A high-resolution map of the brown (b,Tyrp1) deletion complex of mouse Chromosome 4

For over 40 years germ-cell mutagenesis experiments have generated many new mutations at the brown (b or Tyrp1) locus on mouse Chromosome (Chr) 4. These mutations, many of which are deletions, were recovered by the specific-locus mutagenesis technique. Previous analysis of a panel of brown deletions, generated at Oak Ridge, has enabled both a preliminary molecular and a functional map around the locus to be generated. We have used a panel of hybrid DNA from 25 Oak Ridge deletions, where the deleted chromosome was heterozygous with a Mus spretus chromosome, to map polymorphic markers including microclones, microsatellites, and cloned DNA markers. We have generated a fine structure map, based on 25 new markers, of an 8.5-cM region surrounding the brown locus. This map will prove useful in future mapping studies of this region and in the isolation of the genes that lie within it.     

Molecular cloning and mapping of the ecotropic leukemia provirus Emv-23 provides molecular access to the albino-deletion complex in mouse chromosome 7

Genetic analysis of radiation-induced deletion mutations involving the chromosome 7 albino (c) locus has expanded the functional map of this 6 to 11-cM region of the mouse genome. To generate one of many points of molecular access necessary for intensifying the analysis of the genes and phenotypes associated with this particular complex of deletions, we have cloned an endogenous ecotropic leukemia provirus (Emv-23), known to be closely linked to c, along with its flanking chromosome 7 sequences. A unique-sequence probe (23.3), derived from a region immediately 5' to the proviral integration site, was found to map less than 0.5 cM from c in a standard backcross analysis. Southern blot analysis of DNAs from animals carrying homozygous or overlapping albino deletions demonstrated that the 23.3 probe was deleted in several relatively small c-region deletions. The deletion mapping of the 23.3 probe places the Emv-23 locus between c and Mod-2, just proximal to a region important for male fertility and juvenile fitness. Mapping of this locus also provides a refinement of the genetic/deletion map for several mutations within this deletion complex.     

Genetic Analysis of Chromosome Region 63 of Drosophila Melanogaster

The salivary chromosome region including cytological division 63 of Drosophila melanogaster was genetically analyzed in order to (1) characterize this previously unstudied region and (2) attempt to isolate mutations in the hsp82 gene. Seven deletions which span this region were isolated, including four which remove the hsp82 gene. A Minute mutation was mapped to this region and this Minute was used to isolate duplications in the 63 region. These duplications map the Minute to 63B8-C1. F2 screens were initiated using deletions which remove the hsp82 gene. Over 15,000 chromosomes were screened, yielding 40 lethal mutations which comprise 14 complementation groups. Several of these mutations map outside the 63 region and appear to give second site interaction with the Minute locus. Four loci, including the Minute gene, are candidates for hsp82 mutations by cytogenetic mapping. These loci were tested for complementation with a P element carrying the hsp82 gene. However, none of the mutations was rescued.     

Deletions of a tyrosine tRNA gene in S. cerevisiae.

Genetic fine structure analysis of a tyrosine tRNA in yeast revealed that complete deletions of the gene occurred at an unusually high frequency. Among 56 spontaneous mutations at the SUP4 locus, 16 were classified as deletions as judged by their failure to recombine with any other mutations known to map within the gene. Physical analysis of each deletion confirmed the genetic result. The deletions fall into two size classes: ten are 2100 bp deletions and six are 2800 bp deletions. These results imply that the physical structure of the region surrounding the SUP4 locus, which is known to contain short repeated segments, has a direct role in promoting deletions.     

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.     

Point Mutants in the D2a Region of Bacteriophage T4 Fail to Induce T4 Endonuclease IV

We have studied the properties of presumptive point mutants in the D2a region of bacteriophage T4. Dominance tests showed that the D2a mutation was recessive to the wild-type allele. The mutations were shown to map in the D2a region by complementation against rII deletions. The D2a mutations were also located between gene 52 and rIIB by two- and three-factor crosses. The mutants are located at at least two distinct loci in the D2a region. The point mutants grow normally on all hosts tested and none of the mutants makes T4 endonuclease IV. We propose the name "denB" for the D2a locus.     

