataract formation, and its timely repair is essential for maintaining the lens' transparency. Our study firstly found evidence that HSF4 contributes to the repair of DNA strand breaks. Yet, this does not occur with cataract causative mutations in HSF4. We verify that DNA damage repair is mediated by the binding of HSF4 to a heat shock element in the Rad51 promoter, a gene which assists in the homologous recombination (HR) repair of DNA strand breaks. HSF4 up-regulates Rad51 expression while mutations in HSF4 fail, and DNA does not get repaired. Camptothecin, which interrupts the regulation of Rad51 by HSF4, also affects DNA damage repair. Additionally, with HSF4 knockdown in the lens of Zebrafish, DNA damage was observed and the protein level of Rad51 was significantly lower. Our study presents the first evidence demonstrating that HSF4 plays a role in DNA damage repair and may contribute a better understanding of congenital cataract formation.     

Genetic Analysis of 63 Mutations Affecting Maize Kernel Development Isolated from Mutator Stocks

Sixty-three mutations affecting development of the maize kernel were isolated from active Robertson's Mutator (Mu) stocks. At least 14 previously undescribed maize gene loci were defined by mutations in this collection. Genetic mapping located 53 of these defective kernel (dek) mutations to particular chromosome arms, and more precise map determinations were made for 21 of the mutations. Genetic analyses identified 20 instances of allelism between one of the novel mutations and a previously described dek mutation, or between new dek mutations identified in this study; phenotypic variability was observed in three of the allelic series. Viability testing of homozygous mutant kernels identified numerous dek mutations with various pleiotropic effects on seedling and plant development. The mutations described here presumably arose by insertion of a Mu transposon within a dek gene; thus, many of the affected loci are expected to be accessible to molecular cloning via transposon-tagging.     

Ionizing radiation and genetic risks. II. Nature of radiation-induced mutations in experimental mammalian in vivo systems

This paper reviews data on the nature of spontaneous and radiation-induced mutations in the mouse. The data are from studies using a variety of endpoints scorable at the morphological or the biochemical level and include pre-selected as well as unselected loci at which mutations can lead to recessive or dominant phenotypes. The loci used in the morphological recessive specific-locus tests permit the recovery of a wide spectrum of induced changes. Important variables that affect the nature of radiation-induced mutations (assessed primarily using tests for viability of homozygotes) include: germ cell stage, type of irradiation and the locus. Most of the results pertain to irradiated stem cell spermatogonia. The data on morphological specific-locus mutations show that overall, more than two-thirds of the X- or gamma-ray-induced mutations are lethal when homozygous. This proportion may be lower for those that occur spontaneously, but the numbers of tested mutants are small. For spontaneous mutations, there is evidence for the occurrence of mosaics and for proviral insertions. Most or all tested induced enzyme activity variants, dominant visibles (recovered in specific-locus experiments) and dominant skeletal mutations are lethal when homozygous and this is true of 50% of dominant cataract mutations, but again, the numbers of tested mutants are small. Electrophoretic mobility variants, which are known to be due to base-pair changes, are seldom induced by irradiation. At the histocompatibility loci, no radiation-induced mutations have been recovered, presumably because deletions are incompatible with survival even in heterozygotes. All these findings are consistent with the view that in mouse germ cells, most radiation-induced mutations are DNA deletions. Some mutations (in the morphological specific-locus tests) which had previously been inferred to be deletions on the basis of genetic analyses have now been shown to be DNA deletions by molecular methods. However, the possibility cannot be excluded that at least a small proportion of induced mutations may be intragenic changes. The data on the rates of induction of recessive lethals and of dominant skeletal and dominant cataract mutations (and proportions of the latter two which are homozygous lethal) can be used to estimate the proportions of recessive lethals which are expressed as skeletal abnormalities or cataracts. These calculations show that about 10% of recessive lethals manifest themselves as skeletal and less than 0.2% as cataract mutations.(ABSTRACT TRUNCATED AT 400 WORDS)     

A CRYGC gene mutation associated with autosomal dominant pulverulent cataract

Purpose

To describe at molecular level a family with pulverulent congenital cataract associated with a CRYGC gene mutation.

