Covariance among premating, post-copulatory and viability fitness components in Drosophila melanogaster and their influence on paternity measurement.

In polyandrous mating systems, male fitness depends on success in premating, post-copulatory and offspring viability episodes of selection. We tracked male success across all of these episodes simultaneously, using transgenic Drosophila melanogaster with ubiquitously expressed green fluorescent protein (i.e. GFP) in a series of competitive and noncompetitive matings. This approach permitted us to track paternity-specific viability over all life stages and to distinguish true competitive fertilization success from differential early offspring viability. Relationships between episodes of selection were generally not present when paternity was measured in eggs; however, positive correlations between sperm competitive success and offspring viability became significant when paternity was measured in adult offspring. Additionally, we found a significant male × female interaction on hatching success and a lack of repeatability of offspring viability across a focal male's matings, which may underlay the limited number of correlations found between episodes of selection.

Duplicated proteasome subunit genes in Drosophila and their roles in spermatogenesis.

The proteasome is a large, multisubunit complex that acts as the cell's 'protein-degrading machine' in the ubiquitin-mediated proteolytic pathway for regulated protein turnover. Although proteasomes are usually thought of as being homogeneous structures, recent studies have revealed their more dynamic and heterogeneous nature. For example, in a number of plant and animal species, multiple isoforms of several proteasome subunits, encoded by paralogous genes, have been discovered, and in some cases, these alternative isoforms have been shown to be functionally distinct from their conventional counterparts. A particularly striking example of this phenomenon is seen in Drosophila melanogaster, where 12 of the 33 subunits that make up the 26S proteasome holoenzyme are represented in the genome by multiple paralogous genes. Remarkably, in every case, the 'extra' genes are expressed in a testis-specific manner. Here, we describe the extent and nature of these testis-specific gene duplications and discuss their functional significance, and speculate on why this situation might have evolved.

Expression of proteasome subunit isoforms during spermatogenesis in Drosophila melanogaster.

In this study, we sought to identify and characterize all the proteasome genes of Drosophila melanogaster. Earlier work led to the identification of two genes encoding alpha4-type 20S proteasome subunit isoforms that are expressed exclusively in the male germline. Here we extend these results and show that six of the 20S proteasome subunits, and four of the 19S regulatory cap subunits, have gene duplications encoding male-specific isoforms. More detailed analyses of two of these male-specific subunits (Prosalpha3T and Prosalpha6T), using GFP-tagged reporter transgenes, revealed that they are predominantly localized to the nucleus at later stages of spermatogenesis and are present there in mature, motile sperm. These results suggest a possible role of a 'spermatogenesis-specific' proteasome in sperm differentiation and/or function.

Temperature-dependent gene silencing by an expressed inverted repeat in Drosophila.

Posttranscriptional gene silencing (PTGS) induced by double-stranded RNA (dsRNA) is an intriguing phenomenon that has been observed in a variety of organisms, including Drosophila melanogaster. Although PTGS in Drosophila is typically observed following direct injection of the dsRNA into embryos, it is theoretically possible that the in vivo transcription of an inverted repeat transgene might also produce a dsRNA "hairpin" that is capable of triggering PTGS. Here we test this idea, and show that an expressed inverted repeat of a portion of the sex differentiation gene, transformer-2, (tra-2), driven by a GAL4-dependent promoter, does genetically repress the endogenous wild-type tra-2 function, producing a dominant loss-of-function mutant phenotype. Remarkably, this effect is temperature-sensitive, with phenotypic consequences seen at 29 degrees, but not at 22 degrees. Moreover, by altering the dosage of either the transgenes or the endogenous tra2(+) loci, one can vary the effect over a wide range of mutant phenotypes.

Subunit compositions and catalytic properties of proteasomes from developmental temperature- sensitive mutants of Drosophila melanogaster.