Genetic Analysis of Mutants of SACCHAROMYCES CEREVISIAE Resistant to the Herbicide Sulfometuron Methyl

Sulfometuron methyl (SM), a potent new sulfonylurea herbicide, inhibits growth of the yeast Saccharomyces cerevisiae on minimal media. Sixty-six spontaneous mutants resistant to SM were isolated. All of the resistance mutations segregate 2:2 in tetrads; 51 of the mutations are dominant, five are semidominant and ten are recessive. The mutations occur in three linkage groups, designated SMR1, smr2 and smr3. Several lines of evidence demonstrate that the SMR1 mutations (47 dominant and four semidominant) are alleles of ILV2 which encodes acetolactate synthase (ALS), the target of SM. First, SMR1 mutations result in the production of ALS enzyme activity with increased resistance to SM. Second, molecular cloning of the ILV2 gene permitted the isolation of mutations in the cloned gene which result in the production of SM-resistant ALS. Finally, SMR1 mutations map at the ILV2 locus. The smr2 mutations (four recessive, two dominant and one semidominant) map at the pdr 1 (pleiotropic drug resistance) locus and show cross-resistance to other inhibitors, typical of mutations at this locus. The smr3 mutations (six recessive and two dominant) define a new gene which maps approximately midway between ADE2 and HIS3 on the right arm of chromosome XV.     

Identification of a repressor gene involved in the regulation of NAD de novo biosynthesis in Salmonella typhimurium.

Mutations at the nadI locus affect expression of the first two genes of NAD synthesis, nadA and nadB, which are unlinked. Genetic data imply that the regulatory effects of nadI mutations are not due to indirect consequences of physiological alterations. Two types of mutations map in the nadI region. Common null mutations (nadI) show constitutive high-level expression of the nadB and nadA genes. Rare nadIs mutations cause constitutive low-level expression of nadB and nadA. Some nadIs mutations shut off the expression of the biosynthetic genes sufficiently to cause a nicotinic acid auxotrophy. Spontaneous revertants of auxotrophic nadIs mutants have a NadI- phenotype, including some with deletions of the nadI locus. The nadI locus encodes a repressor protein acting on the unlinked nadA and nadB genes.     

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.     

Deletion Mapping of Four Loci Defined by N-Ethyl-N-Nitrosourea-Induced Postimplantation-Lethal Mutations within the Pid-Hbb Region of Mouse Chromosome 7

As part of a long-term effort to refine the physical and functional maps of the Fes-Hbb region of mouse chromosome 7, four loci [l(7)1Rn, l(7)2Rn, l(7)3Rn, l(7)4Rn] 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 mouse chromosome-7 albino (c) locus. Each locus was assigned to a defined subregion of the deletion map at the distal end of the Fes-Hbb interval. Of particular use for this mapping were preimplantation-lethal deletions having distal breakpoints localized between pid and Omp. Hemizygosity or homozygosity for each of the ENU-induced lethals was found to arrest development after uterine implantation; the specific time of postimplantation death varied, and depended on both the mutation itself and on whether it was hemizygous or homozygous. Based on their map positions outside of and distal to deletions that cause death at preimplantation stages, these ENU-induced mutations identify loci, necessary for postimplantation development, that could not have been discovered by phenotypic analyses of mice homozygous for any albino deletion. The mapping of these loci to specific genetic intervals defined by deletion breakpoints suggests a number of positional-cloning strategies for the molecular isolation of these genes. Phenotypic and genetic analyses of these mutations should provide useful information on the functional composition of the corresponding segment of the human genome (perhaps human 11q13.5).     

