Phenotypic and physical analysis of a chemically induced mutation disrupting anterior axial development in the mouse.
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Animals↗
Biomedical subjects
E M Rinchik
Publications and source records attributed to E M Rinchik.
Deficiency of the beta 3 subunit of the type A gamma-aminobutyric acid receptor causes cleft palate in mice.
In addition to its function in the nervous system, gamma-aminobutyric acid (GABA) has been implicated in mouse craniofacial development by the results of both teratological, and genetic studies. We previously reported that disruption of the cleft palate 1 (cp1) locus, closely linked to the pink-eyed dilution (p) locus on mouse chromosome 7, causes a 95% penetrant, recessive, neonatally-lethal cleft palate (CP) in mice homozygous for the p(4THO-II) deletion. We proposed that the beta 3 subunit gene (Gabrb3) of the GABAA receptor might be a candidate for cp1 (ref. 4); our earlier studies had localized cp1 to an interval beginning distal to the gene for the GABAA receptor alpha 5 subunit (Gabra5) and ending within the Gabrb3 coding region. To test the hypothesis that deletion of Gabrb3, and not another gene in the interval, causes CP, we performed an experiment to rescue the CP phenotype by introducing a Gabrb3 transgene into p(4THO-II) homozygotes. We now show that such transgenic mice are phenotypically normal, indicating that Gabrb3 is indeed the cp1 locus.
Animals↗
Molecular analysis of 36 mutations at the mouse pink-eyed dilution (p) locus.
Thirty-six radiation- or chemically induced homozygous-lethal mutations at the p locus in mouse chromosome 7 have been analyzed at 17 loci defined by molecular probes to determine the types of lesions, numbers of p-region markers deleted or rearranged, regions of overlap of deletion mutations, and genetic distances between loci. A linear deletion map of the [Myod1, Ldh3]-[Snrpn, Znf127] region has been constructed from the molecular analyses of the p-locus deletions. The utility of these deletions as tools for the isolation and characterization of the genes specifying the neurological, reproductive, and developmental phenotypes genetically mapped to this region will grow as more detailed molecular analyses continue.
Animals↗
Clustering of six human 11p15 gene homologs within a 500-kb interval of proximal mouse chromosome 7.
Homologs of genes mapping to human chromosome 11p15 are located in three distinct, widely separated regions of mouse chromosome 7 (Mmu7). To date, six genes have been localized to the most proximal HSA11p15/Mmu7 homology region, including Ldh3 (encoding lactate dehydrogenase C), Ldh1 (lactate dehydrogenase A), Myod1 (myogenic differentiation factor-1), Tph (tryptophan hydroxylase), Saa1 (serum amyloid-A-1), and Kcnc1 (encoding a Shaw-type voltage-gated potassium channel). To define the overall size and organization of this region of Mmu7, we have established a long-range physical map including the murine Ldh1, Ldh3, Saa, Tph, Kcnc1, and Myod1 genes. Our results demonstrate that these six genes are physically clustered and are distributed throughout a 500-kb interval located just proximal of the pink-eyed dilution (p) locus. These data, together with recent mapping studies within the related region of HSA11p15, demonstrate that gene content and organization within this proximal homology segment have been highly conserved throughout evolution.
Animals↗
Phenotypic consequences of deletion of the gamma 3, alpha 5, or beta 3 subunit of the type A gamma-aminobutyric acid receptor in mice.
