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J Favor

Publications and source records attributed to J Favor.

At least 19 recordsLinked to original sources
PubMed2001

A 76-bp deletion in the Mip gene causes autosomal dominant cataract in Hfi mice.

Hfi is a dominant cataract mutation where heterozygotes show hydropic lens fibers and homozygotes show total lens opacity. The Hfi locus was mapped to the distal part of mouse chromosome 10 close to the major intrinsic protein (Mip), which is expressed only in cell membranes of lens fibers. Molecular analysis of Mip revealed a 76-bp deletion that resulted in exon 2 skipping in Mip mRNA. In Hfi/Hfi this deletion resulted in a complete absence of the wildtype Mip. In contrast, Hfi/+ animals had the same amount of wildtype Mip as +/+. Results from pulse-chase expression studies excluded hetero-oligomerization of wildtype and mutant Mip as a possible mechanism for cataract formation in the Hfi/+. We propose that the cataract phenotype in the Hfi heterozygote mutant is due to a detrimental gain of function by the mutant Mip resulting in either cytotoxicity or disruption in processing of other proteins important for the lens. Cataract formation in the Hfi/Hfi mouse is probably a combined result of both the complete loss of wildtype Mip and a gain of function of the mutant Mip.

Animals↗
PubMed2001

Molecular characterization of Pax6(2Neu) through Pax6(10Neu): an extension of the Pax6 allelic series and the identification of two possible hypomorph alleles in the mouse Mus musculus.

Phenotype-based mutagenesis experiments will increase the mouse mutant resource, generating mutations at previously unmarked loci as well as extending the allelic series at known loci. Mapping, molecular characterization, and phenotypic analysis of nine independent Pax6 mutations of the mouse recovered in mutagenesis experiments is presented. Seven mutations result in premature termination of translation and all express phenotypes characteristic of null alleles, suggesting that Pax6 function requires all domains to be intact. Of major interest is the identification of two possible hypomorph mutations: Heterozygotes express less severe phenotypes and homozygotes develop rudimentary eyes and nasal processes and survive up to 36 hr after birth. Pax6(4Neu) results in an amino acid substitution within the third helix of the homeodomain. Three-dimensional modeling indicates that the amino acid substitution interrupts the homeodomain recognition alpha-helix, which is critical for DNA binding. Whereas cooperative dimer binding of the mutant homeodomain to a paired-class DNA target sequence was eliminated, weak monomer binding was observed. Thus, a residual function of the mutated homeodomain may explain the hypomorphic nature of the Pax6(4Neu) allele. Pax6(7Neu) is a base pair substitution in the Kozak sequence and results in a reduced level of Pax6 translation product. The Pax6(4Neu) and Pax6(7Neu) alleles may be very useful for gene-dosage studies.

Alleles↗
PubMed2000

A temperature-sensitive mutation of Crygs in the murine Opj cataract.

In Opj, an inherited cataract in mice, opacity is associated with a mutation in Crygs, the gene for gammaS-crystallin, the first mutation to be associated with this gene. A single base change causes replacement of Phe-9, a key hydrophobic residue in the core of the N-terminal domain, by serine. Despite this highly non-conservative change, mutant protein folds normally at low temperature. However, it exhibits a marked, concentration-dependent decrease in solubility, associated with loss of secondary structure, at close to physiological temperatures. This is reminiscent of processes thought to occur in human senile cataracts in which normal proteins become altered and aggregate. The Opj cataract is progressive and more severe in Opj/Opj than in Opj/+. Lens histology shows that whereas fiber cell morphology in Opj/+ mice is essentially normal, in Opj/Opj, cortical fiber cell morphology and the loss of maturing fiber cell nuclei are both severely disrupted from early stages. This may indicate a loss of function of gammaS-crystallin which would be consistent with ideas that members of the betagamma-crystallin superfamily may have roles associated with maintenance of cytoarchitecture.