On the Identification of the ROSY Locus DNA in DROSOPHILA MELANOGASTER: Intragenic Recombination Mapping of Mutations Associated with Insertions and Deletions

DNA extracts of several rosy-mutation-bearing strains were associated with large insertions and deletions in a defined region of the molecular map believed to include the rosy locus DNA. Large-scale, intragenic mapping experiments were carried out that localized these mutations within the boundaries of the previously defined rosy locus structural element. Molecular characterization of the wild-type recombinants provides conclusive evidence that the rosy locus DNA is localized to the DNA segment marked by these lesions. One of the mutations, ry2101, arose from a P-M hybrid dysgenesis experiment and is associated with a copia insertion. Experiments are described which suggest that copia mobilizes in response to P-M hybrid dysgenesis. Relevance of the data to recombination in higher organisms is considered.     

Genetic characterization of the glutamate dehydrogenase gene (gdhA) of Salmonella typhimurium.

Salmonella typhimurium mutants, either devoid or glutamate dehydrogenase activity or having a thermolabile glutamate dehydrogenase protein, were used to identify the structural gene (gdhA) for this enzyme. Transductions showed that the mutations producing these phenotypes were linked to both the pncA and nit genes, placing the gdhA locus between 23 and 30 U on the S. typhimurium chromosome. Additional transductions with several Tn10 insertions established the gene order as pncA-gdhA-nit. Since few genetic markers exist in this region of the chromosome, Hfr strains were constructed to orient the pncA-gdhA-nit cluster with outside genes. Conjugation experiments provided evidence for the gene order pyrD-pncA-gdhA-nit-trp. To further characterize gdhA, we used Mu cts d1 (Apr lac) insertions in this gene to select numerous strains containing deletions with various endpoints. Transductions of these deletions with strains containing different gdh mutations and with a mutant having a thermolabile glutamate dehydrogenase protein permitted us to construct a deletion map of the gdhA region.     

Bipartite Structure of the ade3 Locus of SACCHAROMYCES CEREVISIAE

Forty ade3 mutants were examined with respect to their growth requirements, levels of the tetrahydrofolate interconversion enzymes, and/or map positions. Four deletions were detected. Mutations that result in a requirement for adenine and histidine map in one region of the locus; those which result in a requirement for adenine only map in a quite separate region of the locus, a region not disclosed in previous studies. No correlation was observed between growth properties of the strains and enzyme levels.     

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. XI. Heterozygous effects of gene/point mutations of genotype ad-3A or ad-3B.

Previous studies on X-ray-induced irreparable adenine-3 mutants (designated ad-3IR), induced in heterokaryon 12 of Neurospora crassa, showed that they were not recessive, and that they demonstrated heterozygous effects in terms of markedly reduced linear growth rates as compared with a wild-type control (de Serres, 1965, 1988). Homology tests on X-ray-induced irreparable mutants showed that they map, in the main part, as a series of overlapping multilocus deletions that extend both proximally and distally into the immediately adjacent genetic regions, as well as into the 'X' region (a region of unknown, but essential, function) between ad-3A and ad-3B (de Serres, 1969, 1989a). Studies on a larger sample of X-ray-induced multilocus deletion mutations of genotype (ad-3A)IR or (ad-3B)IR (de Serres et al., 1992) demonstrated that heterozygous effects are allele specific and that there was no correlation with genotype, radiation dose or complementation map position. Furthermore, the heterozygous effects of multilocus deletions in the ad-3 region can be modified genetically and biochemically (de Serres and Miller, 1988). In the present paper, the heterozygous effects of X-ray-induced gene/point mutations of genotype ad-3AR or ad-3BR, induced in heterokaryon 12 (Webber and de Serres, 1965; de Serres, 1988, 1989a), were determined. The studies presented in this paper show that 8.1% (3/37) of X-ray-induced ad-3AR mutations exhibit heterozygous effects in terms of reduced linear growth rates in forced dikaryons with a gene/point mutant at the ad-3B locus, and 10.8% (4/37) in forced dikaryons with a multilocus deletion mutation covering the ad-3B locus. In addition, 24.3% (9/37) of ad-3AR mutations exhibit heterozygous effects in terms of enhanced linear growth rates in forced dikaryons with a gene/point mutant at the ad-3B locus. Similar studies with X-ray-induced ad-3BR mutations showed that 54.9% (28/51) exhibit heterozygous effects in terms of reduced growth rates in forced dikaryons with a gene/point mutant at the ad-3A locus and 100.0% (48/48) in forced dikaryons with a multilocus deletion covering the ad-3A locus. These studies have also shown that about a 13-fold higher percentage of X-ray-induced multiple-locus mutations of genotype ad-3AR + RLCL have heterozygous effects resulting in reduced growth rates than X-ray-induced single-locus mutations of genotype ad-3AR. The overall data base on X-ray-induced ad-3 gene/point mutations in the present studies demonstrates that heterozygous effects are allele specific, genotype specific, and locus specific.(ABSTRACT TRUNCATED AT 400 WORDS)     