Two dominant temperature-sensitive (DTS) Drosophila mutants are missense mutations of proteasome genes encoding beta-type subunits beta6/C5 (DTS5) and beta2/Z (DTS7). At nonpermissive temperature (29 degrees C), heterozygotes (DTS5/+ and DTS7/+) develop normally until metamorphosis; pupae fail to mature and die before eclosion. Proteasomes were purified from wild-type (WT) and heterozygous adult flies raised at permissive temperature (25 degrees C). Two-dimensional gel electrophoresis separated at least 28 proteins, 13 of which were identified with monospecific antibodies to alpha6/C2 (five species), alpha2/C3 (three species), alpha7/C8 (three species), alpha5/zeta, and beta1/Y subunits. Both quantitative and qualitative differences were observed between WT and DTS/+ proteasomes, with DTS5/+ deviating more from WT than DTS7/+ proteasomes. In DTS5/+ there was a shift to more acidic species of C2 and C3 and a shift to less acidic species of 32-kDa subunits (#3-#7) recognized by an anti-alpha subunit monoclonal antibody (MCP222) and were losses of two 32-kDa subunits (#2 and #3), decreases in Y (25 kDa; 2-fold) and 31-kDa (#9; 2-fold) subunits, and increases in 52-kDa (#1; 1.9-fold) and 24-kDa (#13; 2.3-fold) subunits. In DTS7/+ there was a less pronounced shift to acidic species of C3 and no pI shift in C2 species and subunits #3-#7 and were decreases in #9 (2.5-fold) and #14 (3-fold) and a loss of #2. The three C8 species were similar between WT, DTS5/+, and DTS7/+ proteasomes. Qualitatively, the most dramatic difference was the appearance of a new 24-kDa subunit (#16) in DTS/+ preparations, with about a 14-fold greater amount of #16 in DTS7/+ than in DTS5/+ proteasomes. Catalytically, WT and DTS/+ proteasomes had similar peptidase activities, although the DTS/+ proteasomes were slightly more sensitive to SDS and elevated temperatures in vitro. The incorporation of DTS subunits apparently altered proteasome assembly and/or processing at permissive temperature with little effect on catalytic activities. These data suggest that at nonpermissive temperature, assembly/processing is more severely affected, producing DTS-containing complexes that lack functions essential for cellular proliferation and differentiation at metamorphosis.

The dominant temperature-sensitive lethal DTS7 of Drosophila melanogaster encodes an altered 20S proteasome beta-type subunit.

Proteasomes are multicatalytic complexes that function as the major proteolytic machinery in regulated protein degradation. The eukaryotic 20S proteasome proteolytic core structure comprises 14 different subunits: 7 alpha-type and 7 beta-type. DTS7 is a dominant temperature-sensitive (DTS) lethal mutation at 29 degrees that also acts as a recessive lethal at ambient temperatures. DTS7 maps to cytological position 71AB. Molecular characterization of DTS7 reveals that this is caused by a missense mutation in a beta-type subunit gene, beta2. A previously characterized DTS mutant, l(3)73Ai1, results from a missense mutation in another beta-type subunit gene, beta6. These two mutants share a very similar phenotype, show a strong allele-specific genetic interaction, and are rescued by the same extragenic suppressor, Su(DTS)-1. We propose that these mutants might act as "poison subunits," disrupting proteasome function in a dosage-dependent manner, and suggest how they may interact on the basis of the structure of the yeast 20S proteasome.

Cloning and characterization of Pros45, the Drosophila SUG1 proteasome subunit homolog.

The proteasome plays essential roles in a variety of cellular processes, including degradation of the bulk of cellular proteins, degradation of short-lived proteins such as cell cycle regulators, generation of antigenic peptides, and mediating programmed cell death. One of the best characterized subunits of the 26S proteasome is encoded by the yeast gene SUG1. We report here the cloning and characterization of the Drosophila homolog of this gene, Pros45. At the protein level, Pros45 is highly conserved with respect to its homologs in a variety of taxa: it shows 74% identity to yeast Sug1; 86% to mouse m56/mSug1/FZA-B; 87% to human Trip1; and 97% to moth 18-56. Using a genomic clone as a probe for in situ hyridization to polytene chromesomes, we demonstrated that Pros45 maps to 19F, near the base of the X chromosome. Use of a pros45 cDNA clone as a probe revealed a second site of hybridization at 99CD. Pros45 mRNA is found in the unfertilized egg and in all cells of the early embryo. By the end of embryogenesis, Pros45 is expressed predominantly in the central nervous system. Targeted expression of Pros45 in a variety of different cells using the Gal4 UAS P-element system failed to generate an overt phenotype. This study provides the foundation for further examination of the role of the 26S proteasome in homeostasis and development in Drosophila.

Evolutionary conservation of a testes-specific proteasome subunit gene in Drosophila.