Amino Acid Sequence↗
PubMed2000

Further genetic analysis of two autosomal dominant mouse eye defects, Ccw and Pax6(coop).

PURPOSE: The work forms part of a major project to study the genetics of mouse cataract mutants found during the course of mutagenesis experiments. The long-term aim is to find the underlying gene mutation in each cataract mutant. Here we report further studies of the mutant cataract and curly whiskers (Ccw), previously mapped to Chromosome 4, and also investigations of the corneal opacity (Coop) mutant, which is shown to involve a mutation in the Pax6 gene. METHODS: For Ccw, the methods included mapping relative to microsatellite markers and histological studies. For the Coop mutant, breeding methods were used to show that Coop was allelic with Pax6. The Pax6 coding region in the mutant was then sequenced. RESULTS: The Ccw locus was mapped to approximately position 45cM on the consensus map of Chr 4. Histologically, progressive degeneration of the lens was seen. In the Coop mutant, a base-pair change C->T was found at position 1033 in the Pax6 gene, which created a stop codon leading to premature termination of translation, and to a truncated Pax6 protein. CONCLUSIONS: The phenotype in Ccw/+ heterozygotes involves a new type of lens degeneration in the mouse. On the basis of the phenotype and the locus position, no candidate gene has yet been identified. The Pax6coop mutant differs in phenotype from known null alleles of Pax6, implying that it is a hypomorph.

Animals↗
PubMed2000

Primary renal hypoplasia in humans and mice with PAX2 mutations: evidence of increased apoptosis in fetal kidneys of Pax2(1Neu) +/- mutant mice.

PAX2 mutations cause renal-coloboma syndrome (RCS), a rare multi-system developmental abnormality involving optic nerve colobomas and renal abnormalities. End-stage renal failure is common in RCS, but the mechanism by which PAX2 mutations lead to renal failure is unknown. PAX2 is a member of a family of developmental genes containing a highly conserved 'paired box' DNA-binding domain, and encodes a transcription factor expressed primarily during fetal development in the central nervous system, eye, ear and urogenital tract. Presently, the role of PAX2 during kidney development is poorly understood. To gain insight into the cause of renal abnormalities in patients with PAX2 mutations, kidney anomalies were analyzed in patients with RCS, including a large Brazilian kindred in whom a new PAX2 mutation was identified. In a total of 29 patients, renal hypoplasia was the most common congenital renal abnormality. To determine the direct effects of PAX2 mutations on kidney development fetal kidneys of mice carrying a Pax2 (1Neu)mutation were examined. At E15, heterozygous mutant kidneys were approximately 60% of the size of wild-type littermates, and the number of nephrons was strikingly reduced. Heterozygous 1Neu mice showed increased apoptotic cell death during fetal kidney development, but the increased apoptosis was not associated with random stochastic inactivation of Pax2 expression in mutant kidneys; Pax2 was shown to be biallelically expressed during kidney development. These findings support the notion that heterozygous mutations of PAX2 are associated with increased apoptosis and reduced branching of the ureteric bud, due to reduced PAX2 dosage during a critical window in kidney development.

Adolescent↗
PubMed2000

Effects of ENU dosage on mouse strains.

The germline supermutagen, N-ethyl-N-nitrosourea (ENU), has a variety of effects on mice. ENU is a toxin and carcinogen as well as a mutagen, and strains differ in their susceptibility to its effects. Therefore, it is necessary to determine an appropriate mutagenic, non-toxic dose of ENU for strains that are to be used in experiments. In order to provide some guidance, we have compiled data from a number of laboratories that have exposed male mice from inbred and non-inbred strains or their F(1) hybrids to ENU. The results show that most F(1) hybrid animals tolerate ENU well, but that inbred strains of mice vary in their longevity and in their ability to recover fertility after treatment with ENU.

Animals↗
PubMed2000

Saturation mutagenesis for dominant eye morphological defects in the mouse Mus musculus.