Methods

One family with several affected members with pulverulent congenital cataract and 230 healthy controls were examined. Genomic DNA from leukocytes was isolated to analyze the CRYGA-D cluster, CX46, CX50 and MIP genes through high-resolution melting curve and DNA sequencing.

Results

DNA sequencing in the affected members revealed the c.143G>A mutation (p.R48H) in exon 2 of the CRYGC gene; 230 healthy controls and ten healthy relatives were also analyzed and none of them showed the c.143G>A mutation. No other polymorphisms or mutations were found to be present.

Conclusion

In the present study, we described a family with pulverulent congenital cataract that segregated the c.143G>A mutation (p.R48H) in the CRYGC gene. A few mutations have been described in the CRYGC gene in autosomal dominant cataract, none of them with pulverulent cataract making clear the clinical heterogeneity of congenital cataract. This mutation has been associated with the phenotype of congenital cataract but also is considered an SNP in the NCBI data base. Our data and previous report suggest that p.R48H could be a disease-causing mutation and not an SNP.     

Dominant cataract and recessive specific locus mutations in offspring of X-irradiated male mice

Male mice were X-irradiated with 3.0 + 3.0 Gy or 5.1 + 5.1 Gy (fractionation interval 24 h). The offspring were screened for dominant cataract and recessive specific locus mutations. In the 3.0 + 3.0-Gy spermatogonial treatment group, 3 dominant cataract mutations were confirmed in 15 551 offspring examined and 29 specific locus mutations were recovered in 18 139 offspring. In the post-spermatogonial treatment group, 1 dominant cataract mutation was obtained in 1120 offspring and 1 recessive specific locus mutation was recovered in 1127 offspring. The induced mutation rate per locus, per gamete, per Gy calculated for recessive specific locus mutations is 2.0 X 10(-5) in post-spermatogonial stages and 3.7 X 10(-5) in spermatogonia. For dominant cataract mutations, assuming 30 loci, the induced mutation rate is 5.0 X 10(-6) in the post-spermatogonial stages and 1.1 X 10(-6) in spermatogonia. In the 5.1 + 5.1-Gy spermatogonial treatment group, 3 dominant cataract mutations were obtained in 11 205 offspring, whereas in 13 201 offspring 27 recessive specific locus mutations were detected in the spermatogonial group. In the post-spermatogonial treatment group no dominant cataract mutation was observed in 425 offspring and 2 recessive specific locus mutations were detected in 445 offspring. The induced mutation rate per locus, gamete and Gy in spermatogonia for recessive specific locus mutations is 2.8 X 10(-5) and for dominant cataract mutations 0.9 X 10(-6). In post-spermatogonial stages, the mutation rate for recessive specific locus alleles is 6.2 X 10(-5). In the concurrent untreated control group, in 11 036 offspring no dominant cataract mutation and in 23 518 offspring no recessive specific locus mutation was observed. Litter size and the number of carriers at weaning have been determined in the confirmation crosses of the obtained dominant cataract mutants as indicators of viability and penetrance effects. Two mutants had a statistically significantly reduced litter size and one mutant had a statistically significantly reduced penetrance.     

Genetic and cytological analyses of three lethal ovule mutants in soybean (Glycine max; Leguminosae).

Soybean partially sterile mutants 2, 3, and 4 (PS-2, PS-3, and PS-4), recovered from a gene-tagging experiment, were studied to clarify their inheritance, linkage, allelism, and reproductive biology. The PS-2, PS-3, and PS-4 mutants were maintained as heterozygotes and upon self-pollination segregated l fertile: l partially-sterile. For inheritance and linkage tests, all three PS mutants were crossed to flower color mutant Harosoy-w4 and to chlorophyll-deficient (CD) mutants CD-1 and CD-5, also recovered from the tagging study. For allelism tests, reciprocal crosses were made among the three partially sterile mutants. Linkage results indicated that the gene for partial sterility in the PS-2, PS-3, and PS-4 mutants was not linked either to the w4 locus or to the genes for chlorophyll deficiency. Studies of pollen development, pollen viability, and pollen-tube germination indicated no difference between normal and partially sterile genotypes. Linkage and allelism tests indicated that the gene in the three partially sterile mutants was not transmitted through the female when they were used as a female parent. A study of megagametogenesis indicated that the ovules from partially sterile plants had normal embryo sac development. Ovule abortion was due to failure of fertilization.