The gene fl(2)d is needed for the sex-specific splicing of transformer pre-mRNA but not for double-sex pre-mRNA in Drosophila melanogaster

In Drosophila melanogaster, regulation of the sex determination genes throughout development occurs by sex-specific splicing of their products. The first gene is Sex-lethal(Sxl). The downstream target of Sxl is the gene transformer (tra): the Sxl protein controls the female-specific splicing of the Tra pre-mRNA. The downstream target of the gene tra is the gene double-sex (dsx): the Tra protein of females, controls the female-specific splicing of the Dsx pre-mRNA. We have identified a gene, female-lethal-2-d fl(2)d, whose function is required for the female-specific splicing of Sxl pre-mRNA. In this report we analyze whether the gene fl(2)d is also required for the sex-specific splicing of both Tra and Dsx pre-mRNAs. We found that the Sxl protein is not sufficient for the female-specific splicing of Tra pre-mRNA, the fl(2)d function also being necessary. This gene, however, is not required for the female-specific splicing of Dsx pre-mRNA.     

The mechanism of sex-specific splicing at the doublesex gene is different between Drosophila melanogaster and Bombyx mori

We have previously reported that Bmdsx, a homologue of the sex-determining gene, doublesex (dsx), was found to be sex-specifically expressed in various tissues at larval, pupal, and adult stages in the silkworm, Bombyx mori, and was alternatively spliced to yield male- and female-specific mRNAs. To reveal sex-specific differences in splicing patterns of Bmdsx pre-mRNA, the genomic sequence was determined and compared with male- and female-specific Bmdsx cDNA sequences. The open reading frame (ORF) consisted of five exons. Exons 3 and 4 were specifically incorporated into the female type of Bmdsx mRNA. On the other hand, exon 2 was spliced to exon 5 to produce the male type mRNA of Bmdsx. As in the case of Drosophila dsx, the OD2 domain was separated by a female-specific intron into sex-independent and sex-dependent regions. Sex-specific splicing occurred in equivalent positions in the Drosophila dsx gene. However, unlike Drosophila dsx, the female-specific introns showed no weak 3′ splice sites, and the TRA/TRA-2 binding site related sequences were not found in the female-specific exon, nor even in any other regions of the Bmdsx gene. Moreover, an in vitro splicing reaction consisting of HeLa cell nuclear extracts showed that the female-type of Bmdsx mRNA represented the default splicing. These findings suggest that the structural features of the sex-specific splicing patterns of Bmdsx pre-mRNA are similar to those of Drosophila dsx but the regulation of sex-specific alternative splicing of Bmdsx pre-mRNA is different.     

Study of the ref(2)P locus of Drosophila melanogaster

The ref(2)P gene of Drosophila melanogaster is implicated in sigma rhabdovirus multiplication. Two common alleles of ref(2)P are known, ref(2)P ⁰ which permits sigma virus multiplication and ref(2)P ^pwhich is restrictive for most sigma virus strains. This gene maps to the cytogenetic region 37E3-F3. Using Df(2L)E55 (=Df(2L)37D2-El;37F5-38A1), we have screened for lethal, semi-lethal and visible mutations following diepoxybutane (DEB) or ethyl methanesulfonate (EMS) mutagenesis. Our data confirm than DEB is mor efficient than EMS at inducing deletions. The mutations obtained in this region define 14 complementation groups. One of them, l(2)37Dh, appears to be a general enhancer of Minute and Minute-like mutations. None of the mutations were allelic to the ref(2)P locus. Loss-of-function alleles of ref(2)P (called null) were selected following DEB mutagenesis. Homozygous or hemizygous ref(2)P ^nullflies are male sterile. These flies, like homozygous or hemizygous ref(2)P ⁰flies, are fully permissive for sigma virus replication. We suggest that the ref(2)P products interact with viral products, but that this interaction is not necessary for an efficient viral cycle.     