We have summarized our extensive series of mutagenesis experiments to isolate dominant mutations in the mouse that express eye morphological defects. Thirty-two experimental groups in which parental mice were exposed to chemical mutagens or irradiation and a historical control group of the laboratory are presented. The largest series of experiments included parental exposure to ethylnitrosourea or irradiation. A total of 203 dominant mutants were confirmed among 456,890 offspring screened, which represents one of the largest collections of mutations in the mouse affecting one organ following a systematic screen of offspring of mutagenized animals. The largest group of mutations (92) was recovered in offspring of parental mice exposed to ethylnitrosourea. The second largest group of mutations (62) was recovered in irradiation experiments. Fifty-six mutations recovered in ethylnitrosourea experiments have been mapped to 22 loci. The affected genes have been identified for a number of the recovered mutations including Cryga, Crygb, Cgyge, Pax6, Pax2, Mitf, Lim2, and Cx50. On the basis of our experiences, a number of considerations when undertaking such screens are discussed, including a) choice of mutagen, b) experimental design, and c) the criteria for such experiments to ensure that mutations at novel loci will be recovered.

Animals↗
PubMed2000

PAX2 suppresses apoptosis in renal collecting duct cells.

PAX2 is a transcription factor belonging to the evolutionarily conserved paired box family and is required during development of the central nervous system and genitourinary axis. Mutations in the PAX2 gene cause a rare autosomal dominant renal-coloboma syndrome, characterized by optic nerve colobomas and renal hypoplasia. Recent analysis of a spontaneous PAX2 mutant mouse model (1Neu) revealed that the major cause of renal hypoplasia is reduced branching of the ureteric bud (UB) and fewer nephrons. We have observed that this abnormality is associated with a striking increase in the number of UB cells undergoing programmed cell death during nephrogenesis. To ascertain whether apoptosis is directly linked to the level of PAX2 expression, we have studied the role of PAX2 in cultured renal cells. We show that mIMCD-3 cells, a murine collecting duct cell line with high endogenous PAX2 expression, undergo apoptosis when transfected with anti-sense PAX2. In contrast, HEK293 cells expressing exogenous PAX2 are protected against apoptotic death induced by caspase-2. PAX2 has no effect on proliferation of embryonic kidney or in cultured kidney cells. Our observations imply a direct role for PAX2 in survival of ureteric bud cells.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Genomic, transcriptional and mutational analysis of the mouse microphthalmia locus.

Mouse microphthalmia transcription factor (Mitf) mutations affect the development of four cell types: melanocytes, mast cells, osteoclasts, and pigmented epithelial cells of the eye. The mutations are phenotypically diverse and can be arranged in an allelic series. In humans, MITF mutations cause Waardenburg syndrome type 2A (WS2A) and Tietz syndrome, autosomal dominant disorders resulting in deafness and hypopigmentation. Mitf mice thus represent an important model system for the study of human disease. Here we report the complete exon/intron structure of the mouse Mitf gene and show it to be similar to the human gene. We also found that the mouse gene is transcriptionally complex and is capable of generating at least 13 different Mitf isoforms. Some of these isoforms are missing important functional domains of the protein, suggesting that they might play an inhibitory role in Mitf function and signal transduction. In addition, we determined the molecular basis for six microphthalmia mutations. Two of the mutations are reported for the first time here (Mitf(mi-enu198) and Mitf(mi-x39)), while the others (Mitf(mi-ws), Mitf(mi-bws), Mitf(mi-ew), and Mitf(mi-di)) have been described but the molecular basis for the mutation not determined. When analyzed in terms of the genomic and transcriptional data presented here, it is apparent that these mutations result from RNA processing or transcriptional defects. Interestingly, three of the mutations (Mitf(mi-x39), Mitf(mi-bws), and Mitf(mi-ws)) produce proteins that are missing important functional domains of the protein identified in in vitro studies, further confirming a biological role for these domains in the whole animal.