Three genes (Gabrg3, Gabra5, and Gabrb3) encoding the gamma 3, alpha 5, and beta 3 subunits of the type A gamma-aminobutyric acid receptor, respectively, are known to map near the pink-eyed dilution (p) locus in mouse chromosome 7. This region shares homology with a segment of human chromosome 15 that is implicated in Angelman syndrome, an inherited neurobehavioral disorder. By mapping Gabrg3 on a panel of p-locus deletions, we have determined that the order of genes within this cluster is centromere-p(D15S12h)-Gabrg3-Gabra5-Gabrb3-telom ere. Like Gabrb3, neither the Gabra5 nor Gabrg3 gene is functionally imprinted in adult mouse brain. Mice deleted for all three subunits die at birth with a cleft palate, although there are rare survivors (approximately 5%) that do not have a cleft palate but do exhibit a neurological abnormality characterized by tremor, jerky gait, and runtiness. We have previously suggested that deficiency of the beta 3 subunit may be responsible for the clefting defect. Most notably, however, in this report we describe mice carrying two overlapping, complementing p deletions that fail to express the gamma 3 transcript, as well as mice from another line that express neither the gamma 3 nor alpha 5 transcripts. Surprisingly, mice from both of these lines are phenotypically normal and do not exhibit any of the neurological symptoms characteristic of the rare survivors that are deleted for all three (gamma 3, alpha 5, and beta 3) subunits. These mice therefore provide a whole-organism type A gamma-aminobutyric-acid receptor background that is devoid of any receptor subtypes that normally contain the gamma 3 and/or alpha 5 subunits. The absence of an overt neurological phenotype in mice lacking the gamma 3 and/or alpha 5 subunits also suggests that mutations in these genes are unlikely to provide useful animal models for Angelman syndrome in humans.
Angelman Syndrome↗
The mouse scurfy (sf) mutation is tightly linked to Gata1 and Tfe3 on the proximal X chromosome.
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Animals↗
Fine structure mapping and deletion analysis of the murine piebald locus.
piebald (s) is a recessive mutation that affects the development of two cell types of neural crest origin: the melanocytes, responsible for pigment synthesis in the skin, and enteric ganglia, which innervate the lower bowel. As a result, mice carrying piebald mutations exhibit white spotting in the coat and aganglionic megacolon. Previously the gene had been localized to the distal half of mouse chromosome 14. To determine its precise location relative to molecular markers, an intersubspecific backcross was generated. Two anchor loci of chromosome 14, slaty and hypogonadal, in addition to simple sequence length repeat markers, were used to localize s to a 2-cM interval defined by the markers D14Mit38 and D14Mit42. The molecular markers were also used to characterize nine induced s alleles. Three of these mutations exhibited no deletions or rearrangements of the flanking markers, whereas the other six had two or more of these markers deleted. The extent of the deletions was found to be consistent with the severity of the homozygous phenotype. The location of deletion breakpoints in the induced alleles, coupled with the recombination breakpoints in the backcross progeny, provide useful molecular landmarks to define the location of the piebald gene.
Animals↗
Molecular genetics of the brown (b)-locus region of mouse chromosome 4. I. Origin and molecular mapping of radiation- and chemical-induced lethal brown deletions.
Over a period of many years, germ-cell mutagenesis experiments using the mouse specific-locus test have generated numerous radiation- and chemical-induced alleles of the brown (b; Tyrp 1) locus in mouse chromosome 4. We describe here the origin, maintenance and initial molecular characterization of 28 b mutations that are prenatally lethal when homozygous. Each of these mutations is deleted for Tyrp 1 sequences, and each of 25 mutations tested further is deleted for at least one other locus defined by molecular clones previously found to be closely linked to b by interspecific backcross analysis. A panel of DNAs from mice carrying a lethal b mutation and a Mus spretus chromosome 4 was used in the fine structure mapping of these molecularly defined loci. The deletional nature of each of these prenatally lethal mutations is consistent with the hypothesis that the null phenotype at b has an effect only on the quality (color) of eumelanin produced in melanocytes. The resulting deletion map provides a framework on which to build future molecular-genetic and biological analyses of this region of mouse chromosome 4.
Alleles↗
Molecular genetics of the brown (b)-locus region of mouse chromosome 4. II. Complementation analyses of lethal brown deletions.
