Analysis of the Cut Locus of DROSOPHILA MELANOGASTER

Mutants of the cut (ct) locus can be divided into two classes: viable and lethal. Most of the viable alleles are characterized by varying degrees of scalloping and notching of the wings. One mutant, kinked femur, exhibits kinking of the femurs and failure of wing expansion, but no other changes in wing structure. In heterozygous combination with the other viable alleles, it exhibits complete complementation, but it fails to complement with lethal ct alleles with respect to its viable phenotype. Similarly, all of the other viable ct alleles express a mutant wing phenotype when heterozygous with lethal ct alleles.-Mapping experiments indicate that the lethal alleles, which comprise the majority of all ct mutations recovered, are confined to a small region at the right end of the locus. That this restriction is real and not an artifact imposed by the limited number of lethal mutations mapped in the locus is supported by an examination of the mutant ct(JC20), a presumptive deficiency for the left-most third of the locus. Despite its behavior as a deletion, ct(JC20) is viable, though mutant, in combination with the lethal alleles. The restriction of the noncomplementary lethals to a small part of the locus, distinct from the other ct mutants, suggests a polarity that may define a segment that functions only in cis within the complex.-Based on the comparison of the data with the prediction of several models, we suggest that the left portion of the locus, which contains the viable alleles, defines a regulatory region controlling the expression of the locus, while the segment encoding a polypeptide product is at the right end and only it is capable of mutating to a lethal state.     

Genetic Analysis of the Cha-1-Unc-17 Gene Complex in Caenorhabditis

In C. elegans, the gene cha-1 is the structural gene for choline acetyltransferase, the enzyme which synthesizes acetylcholine. cha-1 is a complex gene which includes the previously described unc-17 locus; it has been hypothesized that a single protein is encoded which consists of several discrete structural domains. Mutations of the cha-1-unc-17 locus can be assigned to one of four classes on the basis of phenotype and complementation properties. A fine-structure map of this region has now been obtained by recombinational mapping. It is a large locus, spanning at least 0.035 map unit. On the map, the mutations lie in four contiguous, nonoverlapping regions, corresponding exactly to the different classes as defined by complementation and phenotype. Several new cha-1 mutations are described and mapped in the present study, including temperature-sensitive and lethal alleles.     

Four Distinct Regulatory Regions of the Cut Locus and Their Effect on Cell Type Specification in Drosophila

The cut gene in Drosophila is necessary in at least one cell type, the external sensory organs, for proper cell type specification and morphogenesis. It is also expressed in a variety of other tissues, where its function is less well characterized. Previous work has demonstrated that mutations affecting all the tissues map in the transcribed and translated portion of the gene, while mutations that are tissue specific in their effects map in the 140 kb upstream of the most 5' exon known. Within that 140 kb, the mutations fall into four subregions, two of which contain mutations affecting unique sets of tissues and the other two of which contain mutations that affect a third set. Our examination of the defects of mutants, their complementation behavior, and their effect on the distribution of the cut protein in embryos, alters the picture in three important ways. First, some mutations convert the cells of the Malpighian tubules into what appear to be gut cells, suggesting that cut is necessary for cell type specification and morphogenesis in a variety of tissues. Second, mutations in each of the four subregions in the 140 kb of upstream DNA cause a different set of phenotypes, suggesting that the regulatory region contains at least four separate units with different tissue specific functions. And third, mutations have now been identified that map in the transcribed and translated portion of the gene but that have tissue specific effects.     

Genetic identification and cloning of a gene required for developmental cell interactions in Myxococcus xanthus

Developmental mutants of Myxococcus xanthus have been previously described which appear to be defective in required cell-cell interactions. These mutants fall into four phenotypic classes, Asg, Bsg, Csg, and Dsg, each of which is unable to differentiate into spores but can be rescued by extracellular complementation by wild-type cells or by mutants of a different class. We report the identification of one of the loci in which mutations result in a Bsg phenotype. The cloned locus was contained on a 12-kilobase EcoRI fragment and then localized by subcloning and a combination of in vitro and transposon mutagenesis. All mutations in this locus behave as a single complementation group, which we designate bsgA (formerly ssbA). Each of the bsgA mutations results in a nonsporulating phenotype, which can be rescued by extracellular complementation. Furthermore, we report that the bsgA mutants have a distinctive interaction with wild-type cells when vegetatively growing, swarming colonies converge.     