Malsegregation of chromosomes is not a major source of somatic mosaicism in Drosophila melanogaster

Two genotypes were constructed to determine whether some of the mosaic spots, on which the SMART (somatic mutation and recombination test) procedures are based may arise through malsegregation of the chromosomes. Both arms of the metacentric third chromosomes were labelled with marker mutations, and in this way one- and two-arm events (the former representing rearrangements or point mutations, the latter representing malsegregation) could be recorded separately. Although several hundred clones were identified following exposure of larvae to X-rays, colchicine or vinchicine or vincristine (all known inducers of malsegregation), none arose as a consequence of two-arm events. This suggests that malsegregation of the chromosomes plays little, if any role in the formation of mosaic spots. Instead, the clones develop due to mitotic recombinations, deletions or point mutations.     

Regulatory elements in the promoter of the vitelline membrane gene VM32E of Drosophila melanogaster direct gene expression in distinct domains of the follicular epithelium

The Drosophila vitelline membrane protein gene VM32E is expressed according to a precise temporal and spatial program in the follicle cells. Results from germ line transformation experiments using different fragments of the −465/−39 VM32E region fused to the hsp/lacZ reporter gene revealed that the region −348/−39 is sufficient to confer the wild-type expression pattern. Within this segment, distinct cis-regulatory elements control VM32E expression in ventral and dorsal follicle cells. The region between −135/−113 is essential for expression of the VM32E gene in the ventral columnar follicle cells. Expression in the dorsal domain requires the two regions −348/−254 and −118/−39. Furthermore, the region −253/−119 appears to contain a negative element that represses gene activity in anterior centripetal cells. We suggest that the expression of the VM32E gene throughout the follicular epithelium is controlled by specific cis-regulatory elements acting in distinct spatial domains and following a precise developmental program. Received: 22 October 1996 / Accepted: 14 November 1996     

The heterocyst-specific fdxH gene product of the cyanobacterium Anabaena sp. PCC 7120 is important but not essential for nitrogen fixation

To clarify the role of the heterocyst-specific [2Fe-2S] ferredoxin in cyanobacterial nitrogen fixation, mutational analysis of the Anabaena 7120 fdxH gene region was carried out. First, the DNA sequence of the wild-type 3509-bp EcoRI fragment downstream of the fdxH gene was determined. Genes homologous to ORF3 from the fdxH gene regions of A. variabilis and Plectonemaboryanum, the mop genes of Clostridiumpasteurianum encoding molybdo-pterin binding proteins, and ORF3 from the A. variabilis hydrogenase gene cluster were identified within the sequenced region. For mutational analysis the Anabaena 7120 mutant strains LAK4, BMB92, and KSH10 were constructed. In LAK4 the fdxH coding region is disrupted by an interposon, whereas BMB92 is deleted for a 2799-bp NheI fragment encompassing fdxH, ORF3, mop, ORF4, and ORF5. Mutant strain KSH10 is a derivative of BMB92, complemented for fdxH but not for the other genes located further downstream. Analysis of the Nif phenotype of these mutant strains showed that FdxH is necessary for maximum nitrogenase activity and optimal growth under nitrogen-fixing conditions, but not absolutely essential for diazotrophic growth. The role of alternative electron donors for nitrogenase, which might substitute for FdxH, is discussed. Iron concentrations (1μM Fe) sufficient to induce synthesis of the vegetative cell flavodoxin did not stimulate diazotrophic growth of the fdxH mutant strains, suggesting that FdxH was not replaced by a NifJ-flavodoxin system. Comparison of LAK4 and BMB92 indicated that one of the genes located downstream of fdxH might also play a (minor) role in nitrogen fixation. Received: 28 May 1996 / Accepted: 4 October 1996     

Polymorphism and stability of histone gene clusters in Drosophila melanogaster cultured cells

In a previous communication (Saigo, K., Millstein, L. and Thomas, C.A., Jr. (1981) Cold Spring Harbor Symp. Quant. Biol. 45, 815–827), the overall structure of histone genes of Schneider line 2 cells was shown to extensively differ from that of Oregon-R embryo from which the cell line was established, and it was speculated that the histone genes might be reshuffled extensively during either the periods of the establishment, or maintenance of cell lines, or both. To establish the validity of this notion the structure of histone genes was examined in Drosophila melanogaster cultured cells. The overall organization of histone gene clusters was found to be stably maintained in both the periods for the establishment and maintenance of cultured cells, indicating that the previous assumption is inadequate. Instead of an extensive rearrangement, minor structural changes were found to occasionally occur probably by simple base substitutions and/or, deletion or insertion of very short DNA pieces. It was also shown that the extensive variation in structures of histone genes in cultured cells such as Schneider line 2 are attributable to polymorphism on the level of individual flies.     