Proteasomes are large multisubunit particles that act as the proteolytic machinery for the ubiquitin-dependent proteolytic pathway. The core of this complex, the 20S proteasome, is made up of seven alpha-type and seven beta-type subunits, arranged in an (alpha1-alpha7)(beta1-beta7)(beta1-beta7)(alpha1-al pha7) configuration. Previous work had shown that there exist alternative isoforms of the Drosophila melanogaster alpha4-type subunit, encoded by two distinct genes, alpha4t1_dm and alpha4t2_dm, and that these are expressed exclusively in the germline of the testes. We sought to investigate the evolutionary conservation of this phenomenon by screening for orthologs of the alpha4-type gene family in the distantly related Drosophila species, D. virilis. We isolated the D. virilis orthologs of the somatically expressed gene, alpha4_dm, and the testes-specific gene, alpha4t2_dm. We failed to find an ortholog of the other testes-specific gene, alpha4t1_dm. The alpha4_dv gene maps to the X chromosome at 12A-C, its product shares 90% amino acid identity with alpha4_dm, and it is expressed at high levels in both males and females. The other gene, alpha4t_dv, encodes a protein most similar to the testes-specific alpha4t2_dm proteasome subunit (59% a.a. identity), and it maps to position 27 on chomosome 2. The expression of the alpha4t_dv gene is testes-specific, like that of alpha4t2_dm. The existence of testes-specific alpha4-type subunits in two widely diverged subgenera of Drosophila suggests that these subunit isoforms have important functional roles in spermatogenesis.

Cloning of Drosophila GCN5: conserved features among metazoan GCN5 family members.

PCAF and hGCN5 are distinct human genes that encode proteins related to the yeast histone acetyltransferase and transcriptional adapter GCN5. The PCAF protein shares extensive similarity with the 439 amino acids of yGCN5, but it has an approximately 350 amino acid N-terminal extension that interacts with the transcriptional co-activator p300/CBP. Adenoviral protein E1a can disrupt PCAF-CBP interactions and prevent PCAF-dependent cellular differentiation. In this report, we describe the cloning and initial characterization of a Drosophila homolog of yGCN5. In addition to the homology to yGCN5, the Drosophila protein shares sequencesimilarity with the N-terminal portion of human PCAF that is involved in binding to CBP. In the course of characterizing dGCN5, we have discovered that hGCN5 also contains an N-terminal extension with significant similarity to PCAF. Interestingly, in the case of the h GCN5 gene, alternative splicing may regulate the production of full-length hGCN5. The presence of the N-terminal domain in a Drosophila GCN5 homolog and both human homologs suggests that it was part of the ancestral form of metazoan GCN5.

Molecular cloning of the Drosophila melanogaster gene alpha5_dm encoding a 20S proteasome alpha-type subunit.

Proteasomes are large, multisubunit particles that act as the proteolytic machinery for most regulated intracellular protein breakdown in eukaryotic cells. The core proteinase of this complex, known as the 20S proteasome, is a hollow barrel-shaped structure made up of four stacked rings of seven subunits each, with the outer two rings each being made up of seven distinct alpha-type subunits, and the two inner rings composed of seven different beta-type subunits. Here we present the cloning, sequencing and genetic mapping of a Drosophila melanogaster gene, alpha5_dm, encoding one of the proteasome alpha subunits. This gene, which is homologous to the yeast PUP2 and the human Zeta genes, maps to chromosome 2 at position 54B3-5. The map positions of the previously cloned proteasome genes Pros25 and Pros29 were also determined, and found to lie at positions 87B and 57B, respectively. A search for other D. melanogaster alpha5_dm-like genes encoding potential isoforms of this subunit failed to identify any closely related genes.

Duplicated proteasome subunit genes in Drosophila melanogaster encoding testes-specific isoforms.

Using the previously cloned proteasome alpha-type subunit gene Pros28.1, we screened a Drosophila melanogaster genomic library using reduced stringency conditions to identify closely related genes. Two new genes, Pros28.1A (map position 92F) and Pros28.1B (map position 60D7), showing high sequence similarity to Pros28.1, were identified and characterized. Pros28.1A encodes a protein with 74% amino acid identity to PROS28.1, while the Pros28.1B gene product is 58% identical. The Pros28.1B gene has two introns, located in exactly analogous positions as the two introns in Pros28.1, while the Pros28.1A gene lacks introns. Northern blot analysis reveals that the two new genes are expressed only in males, during the pupal and adult stages. Tissue-specific patterns of expression were examined using transgenic flies carrying lacz-fusion reporter genes. This analysis revealed that both genes are expressed in germline cells during spermatogenesis, although their expression patterns differed. Pros28.1A expression is first detected at the primary spermatocyte stage and persists into the spermatid elongation phase of spermiogenesis, while Pros28.1B expression is prominent only during spermatid elongation. These genes represent the most striking example of cell-type-specific proteasome gene expression reported to date in any system and support the notion that there is structural and functional heterogeneity among proteasomes in metazoans.

