The genetics and molecular biology of zeste in Drosophila melanogaster.
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Amino Acid Sequence↗
Biomedical subjects
V Pirrotta
Publications and source records attributed to V Pirrotta.
Interactions of the Drosophila gap gene giant with maternal and zygotic pattern-forming genes.
The Drosophila gene giant (gt) is a segmentation gene that affects anterior head structures and abdominal segments A5-A7. Immunolocalization of the gt product shows that it is a nuclear protein whose expression is initially activated in an anterior and a posterior domain. Activation of the anterior domain is dependent on the maternal bicoid gradient while activation of the posterior domain requires maternal nanos gene product. Initial expression is not abolished by mutations in any of the zygotic gap genes. By cellular blastoderm, the initial pattern of expression has evolved into one posterior and three anterior stripes of expression. The evolution, position and width of these stripes are dependent on interactions between gt and the other gap genes. In turn, gt activity in these domains affects the expression of the other gap genes. These interactions, typical of the cross-regulation previously observed among gap genes, confirm that gt is a member of the gap gene class whose function is necessary to establish the overall pattern of gap gene expression. After cellular blastoderm, gt protein continues to be expressed in the head region in parts of the maxillary and mandibular segments as well as in the labrum. Expression is never detected in the labial or thoracic segment primordia but persists in certain head structures, including the ring gland, until the end of embryonic development.
Animals↗
Transvection and long-distance gene regulation.
Numerous genes contain regulatory elements located many tens of kilobases away from the promoter they control. Specific mechanisms must be required to ensure that such distant elements can find and interact with their proper targets but not with extraneous genes. This review explores the connections between transvection phenomena, the activation of domains of homeotic gene expression, position effect variegation and silencers. These various examples of long-distance effects suggest that, in all cases, related forms of chromatin packaging may be involved.
Animals↗
Self-association of the Drosophila zeste protein is responsible for transvection effects.
The zeste gene product is required for transvection effects that imply the ability of regulatory elements on one chromosome to affect the expression of the homologous gene in a somatically paired chromosome. The z1 mutation causes a pairing dependent inhibition of the expression of the white gene. Both of these phenomena can be explained by the tendency of zeste protein, expressed in bacteria or in flies, to self-associate, forming complexes of several hundred monomers. These large aggregates bind to DNA and are found in nuclear matrix preparations, probably because they co-sediment with the matrix. The principal determinants of this self-association are located in the C-terminal half of the protein but some limited aggregation is obtained also with the N-terminal half, which contains the DNA binding domain. The z1 and zop2 mutant proteins aggregate to the same degree as the wild type but the z11G3 product, a pseudorevertant of z1, has a reduced tendency to aggregate. This mutation, which in vivo is antagonistic to z1 and does not support transvection effects, can be made to revert its phenotype when the mutant protein is over-produced under the control of the heat shock promoter. These results indicate that both the zeste-white interaction and transvection effects require the formation of high order aggregates. When the z1 protein is over-produced in vivo, it reduces the expression of an unpaired copy of white, indicating that the normal requirement for chromosome pairing is simply a device to increase the size of the aggregate bound to the white regulatory region.
Amino Acid Sequence↗
[Molecular-genetic analysis of of the band 10A1-2 in the Drosophila melanogaster chromosome X].
With the help of in situ hybridization respective location of 33 microclones from the genomic library of the Drosophila melanogaster large band 10A1-2, 4 clones obtained by chromosomal walking and 18 chromosomal rearrangements with breakpoints in the limits of this band was determined. A DNA fragment homologous to poly(A)+ RNA from the 3rd instar larvae has been revealed. Summarizing the obtained and published data, at least 3 genes and 6 transcriptionally active fragments appear to be located in the 10A1-2 band. Using DNA clones from different regions of Drosophila melanogaster 10A1-2 as probes in some Diptera species, the 10A1-2 distal clone, carrying vermilion gene, in D. virilis was shown to be located in a very thin band of the 5A region, while the proximal clones of the 10A1-2 band were mapped in a large band of the 2B region. In D. paranaensis a sequence homologous to the vermilion gene DNA was mapped in a large band, whereas the proximal clones of the 10A1-2 band were localized in a different region. These results evidence for the fact that DNA sequences are not evolutionary fixed, i. e. in different species they may belong to different chromomeres.