Numerous new mutations at the brown (b) locus in mouse chromosome 4 have been recovered over the years in germ-cell mutagenesis experiments performed at the Oak Ridge National Laboratory. A large series of radiation- and chemical-induced b mutations known to be chromosomal deletions, and also known to be prenatally lethal when homozygous, were analyzed by pairwise complementation crosses as well as by pseudodominance tests involving flanking loci defined by externally visible phenotypes. These crosses were designed to determine the extent of each deletion on the genetic and phenotype map of the chromosomal region surrounding the b locus; the crosses also provided basic data that assigned deletions to complementation groups and defined four new loci associated with aberrancies in normal development. Specifically, the pseudodominance tests identified deletions that include the proximally mapping whirler (wi) and the distally mapping depilated (dep) genes, thereby bracketing these loci defined by visible developmental abnormalities with landmarks (deletion breakpoints) that are easily identified on the physical map. Furthermore, the complementation crosses, which were supplemented with additional crosses that allowed determination of the gross time of lethality of selected deletions, defined four new loci required for normal development. Homozygous deletion of one of these loci (b-associated fitness, baf) results in a runting syndrome evident during postnatal development; deletion of one locus [l(4)2Rn] causes death in the late gestation/neonatal period; and deletion of either of two loci [l(4)1Rn or l(4)3Rn] results in embryonic death, most likely in pre-, peri- or postimplantation stages. The placement of these new functionally defined loci on the evolving molecular map of the b region should be useful for continuing the analysis of the roles played in development by genes in this segment of chromosome 4.
Animals↗
Concordance between isolated cleft palate in mice and alterations within a region including the gene encoding the beta 3 subunit of the type A gamma-aminobutyric acid receptor.
Genetic and molecular analyses of a number of radiation-induced deletion mutations of the pink-eyed dilution (p) locus in mouse chromosome 7 have identified a specific interval on the genetic map associated with a neonatally lethal mutation that results in cleft palate. This interval, closely linked and distal to p, and bracketed by the genes encoding the alpha 5 and beta 3 subunits of the type A gamma-aminobutyric acid receptor (Gabra5 and Gabrb3, respectively), contains a gene(s) (cp1; cleft palate 1) necessary for normal palate development. The cp1 interval extends from the distal breakpoint of the prenatally lethal p83FBFo deletion to the Gabrb3 locus. Among 20 p deletions tested, there was complete concordance between alterations at the Gabrb3 transcription unit and inability to complement the cleft-palate defect. These mapping data, along with previously described in vivo and in vitro teratological effects of gamma-aminobutyric acid or its agonists on palate development, suggest the possibility that a particular type A gamma-aminobutyric acid receptor that includes the beta 3 subunit may be necessary for normal palate development. The placement of the cp1 gene within a defined segment of the larger D15S12h (p)-D15S9h-1 interval in the mouse suggests that the highly homologous region of the human genome, 15q11-q13, be evaluated for a role(s) in human fetal facial development.
Animals↗
Evaluation of potential models for imprinted and nonimprinted components of human chromosome 15q11-q13 syndromes by fine-structure homology mapping in the mouse.
Prader-Willi and Angelman syndromes are complex neurobehavioral contiguous gene syndromes whose expression depends on the unmasking of genomic imprinting for different genetic loci in human chromosome 15q11-q13. The homologous chromosomal region in the mouse genome has been fine-mapped by using interspecific (Mus spretus) crosses and overlapping, radiation-induced deletions to evaluate potential animal models for both imprinted and nonimprinted components of these syndromes. Four evolutionarily conserved sequences from human 15q11-q13, including two cDNAs from fetal brain (DN10, D15S12h; DN34, D15S9h-1), a microdissected clone (MN7; D15F37S1h) expressed in mouse brain, and the gene for the beta 3 subunit of the gamma-aminobutyric acid type A receptor (Gabrb3), were mapped in mouse chromosome 7 by analysis of deletions at the pink-eyed dilution (p) locus. Three of these loci are deleted in pre- and postnatally lethal p-locus mutations, which extend up to 5.5 +/- 1.7 centimorgans (cM) proximal to p; D15S9h-1, which maps 1.1 +/- 0.8 cM distal to p and is the mouse homolog of the human gene D15S9 (which shows a DNA methylation imprint), is not deleted in any of the p-locus deletion series. A transcript from the Gabrb3 gene, but not the transcript detected by MN7 at the D15F37S1h locus, is expressed in mice homozygous for the p6H deletion, which have an abnormal neurological phenotype. Furthermore, the Gabrb3 transcript is expressed equally well from the maternal or paternal chromosome 7 and, therefore, its expression is not imprinted in mouse brain. Deletions at the mouse p locus should serve as intermediate genetic reagents and models with which to analyze the genetics and etiology of individual components of human 15q11-q13 disorders.