Alleles↗
PubMed2000

A comparison of enzyme activity mutation frequencies in germ cells of mice (Mus musculus) and golden hamsters (Mesocricetus auratus) after exposure to 2 + 2 Gy gamma-irradiation.

The radiation-induced germ cell mutation rate has been investigated in two species of mammals. Mice and golden hamsters of both sexes were exposed to 2 + 2 Gy gamma-irradiation with a 24 h fractionation interval and mated to untreated partners. In mice, specific locus mutations were examined as positive controls and the obtained mutation rates (per locus and gamete x10(-5)) were 51.4, 10.1, 13.6 and 17.4 for irradiated post-spermatogonia, spermatogonia and 1-7 and >7 days post-treatment oocytes, respectively. Offspring of mice and golden hamsters were screened for activity alterations of 10 erythrocyte enzymes coded by at least 14 loci. The observed mutation rates per locus per gamete x10(-5) for treated post-spermatogonial stages, spermatogonia and oocytes 1-7 and >7 days post-treatment were 6.5, 1.5, 8.8 and 7.0, respectively, for mice and 16.7, 0, 7.6 and 0, respectively, for golden hamsters. There is a significant difference for mutation rates in mouse oocytes 1-7 days post-treatment compared with the control. No differences in the frequencies of mutations in the various germ cell stages could be observed between mice and golden hamsters. A critical assumption for the extrapolation of experimental mutagenesis studies to humans is that no species effects exist in sensitivity to mutation induction by irradiation. Our results do not contradict this assumption.

Animals↗
PubMed1999

Mechanisms of mutation induction in germ cells of the mouse as assessed by the specific locus test.

Mouse germ cell specific locus mutagenesis data and a molecular characterization of mutant alleles have been reviewed to arrive at an understanding of the mechanism of mutation induction in mammals. (a) The spermatogenic stage specificity for the sensitivity to mutation induction by 20 chemical mutagens is considered. (b) The effects of a saturable repair process and its recovery over time are examined for the mutagenic efficiency of ethylnitrosourea. (c) The mutagenic events following methylnitrosourea and chlorambucil are shown to be mainly deletions. In contrast the mutations recovered after ethylnitrosourea treatment are almost exclusively base pair substitutions. (d) It is emphasized that to date very few specific locus experiments have been designed to test for mutagenic events outside the interval stem cell spermatogonia-mature spermatozoa. A specific locus mutation has recently been shown to be due to loss of heterozygosity via mitotic recombination in an early zygote stage and suggests a broader range of possible mechanisms of mutation when these stages are considered. (e) With the cloning of all 7 marker loci mutation analysis at the molecular level will allow a more direct assessment of the mutation process in future studies.

Alleles↗
PubMed1998

The mutagenic activity of ethylnitrosourea at low doses in spermatogonia of the mouse as assessed by the specific-locus test.

Ethylnitrosourea is the most efficient chemical mutagen in spermatogonial stem cells of the mouse and its mutagenic activity has been intensively studied. The pertinent specific-locus mutation test results for a discussion of low dose-effect studies have been summarized and indicate: (1) A threshold dose response best characterizes the relationship between dose and mutation rate. (2) The reduced effectiveness of ethylnitrosourea in the low dose range is likely due to a saturable repair process. (3) The recovery of the saturable repair process as assessed in fractionated dose experiments is long (ca. 168 h). The dynamics of stem cell spermatogonia suggests a long time interval before the cell population passes through at least one cell division and this may be relevant to an interpretation of the fractionation effects. (4) There is a slight but important discrepancy between the predicted and observed mutagenic activity of ethylnitrosourea in the low dose range. This is interpreted to be due to the differences between a mathematical abstraction and the biological realities of the system being studied.

Animals↗
PubMed1998

Identification of mouse crystallins in 2D protein patterns by sequencing and mass spectrometry. Application to cataract mutants.