Detection of a new tissue-specific enhancer in the ct6 region of the regulatory region of the drosophila cut locus]

The cut locus of Drosophila is an interesting example of a complex eukaryotic locus responsible for the development of many tissues and organs. Most of this locus is regulatory. The entire locus was cloned by Tchurikov et al. in 1986 and Blochlinger et al. in 1988. The wing ctn enhancer located 80 kb upstream of the promoter was earlier found in a 2.7 kb EcoRI-BamHI DNA fragment. The locus region 65-80 kb remote from the promoter was assumed to control the development of wings and vibrissae. We have found a new enhancer region in the ct6 region of the locus, which was in a 5 kb BamHI-EcoRI DNA fragment adjacent to the ctn enhancer. This region is responsible for the expression of the reporter lacZ gene in many tissues and organs at all stages of Drosophila development (at least in the intestine, Malpighian tubules, thoracic and abdominal sensory organs, thoracic ganglia and in ring glands). Thus, the region located 75 kb upstream of the promoter has some properties of the locus control region (LCR).     

Superunstable systems in Drosophila melanogaster. Analysis of mutations in signed and cut loci

The lines of the M'-cytotype characterized by a long-term instability (which was shown to be conditioned by transpositions of the new mobile element, Stalker) were hybridized with the P-line. This resulted in the appearance of a number of superunstable mutations at the yellow, white, singed, ocelliless and some other loci. The authors analyzed four independently obtained families of superunstable mutations at the singed locus. A wide spectrum of derivatives and high frequency of mutations were demonstrated, as well as the regularities of allelic transitions. Besides this, mutagenesis at the cut locus was observed in the chromosomes carrying sn mutations with frequency of 5.05 x 10(4). By means of the blot analysis it has been shown that most of ct mutations are intragenic deficiencies, ranging from 1.3 to 3 Kb, whose appearance is, conceivably, attributed to the inaccuracy of the insertion excision (the insertion is present but fails to alter the phenotype) at the cut locus of the chromosomes with the superunstable sn-alleles. In the lines with the sn- and ct-mutations the transpositions of the P-element and the Stalker were found, which indicates their involvement in mutagenesis. The authors discuss possible effects of inserting the complicated constructions, based on the combinations of P-element and the Stalker, on the induction of superinstability.     

Transposition bursts in analysis of repeated mutations in unstable strains of Drosophila melanogaster

The phenomenon of transposition memory was earlier demonstrated for the cut locus and mdg4. This work has been aimed at finding out, in what way the transposition memory can be realized. An unstable stock cmMR17ctMRpN17 was analysed which had high frequency of double cm+ct+ reversions and cmMRctMRpN repeated mutations. A series of five such transpositions could be followed. The ctMRpN17 mutation is a result of insertion at the cut locus mdg4 with the jockey element inserted within it. As seen from in situ hybridization analysis, transitions to the normal phenotype correlate, as a rule, with the excision of mdg4 and the jockey from the cut locus. Analysis of distribution of mdg1, mdg2, mdg3 and jockey on the X-chromosome of unstable revertants and repeated mutants indicated that not only transpositions of mdg4 and jockey, but also those of all mobile elements tested occur. So, we propose that the transposition memory in our genetic system is manifested in the process of transposition bursts.     