Protein-protein interactions among components of the Drosophila primary sex determination signal.

Sex determination in Drosophila melanogaster is initiated in the early embryo by a signal provided by three types of genes: (1) X-linked numerator elements [e.g., sisterless-a (sis-a) and sisterless-b (sis-b)], (2) autosomally linked denominator elements [e.g., deadpan (dpn)], and (3) maternal factors [e.g., daughterless (da)]. This signal acts to stimulate transcription from an embryo-specific promoter of the master regulatory gene Sex-lethal (Sxl) in embryos that have two X chromosomes (females), while it fails to activate Sxl in those with only one X (males). It has been previously proposed that competitive dimerizations among the components of this signal might provide the molecular basis for this sex specificity. Here, we use the yeast two-hybrid system to demonstrate specific protein-protein interactions among the above-mentioned factors, and to delimit their interacting domains. These results support and extend the model of the molecular basis of the X/A ratio signal.

Isolation of the rat F1-ATPase inhibitor gene and its pseudogenes.

Multiple mitochondrial ATPase inhibitor genes have been identified in the rat-genome. The sequences of two genomic clones indicate that one encodes the functional gene, and the other is a processed pseudogene. The ATPase inhibitor gene isolated is about 1.5 kb long and the coding region contains three exons and two introns. The presence of multiple pseudogenes in the rat is suggested by this study and this is unique since in the bovine genome only a single gene has been found, which is also confirmed here. The presence of multiple inhibitor transcripts in the rat suggests that the functional gene might have multiple transcriptional start sites.

Determination of the molecular lesions associated with loss-of-function transformer alleles of Drosophila melanogaster.

Three mutant alleles of the transformer locus, trav1, trav2 and traenu, were analyzed genetically, and all were found to be functionally null alleles. The DNA sequences of these mutants revealed that they represent nonsense mutations that potentially encode truncated Tra polypeptides of 12, 61, and 90 amino acids, respectively.

Identification of an essential gene, l(3)73Ai, with a dominant temperature-sensitive lethal allele, encoding a Drosophila proteasome subunit.

Proteasomes are multicatalytic proteinase complexes that function as a major nonlysosomal proteolytic system in all eukaryotes. These particles are made up of 13-15 nonidentical subunits, and they exhibit multiple endopeptidase activities that promote the intracellular turnover of abnormal polypeptides and short-lived regulatory proteins. Although the biochemical characterization of proteasomes has been quite extensive, and although a number of the genes encoding proteasome subunits have been cloned from various organisms, there is still much to be learned about their function in vivo and what role(s) they might play during development. Here, we report the identification of the l(3)73Ai1 allele of Drosophila melanogaster as a dominant temperature-sensitive lethal mutation in a gene encoding a component of the proteasome, thus opening the way for future genetic and developmental studies on this important proteolytic system in a higher eukaryote.

Interspecific comparison of the transformer gene of Drosophila reveals an unusually high degree of evolutionary divergence.

The transformer (tra) gene of Drosophila melanogaster occupies an intermediate position in the regulatory pathway controlling all aspects of somatic sexual differentiation. The female-specific expression of this gene's function is regulated by the Sex lethal (Sxl) gene, through a mechanism involving sex-specific alternative splicing of tra pre-mRNA. The tra gene encodes a protein that is thought to act in conjunction with the transformer-2 (tra-2) gene product to control the sex-specific processing of doublesex (dsx) pre-mRNA. The bifunctional dsx gene carries out opposite functions in the two sexes, repressing female differentiation in males and repressing male differentiation in females. Here we report the results from an evolutionary approach to investigate tra regulation and function, by isolating the tra-homologous genes from selected Drosophila species, and then using the interspecific DNA sequence comparisons to help identify regions of functional significance. The tra-homologous genes from two Sophophoran subgenus species, Drosophila simulans and Drosophila erecta, and two Drosophila subgenus species, Drosophila hydei and Drosophila virilis, were cloned, sequenced and compared to the D. melanogaster tra gene. This comparison reveals an unusually high degree of evolutionary divergence among the tra coding sequences. These studies also highlight a highly conserved sequence within intron one that probably defines a cis-acting regulator of the sex-specific alternative splicing event.