Angelman Syndrome↗
A gene for the mouse pink-eyed dilution locus and for human type II oculocutaneous albinism.
The mouse pink-eyed dilution (p) locus on chromosome 7 is associated with defects of skin, eye and coat pigmentation. Mutations at p cause a reduction of eumelanin (black-brown) pigment and altered morphology of black pigment granules (eumelanosomes), but have little effect on pheomelanin (yellow-red) pigment. We show here that the human complementary DNA DN10, linked to the p locus in mice, identifies the human homologue (P) of the mouse p gene, and appears to encode an integral membrane transporter protein. The expression pattern of this gene in various p mutant mice correlates with the pigmentation phenotype; moreover, an abnormally sized messenger RNA is detected in one mutant, p(un), which reverts to the normal size in p(un) revertants. The human P gene corresponds to the D15S12 locus within the chromosome segment 15q11-q13, which is typically deleted in patients with Prader-Willi and Angelman syndrome (see ref. 5 for review). These disorders are phenotypically distinct, depending on the parent of origin of the deleted chromosome, but both syndromes are often associated with hypopigmentation of the skin, hair and eyes (see ref. 8 for review), and deletion of the P gene may be responsible for this hypopigmentation. In addition, we report a mutation in both copies of the human P gene in one case of tyrosinase-positive (type II) oculocutaneous albinism, recently linked to 15q11-q13 (ref. 9).
Albinism, Oculocutaneous↗
High-frequency induction of chromosomal rearrangements in mouse germ cells by the chemotherapeutic agent chlorambucil.
Recent mutagenesis studies have demonstrated that the chemotherapeutic agent, chlorambucil (CHL), is highly mutagenic in male germ cells of the mouse. Post-meiotic germ cells, and especially early spermatids, are the most sensitive to the cytotoxic and mutagenic effects of this agent. Genetic, cytogenetic and molecular analyses of many induced mutations have shown that, in these germ-cell stages, CHL induces predominantly chromosomal rearrangements (deletions and translocations), and mutation-rate studies show that, in terms of tolerated doses, CHL is perhaps five to ten times more efficient in inducing rearrangements than is radiation exposure. Appropriate breeding protocols, along with knowledge of the advantages and limitations associated with the use of CHL, can be used to expand the current resource of chromosomal rearrangements in the mouse and to provide new phenotype-associated mutations amenable to positional-cloning techniques. The analysis of CHL-induced mutations has also contributed to understanding the factors that affect the yield and nature of chemically induced germline mutations in mammals.
Animals↗
N-ethyl-N-nitrosourea-induced prenatally lethal mutations define at least two complementation groups within the embryonic ectoderm development (eed) locus in mouse chromosome 7.
Two loci [l(7)5Rn and l(7)6Rn] defined by N-ethyl-N-nitrosourea (ENU)-induced, prenatally lethal mutations were mapped by means of trans complementation crosses to mice carrying lethal deletions of the albino (c) locus in Chromosome (Chr) 7. Both loci were found to map to the subregion of the Mod-2-sh-1 interval that contains the eed (embryonic ectoderm development) locus, eed has been defined by the inability of embryos homozygous for certain c deletions to develop beyond the early stages of gastrulation. Evidence for at least two loci necessary for normal prenatal development, rather than one locus, that map within the eed interval came from the observation that two prenatally lethal mutations, 3354SB [l(7)5Rn3354SB] and 4234SB [l(7)6Rn4234SB], could complement each other in trans, but could not each be complemented individually by c deletions known to include the eed locus. A somewhat leaky allele of l(7)5Rn [l(7)5Rn1989SB] was also recovered, in which hemizygotes are often stillborn and homozygotes exhibit variable fitness and survival. The mapping of the loci defined by these mutations is likely to be useful for genetic, molecular, and phenotypic characterization of the eed region, and mutations at either locus (or both loci) may contribute to the eed phenotype.