Animals↗
The claret locus in Drosophila encodes products required for eyecolor and for meiotic chromosome segregation.
The claret (ca) locus in Drosophila encodes products that are needed both for wild-type eyecolor and for correct meiotic chromosome segregation. Mutants described previously provide evidence that two mutationally independent coding regions are present at ca. We have recovered six new P element-induced and one spontaneous ca mutant. Four of these new mutants affect both eyecolor and chromosome segregation. The high frequency of co-mutation of these two functions suggests that the corresponding genes are closely adjacent to one another. We recovered genomic DNA sequences corresponding to the ca locus by chromosome walking, and showed using revertant analysis that the cloned region encodes ca+. Transformation experiments demonstrate that the mutant effect resulting in meiotic chromosome non-disjunction (nd) and loss is fully rescued by DNA from the cloned region. Two RNAs of 7.4 and 2.2 kb have been identified by Northern blot analysis as the putative eyecolor and segregational products. Expression of the RNAs with respect to males and females, and their presence or absence in ca and nd mutants indicate that the 7.4 kb RNA corresponds to the product needed for wild-type eyecolor and the 2.2 kb RNA is the product required for normal chromosome segregation. These RNAs are transcribed in opposite directions to one another. Alleles that affect both eyecolor and chromosome segregation are deletion mutants that affect both transcripts. Thus, the putative eyecolor and segregational products are encoded by separate genes. Mutants that affect both eyecolor and chromosome segregation apparently do so because they delete essential regions of both genes.
Alleles↗
A novel spatial transcription pattern associated with the segmentation gene, giant, of Drosophila.
The segmentation gene, giant, is located in 3A1 within a cloned chromosome region surrounding the zeste locus. Rearrangement breakpoints associated with giant mutations were localized on the genomic clone map, and nearby transcription units were identified. One transcription unit is active during early embryogenesis and its transcripts are spatially localized from blastoderm into extended germband stages, consistent with expected expression patterns predicted by the 'gap' phenotype of giant mutants. Germ line transformation experiments using a 10-kb DNA fragment containing this transcription unit gave complete rescue of the abdominal giant defect but only partial correction of the head defect. The effect of mutations in three other gap loci, Kr, kni and hb, were also analyzed.
Alleles↗
The Drosophila zeste protein binds cooperatively to sites in many gene regulatory regions: implications for transvection and gene regulation.
The Drosophila zeste protein binds in vitro to several sites in the white, Ultrabithorax, decapentaplegic, Antennapedia, and engrailed genes and to at least one site in the zeste gene itself. The distribution of these sites corresponds often with that of regulatory elements in these genes as defined by mutations or, in the case of white, by molecular analysis. A zeste binding site is frequently found in the immediate vicinity of the promoter. zeste binding sites are composed of two or more zeste recognition sequences T/CGAGT/CG. Isolated consensus sequences do not bind or footprint. Cooperative interactions are involved both in binding to a given site and between proteins bound at independent sites. zeste bound to one DNA molecule can in fact bind simultaneously to another DNA molecule. These results suggest a general role for zeste in bringing together distant regulatory elements controlling the activity of a target gene. In this model, transvection effects are a by-product of normal intragenic zeste action.
Animals↗
Zeste encodes a sequence-specific transcription factor that activates the Ultrabithorax promoter in vitro.
Zeste is a Drosophila regulatory gene that is required for transvection at the bithorax complex. Here we find that purified zeste protein binds to multiple sites just 5' of the initiation site of Ubx RNA. Zeste protein purified from Drosophila cells or from E. coli expressing the zeste gene activates Ubx transcription in vitro. This activation is dependent on the presence of zeste protein binding sites, as it is not observed with a Ubx promoter lacking these sites or with an Adh promoter. These results suggest that transvection involves regulatory elements that act at the level of transcriptional initiation and may be mechanistically similar to activation of transcription by enhancer elements, except that transvection occurs across paired chromosomes. These findings are consistent with the hypothesis that zeste may play a more important role in the normal regulation of Ubx and its other target genes than current genetic evidence implies.