The eye lens proteins of the mouse were separated into 1940 polypeptide spots by two-dimensional electrophoresis in large gels. All 16 crystallins ubiquitous in mammals were identified by protein sequencing and mass spectrometry except for (gamma)-F, which shows an almost identical sequence with (gamma)-E. Two crystallins, (beta)-A2 and (gamma)-S, were shown for the first time to occur in the mouse lens. An investigation of the murine cataract mutant Cat2(nop)((gamma)-B gene) demonstrated that a monogenic mutation might affect a broad spectrum of proteins.

Amino Acid Sequence↗
PubMed1998

Three murine cataract mutants (Cat2) are defective in different gamma-crystallin genes.

A number of murine cataract mutations have been localized to chromosome 1 close to the gamma-crystallin gene cluster (Cryg) (Everett et al., 1994, Genomics 20: 429-434; Löster et al., 1994, Genomics 23: 240-242). Based on the size of the mapping or allelism tests they have not been shown to be genetically distinct and have been assigned to locus symbol Cat2. Here we assign three mutations to the respective gamma-crystallin gene. Using a systematic candidate gene approach to analyze the entire Cryg cluster, an A-->G transition was found in exon 2 of Cryga for the ENU-436 mutation and is designated Cryga1Neu. The mutant allele Crygbnop (formerly Cat2(nop)) is caused by a replacement of 11 bp by 4 bp in the third exon of Crygb, while a C-->G transversion in exon 3 of Cryge has been found for the Cryget (formerly Cat2(t)) mutation. For the mutation Cryga1Neu, an Asp-->Gly exchange is deduced, whereas the mutations Crygbnop and Cryget lead to the formation of in-frame stop codons and give rise to truncated proteins of 144 and 143 amino acids, respectively. The effects of the mutations upon gamma-crystallin structure are likely to be quite different. The Cryga1Neu mutation is expected to affect the link between Greek-key motifs 2 and 3, whereas both Crygbnop and Cryget mutations are supposed to truncate the fourth Greek-key motif. All three mutations are predicted to alter protein folding of the gamma-crystallins and result in lens cataract, but the phenotype for each is quite distinctive.

Amino Acid Sequence↗
PubMed1998

Recommendations for statistical designs of in vivo mutagenicity tests with regard to subsequent statistical analysis.

A workshop was held on September 13 and 14, 1993, at the GSF, Neuherberg, Germany, to start a discussion of experimental design and statistical analysis issues for three in vivo mutagenicity test systems, the micronucleus test in mouse bone marrow/peripheral blood, the chromosomal aberration tests in mouse bone marrow/differentiating spermatogonia, and the mouse dominant lethal test. The discussion has now come to conclusions which we would like to make generally known. Rather than dwell upon specific statistical tests which could be used for data analysis, serious consideration was given to test design. However, the test design, its power of detecting a given increase of adverse effects and the test statistics are interrelated. Detailed analyses of historical negative control data led to important recommendations for each test system. Concerning the statistical sensitivity parameters, a type I error of 0.05 (one tailed), a type II error of 0.20 and a dose related increase of twice the background (negative control) frequencies were generally adopted. It was recommended that sufficient observations (cells, implants) be planned for each analysis unit (animal) so that at least one adverse outcome (micronucleus, aberrant cell, dead implant) would likely be observed. The treated animal was the smallest unit of analysis allowed. On the basis of these general consideration the sample size was determined for each of the three assays. A minimum of 2000 immature erythrocytes/animal should be scored for micronuclei from each of at least 4 animals in each comparison group in the micronucleus assays. A minimum of 200 cells should be scored for chromosomal aberrations from each of at least 5 animals in each comparison group in the aberration assays. In the dominant lethal test, a minimum of 400 implants (40-50 pregnant females) are required per dose group for each mating period. The analysis unit for the dominant lethal test would be the treated male unless the background frequency of dead implants (DI) is so low that multiple males would need to be integrated to meet the minimum observation of one adverse outcome (DI) per analysis unit. A three-step strategy of data analysis was proposed for the cytogenetic assays. Use of negative historical controls was allowed in certain circumstances for interpretation of results from micronucleus tests and chromosomal aberration tests.