The KEULE gene is involved in cytokinesis in Arabidopsis

 We present evidence to show that the KEULE gene of Arabidopsis is involved in cytokinesis. Mutant keule embryos have large multinucleate cells with gapped or incomplete cross walls, as well as cell wall stubs that are very similar to those observed upon caffeine inhibition of cytokinesis in plants. These defects are observed in all populations of dividing cells in the mutant, including calli, but less frequently in mature cells. Cell division appears to be slowed down, and the planes of cell division are often misoriented. In late embryos and seedlings, cross-wall formation usually appears complete, suggesting that the requirement for KEULE during cytokinesis is not absolute. Nonetheless, keule mutants die as seedlings with large polyploid cells. The bloated surface layer of keule seedlings does not uniformly behave like wild-type epidermis, and patches of this layer assume characteristics of the underlying ground tissue. The cytokinesis defect of keule mutants may influence aspects of cellular differentiation. Received: 24 April 1996 / Accepted: 11 June 1996     

The influence of heavy water (D2O) on the thermopreferendum in Drosophila melanogaster

1. 1. Thermopreferendum of contron Drosophila melanogaster flies, fed sugared water was compared with that of flies, fed sugared water containing 50% D₂O.

2. 2. Deuterium oxide increased not only the thermoresistance of some proteins, cells and the organisms, but also organism thermophilly.

Expression of the seqA gene is negatively modulated by the HU protein in Escherichia coli

The SeqA protein acts as a regulator of chromosomal replication initiation in Escherichia coli by sequestering hemi-methylated oriC, effectively blocking methylation and therefore preventing rapid re-initiation. The level of SeqA protein is maximal at mid-log phase and decreases when cells enter late-log phase. In hup mutants that lack the HU protein, the maximal seqA expression is also seen at mid-log phase, but seqA expression, as well as SeqA levels and activity, is increased by up to four fold relative to that in the wild type. These results suggest that the HU protein functions as a negative modulator of seqA expression. Received: 23 June 2000 / Accepted: 29 September 2000 / Published online: 11 November 2000     

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.     

The novel function of the Saccharomyces cerevisiaeCBP2 gene as a splicing factor essential to excision of the Saccharomyces douglasiiLSU intron in vivo

In the yeast Saccharomyces cerevisiae, the product of the nuclear gene CBP2 is required exclusively for the splicing of the terminal intron of the mitochondrial cytochrome b gene. The homologous gene from the related yeast, Saccharomyces douglasii, has been shown to be essential for respiratory growth in the presence of a wild-type S. douglasii mitochondrial genome and dispensable in the presence of an intronless mitochondrial genome. The two CBP2 genes are functionally interchangeable although the target intron of the S. cerevisiaeCBP2 gene is absent from the S. douglasii mitochondrial genome. To determine the function of the CBP2 gene in S. douglasii mitochondrial pre-RNA processing we have constructed and analyzed interspecific hybrid strains between the nuclear genome of S. cerevisiae carrying an inactive CBP2 gene and S. douglasii mitochondrial genomes with different intron contents. We have demonstrated that inactivation of the S. cerevisiaeCBP2 gene affects the maturation of the S. douglasii LSU pre-RNA, leading to a respiratory-deficient phenotype in the hybrid strains. We have shown that the CBP2 gene is essential for excision of the S. douglasii LSU intron in vivo and that the gene is dispensable when this intron is deleted or replaced by the S. cerevisiae LSU intron. Received: 1 October 1997 / Accepted: 18 November 1997