Retrotransposon-Induced Ectopic Expression of Cut Causes the Om(1a) Mutant in Drosophila Ananassae

Optic morphology (Om) mutations in Drosophila ananassae map to at least 22 loci scattered throughout the genome. They are semidominant, neomorphic, nonpleiotropic, and are associated with the insertion of a retrotransposon, tom. The Om(1A) gene, which is cytogenetically linked to the cut locus, was cloned using a DNA fragment of the cut locus of Drosophila melanogaster as a probe. Three of the eight alleles of Om(1A) examined have insertion of the tom element within a putative cut region. The γ-ray-induced revertants of Om(1A) are accompanied with cut lethal mutations and rearrangements within the cut coding region. In the eye imaginal discs of the Om(1A) mutants, differentiation of photoreceptor clusters is suppressed, abnormal cell death occurs in the center and the cut protein is expressed ectopically. D. melanogaster flies transformed with a chimeric cut gene under the control of a heat-inducible promoter show excessive cell death in the region anterior to the morphogenetic furrow, suppressed differentiation to photoreceptor clusters and defect in the imaginal eye morphology when subjected to temperature elevation. These findings suggest that the tom element inserted within the Om(1A) region induces ectopic cut expression in the eye imaginal discs, thus resulting in the Om(1A) mutant phenotype.     

The Doublesex Locus of Drosophila Melanogaster and Its Flanking Regions: A Cytogenetic Analysis

The region of the third chromosome (84D-F) of Drosophila melanogaster that contains the doublesex (dsx) locus has been cytogenetically analyzed. Twenty nine newly induced, and 42 preexisting rearrangements broken in dsx and the regions flanking dsx have been cytologically and genetically characterized. These studies established that the dsx locus is in salivary chromosome band 84E1-2. In addition, these observations provide strong evidence that the dsx locus functions only to regulate sexual differentiation and does not encode a vital function. To obtain new alleles at the dsx locus and to begin to analyze the genes flanking dsx, 59 lethal and visible mutations in a region encompassing dsx were induced. These mutations together with preexisting mutations in the region were deficiency mapped and placed into complementation groups. Among the mutations we isolated, four new mutations affecting sexual differentiation were identified. All proved to be alleles of dsx, suggesting that dsx is the only gene in this region involved in regulating sexual differentiation. All but one of the new dsx alleles have equivalent effects in males and females. The exception, dsxEFH55, strongly affects female sexual differentiation, but only weakly affects male sexual differentiation. The interactions of dsxEFH55 with mutations in other genes affecting sexual differentiation are described. These results are discussed in terms of the recent molecular findings that the dsx locus encodes sex-specific proteins that share in common their amino termini but have different carboxyl termini. The 72 mutations in this region that do not affect sexual differentiation identify 25 complementation groups. A translocation, T(2;3)Es that is associated with a lethal allele in one of these complementation groups is also broken at the engrailed (en) locus on the second chromosome and has a dominant phenotype that may be due to the expression of en in the anterior portion of the abdominal tergites where en is not normally expressed. The essential genes found in the 84D-F region are not evenly distributed throughout this region; most strikingly the 84D1-11 region appears to be devoid of essential genes. It is suggested that the lack of essential genes in this region is due to the region (1) containing genes with nonessential functions and (2) being duplicated, possibly both internally and elsewhere in the genome.     

Fine mapping of the human biotinidase gene and haplotype analysis of five common mutations

Biotinidase deficiency is an autosomal recessive defect in the recycling of biotin that can lead to a variety of neurologic and cutaneous symptoms. The disease can be prevented or effectively treated with exogenous biotin. The biotinidase locus (BTD) has been maped to 3p25 by in situ hybridization. The gene has been cloned, the coding region sequenced, the genomic organization determined, and a spectrum of mutations has been characterized in more than 90 individuals with profound or partial biotinidase deficiency. We have conducted haplotype analysis of 10 consanguineous and 39 nonconsanguineous probands from the United States and 8 consanguineous probands from Turkey to localize BTD with respect to polymorphic markers on 3p and to investigate the origins of five common mutations. The inbred probands were homozygous for overlapping regions of 3p ranging in size from 1.1 to 80 cM which were flanked most narrowly by D3S1259 and D3S1293. Radiation hybrids and haplotype analysis of markers within this region suggest that BTD is located within a 0.1-cM region flanked by D3S3510 and D3S1286. The radiation hybrid data suggest that the BTD gene is oriented 5' to 3' between the centromere and the 3p telomere. Association studies indicate that the gene is closer to a third locus D3S3613 than D3S3510, two markers which cannot be resolved by existing linkage data. The BTD locus and D3S3613 must therefore lie between D3S3510 and D3S1286. Comparison of haplotypes reveals evidence for possible founder effects for four of the five common mutations.     