Animals↗
PubMed1998

Aphakia (ak), a mouse mutation affecting early eye development: fine mapping, consideration of candidate genes and altered Pax6 and Six3 gene expression pattern.

The homozygous mouse mutant aphakia (ak) has been characterized by bilaterally aphakic eyes without a pupil [Varnum DS, Stevens, LC (1968): J Hered 59:147-150]. The mutation was mapped to chromosome 19 [Varnum DS, Stevens, LC (1975): Mouse News Lett 53:35]. Our linkage studies yielded a precise localization of the ak gene 0.6 +/- 0.3 cM proximal to the microsatellite marker D19Mit10 and 0.7 +/- 0.4 cM distal to D19Mit4 and D19Mit91. No recombination was found with the marker D19Mit9 among 418 backcross offspring tested. The developmental control gene Pax2 mapped 11.0 +/- 3.5 cM proximal to ak and is excluded as a candidate gene. Sequence analysis of Fgf8 and Chuk1, which are localized close to the marker D19Mit10, detected no mutations in the ak/ak mutants. Histological analysis of homozygous mutants suggested the arrest of lens development at the lens stalk stage, a transient morphological structure during the formation of the lens vesicle. In the lens remnants, Pax6 and Six3 are expressed, whereas in the persisting lens stalk only Pax6 was detected. The expression pattern of Pax2 appeared normal; Cryaa expression could not be detected. As a consequence of the arrested lens development, other ocular tissues that require for their development information from the intact lens, such as iris, ciliary muscle, retina, and vitreous body, are absent or formed abnormally.

Animals↗
PubMed1998

Molecular, genetic and biochemical characterization of lactate dehydrogenase-A enzyme activity mutations in Mus musculus.

Four independent heterozygous lactate dehydrogenase (LDH) mutations with approximately 60% of wild-type enzyme activity in whole blood have been recovered. The mutant line Ldh1a2Neu proved to be homozygous lethal, whereas for the three lines Ldh1a7Neu, Ldh1a11Neu, and Ldh1a12Neu homozygous mutants with about 20% residual activity occurred in the progeny of heterozygous inter se matings. However, the number of homozygous mutants was less than expected, suggesting an increased lethality of these animals. Various physicochemical and kinetic properties of LDH are altered. Exons of the Ldh1 gene were PCR amplified and sequenced to determine the molecular lesion in the mutant alleles. Ldh1a2Neu carried an A/T-->G/C transition in codon 112 (in exon 3), resulting in an Asn-->Asp substitution; Asn112 is part of the helix alpha D, which is involved in the coenzyme-binding domain. Ldh1a7Neu contained an A/T-->C/G transversion within the codon for residue 194 in exon 4, causing an Asp-->Ala substitution, which may affect the arrangement of the substrate-binding site. Three base substituions were discovered for the mutation Ldh1a11Neu in exon 7: the transition C/G-->T/A, a silent mutation, and two transversions C/G-->A/T and C/G-->G/C, both missense mutations, which led to the amino acid replacements A1a319-->Glu and Thr321-->Ser, respectively, located in the alpha H helix structure of the COOH tail of LDHA. We suggest that the mutation in the result of a gene conversion event between Ldh1a wild-type gene and the pseudogene Ldhl-ps. The alteration Ile-->Thr of codon 241 in exon 6 caused by the base pair change T/A-->C/G was identified in the mutation Ldh1a12Neu; Ile241 is included in the helix alpha 2G, a structure that is indirectly involved in coenzyme binding. Each of the sequence alterations has a potential impact on the structure of the LDHA protein, which is consistent with the decreased LDH activity and biochemical and physiological alterations.

Amino Acid Sequence↗