Cytogenetic analysis of chromosome region 73AD of Drosophila melanogaster.

The 73AD salivary chromosome region of Drosophila melanogaster was subjected to mutational analysis in order to (1) generate a collection of chromosome breakpoints that would allow a correlation between the genetic, cytological and molecular maps of the region and (2) define the number and gross organization of complementation groups within this interval. Eighteen complementation groups were defined and mapped to the 73A2-73B7 region, which is comprised of 17 polytene bands. These complementation groups include the previously known scarlet (st), transformer (tra) and Dominant temperature-sensitive lethal-5 (DTS-5) genes, as well as 13 new recessive lethal complementation groups and one male and female sterile locus. One of the newly identified lethal complementation groups corresponds to the molecularly identified abl locus, and another gene is defined by mutant alleles that exhibit an interaction with the abl mutants. We also recovered several mutations in the 73C1-D1.2 interval, representing two lethal complementation groups, one new visible mutant, plucked (plk), and a previously known visible, dark body (db). There is no evidence of a complex of sex determination genes in the region near tra.

The control of sexual development in Drosophila melanogaster: genetic and molecular analysis of a genetic regulatory hierarchy--a minireview.

Genetic analyses of mutants affecting the sexual development of the fruitfly, Drosophila melanogaster, have led to a genetic model that describes how the wild-type genes interact with one another as parts of a single regulatory pathway. More recent molecular studies on some of these sex differentiation regulatory genes have lent support to this genetic model, and have defined the molecular nature of some of these genetic interactions. One surprising feature of the regulatory hierarchy to emerge from these molecular studies is that many of the sex-specific genetic switches in this pathway are not transcriptional regulation events but, rather, are based on sex-specific alternative RNA processing events (pre-mRNA splicing and/or polyadenylation site selection).

Sex-specific alternative splicing of RNA from the transformer gene results from sequence-dependent splice site blockage.

Sex-specific alternative splicing of RNA from the Drosophila transformer gene involves competition between two 3' splice sites. In the absence of Sex-lethal activity (as in males), only one site functions; in the presence of Sex-lethal activity (as in females), both sites function. Information for sex-specific splice site choice is contained within the intron itself. Deletions of the splice site used in males lead to Sex-lethal-independent use of the otherwise female-specific site. The relative amounts of unspliced and spliced RNA derived from these mutant genes do not change with changes in Sex-lethal activity. Specific nucleotide changes in the non-sex-specific splice site do not affect splicing activity but eliminate Sex-lethal-induced regulation. A deletion removing material between the two splice sites does not eliminate sex-specific regulation, while a deletion of the female splice site leads to a female-specific increase in unspliced RNA. These results are consistent with a model in which female-specific factors block the function of the non-sex-specific 3' splice site.

Cloning and characterization of the scarlet gene of Drosophila melanogaster.

DNA from the scarlet (st) region of Drosophila melanogaster has been cloned by chromosome walking, using the breakpoints of a new X-ray-induced third chromosome inversion (In(3LR)st-a27) which breaks in the scarlet (73A3.4) and rosy (87D13-14) regions. Two spontaneous mutants of st(st1 and stsp) contain insertions of non-st DNA located within 3.0 kb of the site of the inversion breakpoint used to isolate the gene, and a second scarlet inversion breaks within 6.5 kb of this site. However no changes detectable by Southern blotting were found in 5 X-ray-induced st mutants with cytologically normal third chromosomes. A 2.3-kb transcript arising from the st gene region (as defined by mutant analysis and DNA transformation) has been detected. This transcript is present throughout development at low levels, with a peak level during the early to mid-pupal stage. The size and amount of this transcript is altered in st1, and its amount is drastically reduced in stsp. Flies carrying the white1 mutation show normal levels of expression of the st transcript, suggesting that the w+ gene does not regulate transcription of the st+ gene. Nucleotide homology between sequences from the st transcription unit and a fragment carrying coding information from the white gene has been detected. This suggests that the st and w proteins are related; they appear to belong to a family of membrane-spanning, ATP-binding proteins involved in the transport of pigment precursors into cells.