Alleles↗
Deletion mapping of the chocolate (cht) locus within the Fes-Hbb region of mouse chromosome 7.
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Animals↗
Reassignment of the H-ras-1 gene to the Hbb-terminus region of mouse chromosome 7.
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Animals↗
Structural differences between specific-locus mutations induced by different exposure regimes in mouse spermatogonial stem cells.
It was first shown by W.L. Russell (1962), and confirmed by him and others, that a 24-h interval between dose fractions (but not shorter or longer ones) elevates the rate of radiation-induced spermatogonial specific-locus mutations to levels considerably above the linear extrapolation made from lower-dose results. We have now analyzed the nature of mutations induced either in previously undisturbed or in "sensitized" spermatogonial stem cells, i.e., those that received a challenging dose of X-rays 24 h following a priming dose. Results are based on molecular studies of a large set of viable albino mutations [using probes derived from the tyrosinase (c) gene and from the regions surrounding c], and on retrospective classifications of mutations at c and two additional loci into LL (large lesions), IG (intragenic mutations), and OL (other lesions), utilizing criteria developed earlier. A significant difference (P = 0.016) was found between previously undisturbed and sensitized stem-cell spermatogonia; the latter have a higher LL/IG ratio, similar to the ratio observed for mutations induced in poststem-cell stages. This finding of a qualitative difference indicates that the additional mutations produced by a 24-h fractionated treatment are the result of the second (challenging) dose. The qualitative difference, further, indicates that the mutation-rate-augmenting effects of 24-h fractionation are not due, merely, to an increase (caused by the priming dose) of a normally responsive component of the spermatogonial population. The finding that the additional mutations that are produced by the challenging dose are primarily large DNA lesions suggests that the nuclear state of sensitized stem-cell spermatogonia may be different from the state of previously undisturbed spermatogonia. This state, which appears to be similar to that of postspermatonial stages, may be conducive to the formation of LLs, even by agents that are not LL inducers in other systems. The results further indicate that the relative paucity of LLs characteristic of treated (previously undisturbed) spermatogonial stem cells is probably not the result of selection against such mutations during subsequent germ-cell development.
Animals↗
Molecular analysis of viable spontaneous and radiation-induced albino (c)-locus mutations in the mouse.
Thirty-one homozygous-viable, radiation-induced or spontaneous mutations at the albino (c) locus in mouse chromosome 7 were analyzed by Southern blot analysis with a tyrosine cDNA clone and with probes derived from the closely linked proviral integration sites Pmv-31 and Emv-23, which flank the tyrosinase gene on the proximal and distal sides, respectively. Thirteen of 27 radiation-induced and one of four spontaneous mutations manifested deletions or rearrangements for the tyrosinase gene. The sizes of four deletions found to break within the tyrosinase gene itself were estimated to be < or = 36 kb, < or = 40 kb, approximately 260 kb, and approximately 480 kb. Two homozygous-viable deletions were found to include flanking proviral loci, suggesting that they could be from 1500-2000 kb in length, if not longer. The existence of these very large, homozygous-viable deletions suggests that the one-to-two megabases including and surrounding the c locus harbor no genes essential for normal viability or fertility, although genes controlling more subtle (or "nonessential") phenotypes are likely to be present. These data thus provide some insight into the molecular structure of a number of viable c-locus mutations, whose nature could not be predicted solely on the basis of genetic analysis, as could be done for either lethal or reduced-pigment c mutations.
Albinism↗