A high-resolution genetic map of mouse Chromosome 15 encompassing the Dominant megacolon (Dom) locus

Dominant megacolon (Dom) is one of four mutations in the mouse that can produce a phenotype similar to Hirschsprung disease in human. The Dom gene product is not known, and no candidate region has been defined for a possible human homolog. In this publication we report mapping the Dom locus with high definition, using several intra- and interspecific crosses and a set of 16 Chr 15-specific microsatellites flanking this locus.     

Genomic and cDNA clones of the homeotic locus Antennapedia in Drosophila.

Homeotic genes are involved in the control of developmental pathways: dominant mutations at the Antennapedia locus of Drosophila, for example, lead to replacement of the antennae on the head of the fly by mesothoracic legs. Using a combination of chromosome walking and jumping, we have cloned a DNA region from Drosophila containing Antennapedia. Five DNA inversion rearrangements which are associated with the Antennapedia mutant phenotype were localized within a 25-kb region. Genomic DNA sequences from this area were used as hybridization probes to screen cDNA libraries prepared from Drosophila embryonic and pupal poly(A)+ RNA. A 2.2-kb cDNA sequence (903) was isolated which appears to derive from at least four non-contiguous chromosomal regions that span 100 kb. It includes the positions of the inversion breakpoints. A second cDNA of 2.9 kb (909) is composed of sequences from at least three chromosomal regions, two of which are similar or identical to sequences contained in the 903 clone but the third is derived from genomic DNA within a putative 903 intron. The unusual size and complexity of this locus are discussed.     

Genetic and Physical Mapping of the Mouse Ulnaless Locus

The mouse Ulnaless locus is a semidominant mutation which displays defects in patterning along the proximal-distal and anterior-posterior axes of all four limbs. The first Ulnaless homozygotes have been generated, and they display a similar, though slightly more severe, limb phenotype than the heterozygotes. To create a refined genetic map of the Ulnaless region using molecular markers, four backcrosses segregating Ulnaless were established. A 0.4-cM interval containing the Ulnaless locus has been defined on mouse chromosome 2, which has identified Ulnaless as a possible allele of a Hoxd cluster gene(s). With this genetic map as a framework, a physical map of the Ulnaless region has been completed. Yeast artificial chromosomes covering this region have been isolated and ordered into a 2 Mb contig. Therefore, the region that must contain the Ulnaless locus has been defined and cloned, which will be invaluable for the identification of the molecular nature of the Ulnaless mutation.     

Nucleotide sequence and complementation analysis of a polycistronic sporulation operon, spoVA, in Bacillus subtilis

We have determined the nucleotide sequence of a 3706 bp stretch of Bacillus subtilis chromosomal DNA that complements all known spoVA mutations. The sequence contains five consecutive large open reading frames capable of encoding proteins of molecular weights ranging from approximately 15000 to 36000. Analysis using integrational plasmids suggests that the region is likely to be transcribed as a single mRNA. A novel form of complementation analysis, based on derivatives of bacteriophage phi 105 carrying the cloned spoVA locus, has been used to define four distinct complementation groups among the eight previously characterized spoVA mutations. The spoVA locus is the largest polycistronic sporulation operon yet characterized.     

Mobile elements and transposition events in the cut locus of Drosophila melanogaster

We have cloned from the Oregon R strain of Drosophila melanogaster a 240 kb segment of DNA that contains the cut (ct) locus, and characterized the region for the presence of repetitive elements. Within this region at least five copies of the suffix element were detected, as well as several putatively novel mobile elements. A number of mutations obtained from the unstable ct ^MR2 strain and its derivatives were mapped within the cut locus. Comparison between parental and daughter strains indicates that frequently two or more independent transposition events involving the cut locus occur simultaneously within a single germ cell, thus providing a molecular basis for the transposition explosion phenomenon.