Animals↗
Vectors for P-mediated transformation in Drosophila.
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Animals↗
Microcloning reveals a high frequency of repetitive sequences characteristic of chromosome 4 and the beta-heterochromatin of Drosophila melanogaster.
Microdissection and microcloning of the euchromatin-heterochromatin transition region of the Drosophila melanogaster polytene X chromosome and part of the euchromatin of chromosome 4 reveals that they share certain features characteristic of beta-heterochromatin, which is morphologically defined as the loosely textured material at the bases of some polytene chromosome arms. Both are mosaics of many different middle-repetitive DNA sequences interspersed with single-copy DNA sequences. Sixty percent of cloned inserts derived from division 20 and about 40 percent from subdivisions 19EF of the X chromosome harbor at least one repetitive DNA sequence in an average insert of 4.5 kilobases. No repeats have significant cross-hybridization to any of the eleven satellite DNAs, or to the clustered-scrambled sequences present in pDm1. The repetitive elements are, in general, confined to the beta-heterochromatic regions of polytene chromosomes, but some are adjacent to nomadic elements. Chromosome 4, however, has some repeats spread throughout its entire euchromatin. These data have implications for the structure of transition zones between euchromatin and heterochromatin of mitotic chromosomes and also provide a molecular basis for reexamining some of the unusual classical properties of chromosome 4.
Animals↗
Developmental expression of the Drosophila zeste gene and localization of zeste protein on polytene chromosomes.
The expression of the zeste gene varies through the life cycle of the fly. Its transcription is most abundant in maternal RNA, declines to very low levels during larval growth, but rises again in late third instar larvae and pupae. Using transposons containing a zeste-lacZ gene, we found a corresponding variation in the tissue distribution of zeste from stage to stage. Nearly ubiquitous expression of the zeste-lacZ gene is found in late embryos and first instar larvae, but disappears almost completely except in brain and gonads by third instar larva. Shortly before pupation expression rises again in imaginal discs, Malpighian tubules, and salivary glands and again becomes nearly ubiquitous in pupae. zeste continues to be expressed in adult brain and gonads. We constructed flies carrying a zeste gene controlled by the heat shock promoter and studied the distribution of zeste protein in their polytene chromosomes as well as those of wild-type flies. Using affinity-purified anti-zeste antibodies, we find that wild-type salivary gland chromosomes contain about 60 strong bands of zeste immunofluorescence at specific cytological locations. After heat induction of larvae containing the hs-zeste gene, many hundreds of bands appear. These results suggest the involvement of zeste in the expression of a wide variety of genes at different developmental stages.
Animals↗
The tko locus, site of a behavioral mutation in D. melanogaster, codes for a protein homologous to prokaryotic ribosomal protein S12.
The tko (technical knockout) mutation is one of a family of behavioral mutations that cause "bang sensitivity" in D. melanogaster. Using P-element-mediated transformation, we show that a 3.1 kb piece of genomic DNA complements tko. This fragment contains only one complete transcript, 0.68 kb in length. This transcript is abundantly expressed through all stages of the life cycle, and we have isolated cDNAs corresponding to this transcript. Their sequence implies a protein product composed of 140 amino acids, which exhibits considerable sequence similarity to ribosomal protein S12 from both Euglena gracilis chloroplasts and E. coli. We suggest that tko codes for a mitochondrial ribosomal protein and that the tko phenotype results from defective mitochondria.
Amino Acid Sequence↗
The product of the Drosophila zeste gene binds to specific DNA sequences in white and Ubx.
Three different segments of the zeste coding sequence were inserted in an expression vector and antibodies were raised against the resulting zeste-beta galactosidase hybrid proteins. The antibodies were used to analyse the zeste protein produced in bacteria from a different expression vector containing the entire zeste coding region. The major products made in bacteria as well as the products of in vitro translation of zeste RNA migrate anomalously upon SDS--acrylamide gel electrophoresis. Specific DNA fragments from the white and Ubx gene co-immunoprecipitate with zeste protein. At least two independent zeste binding sites are found in a 250-bp interval of the white regulatory region that contains also the sites of wsp mutations, which are known to be deficient in zeste interaction.