3 linkage groups of the genes of riboflavin biosynthesis in Escherichia coli

Using transposon Tn5 inactivation technology a collection of Escherichia coli mutants defective in riboflavine biosynthesis was obtained. All mutations were distributed within three linkage groups. With the help of P1-transduction mapping, group I mutations (ribA locus) were localized near cysB locus (28 min of the standard 100 min E. coli map) and mutations of group II (ribB locus) were mapped near tolC locus (66 min). The location of group III mutations was approximately determined by the F' complementation analysis: this linkage group lies in the region of 56-60 min of the E. coli map.     

Genetic analysis of the X-chromosomal region 1E-2A of Drosophila melanogaster

Reversion mutagenesis of three single P elements located in the cytogenetic interval 1E-2A at the tip of the X chromosome of Drosophila melanogaster was used to recover new deletions in this chromosomal region. The deletions obtained include small aberrations within region 2A and larger lesions extending from 2A into 1E and 1B. All three screens also yielded terminal deficiencies. The new deficiencies, together with previously characterized rearrangements, were analyzed for their complementation behaviour with the maternal effect locus fs(1)Nasrat and lethal loci in the region. These analyses provide an overall genetic map of the interval 1E-2A. In addition, the smaller deletions were physically mapped within cloned genomic DNA of the 2A region.     

Genetic analysis of the X-chromosomal region 1E-2A of Drosophila melanogaster

Reversion mutagenesis of three single P elements located in the cytogenetic interval 1E-2A at the tip of the X chromosome of Drosophila melanogaster was used to recover new deletions in this chromosomal region. The deletions obtained include small aberrations within region 2A and larger lesions extending from 2A into 1E and 1B. All three screens also yielded terminal deficiencies. The new deficiencies, together with previously characterized rearrangements, were analyzed for their complementation behaviour with the maternal effect locus fs(1)Nasrat and lethal loci in the region. These analyses provide an overall genetic map of the interval 1E-2A. In addition, the smaller deletions were physically mapped within cloned genomic DNA of the 2A region.     

Molecular Analysis of Radiation-Induced Albino (C)-Locus Mutations That Cause Death at Preimplantation Stages of Development

Deletion mutations at the albino (c) locus have been useful for continuing the development of fine-structure physical and functional maps of the Fes-Hbb region of mouse chromosome 7. This report describes the molecular analysis of a number of radiation-induced c deletions that, when homozygous, cause death of the embryo during preimplantation stages. The distal extent of these deletions defines a locus, pid, (preimplantation development) genetically associated with this phenotype. The proximal breakpoints of eight of these deletions were mapped with respect to the Tyr (tyrosinase; albino) gene as well as to anonymous loci within the Fah-Tyr region that are defined by the Pmv-31 viral integration site and by chromosome-microdissection clones. Rearrangements corresponding to the proximal breakpoints of two of these deletions were detected by Southern blot analysis, and a size-altered restriction fragment carrying the breakpoint of one of them was cloned. A probe derived from this deletion fusion fragment defines a locus, D7Rn6, which maps within (or distal to) the pid region, and which discriminates among the distal extents of deletions eliciting the pid phenotype. Extension of physical maps from D7Rn6 should provide access both to the pid region and to loci mapping distal to pid that are defined by N-ethyl-N-nitrosourea-induced lethal mutations.     

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)5Rn ^3354SB ] and 4234SB [l(7)6Rn ^4234SB ], 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)5Rn ^1989SB ] 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.