Animals↗
Structure and sequence of the Drosophila zeste gene.
The zeste gene of Drosophila affects the expression of other genes in a manner that depends on the homologous pairing of the chromosomes bearing the target gene. Zeste mediates transvection effects, the ability of one gene to control the expression of its homologous copy on another chromosome. We have determined the structure of the zeste gene and several mutants bearing partial deletions and the sequence of the z+, z1, zop6 and z11G3 alleles. The predicted zeste protein has an unusual structure including runs of Gln, Ala and alternating Gln Ala. Contrary to expectations the z1, zop6 and z11G3 mutations can each be attributed to single amino acid changes. The analysis of the mutants suggests that the zeste gene product is required for normal expression of at least some genes and we argue that za mutants may have residual function.
Amino Acid Sequence↗
The sex-determining gene tra of Drosophila: molecular cloning and transformation studies.
In Drosophila, the primary signal for sex determination is given by the ratio of X chromosomes to sets of autosomes (X:A). The primary signal is read by a key gene (Sxl) and transmitted down to the differentiation genes by the subordinate control genes tra, tra-2, ix and dsx. Mutations in tra transform chromosomal females (X/X; tra/tra) into sterile males (pseudomales). We have cloned the tra region by microdissection and chromosomal walking. We identified the gene using deficiency breakpoints, DNA aberrations in three different alleles of tra and by P-mediated transformation. A 3.8-kb fragment perfectly rescued the mutant phenotype of X/X; tra/tra flies, showing that it contained all the necessary information to restore female-specific functions in the mutant flies. We present evidence that most of the function of tra can be provided by a subsegment of 2 kb that is differentially transcribed or processed in males and females.
Animals↗
P transposons controlled by the heat shock promoter.
We have transformed Drosophila melanogaster with modified P-element transposons, which express the transposase function from the heat-inducible hsp70 heat shock promoter. The Icarus transposon, which contains a direct hsp70-P fusion gene, behaved like a very active autonomous P element even before heat shock induction. Although heat shock led to abundant somatic transcription, transposition of the Icarus element was confined to germ line cells. To reduce the constitutive transposase activity observed for the Icarus element, we attenuated the translational efficiency of the transposase RNA by inserting the transposon 5 neomycin resistance gene between the hsp70 promoter and the P-element sequences. The resulting construct, called Icarus-neo, conferred resistance to G418, and its transposition was significantly stimulated by heat shock. Heat shocks applied during the embryonic or third instar larval stage had similar effects, indicating that transposition of P elements is not restricted to a certain developmental stage. Both Icarus and Icarus-neo destabilized snw in a P-cytotype background and thus at least partially overcome the repression of transposition. Our results suggest that the regulation of P-element transposition occurs at both the transcriptional and posttranscriptional levels.
Animals↗
Expression of the Drosophila white gene under the control of the hsp70 heat shock promoter.
We have used P-mediated transformation to introduce into the Drosophila genome transposons containing the hsp-70 heat shock promoter (hsp) placed at different distances upstream of the white gene. Using these constructions we found a strong terminator 250 nucleotides upstream of the white transcription start. When all white upstream sequences are deleted and the hsp is fused to the white leader region, white transcription is under hsp control, however, flies carrying this construction have red eyes even at low temperature, due to the basal level of activity of the hsp. High levels of transcription are obtained after heat shock but about half of the transcripts are incompletely spliced. One transformed line carries the transposon integrated near the heterochromatin. These flies have very pale eyes but develop strongly pigmented, variegated eyes if heat shocked in the first 2 days of the pupal stage. Heat shocks after eclosion have no effect on eye pigmentation. The properties of these transposons also indicated that the control sequences required for Zeste interaction lie > 1 kb upstream of the white transcription start while those required for dosage compensation are close to the gene.
Journal Article↗