Egg production and fertility in Drosophila depend upon the number of yolk-protein gene copies

Summary The yolk proteins of Drosophila melanogaster comprise a family of three related yolk polypeptides each encoded by a single-copy gene. We show by genetic crosses that each gene makes an equivalent contribution to the fecundity and fertility of the female and they do not individually provide unique functions to the embryo. We show that the number of eggs laid by a female depends upon the number of genes encoding yolk polypeptides present in the genome and furthermore that the probability of an egg hatching into an adult also critically depends upon the number of yolk protein genes present in the mother. This suggests that the three yolk protein-encoding genes in Drosophila melanogaster may have arisen by duplication, then been maintained for quantitative reasons because they increased egg production and fertility, rather than each protein evolving a different function as is the case with most small gene families, such as tubulins and collagen genes.     

Evolution of DNA in Heterochromatin: the Drosophila Melanogaster Sibling Species Subgroup as a Resource

The Drosophila melanogasterspecies subgroup is a closely-knit collection of eight sibling species whose relationships are well defined. These species are too close for most evolutionary studies of euchromatic genes but are ideal to investigate the major changes that occur to DNA in heterochromatin over short periods during evolution. For example, it is not known whether the locations of genes in heterochromatin are conserved over this time. The 18S and 28S ribosomal RNA genes can be considered as genuine heterochromatic genes. In D. melanogasterthe rRNA genes are located at two sites, one each on the X and Y chromosome. In the other seven sibling species, rRNA genes are also located on the sex chromosomes but the positions often vary significantly, particularly on the Y. Furthermore, rDNA has been lost from the Y chromosome of both D. simulansand D. sechellia, presumably after separation of the line leading to present-day D. mauritiana.We conclude that changes to chromosomal position and copy number of rDNA arrays occur over much shorter evolutionary timespans than previously thought. In these respects the rDNA behaves more like the tandemly repeated satellite DNAs than euchromatic genes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Hybrid lethal systems in theDrosophila melanogaster species complex

Lethal phases of the hybrids betweenDrosophila melanogaster and its sibling species,D. simulans are classified into three types: (1) embryonic lethality in hybrids carryingD. simulans cytoplasm andD. melanogaster X chromosome, (2) larval lethality in hybrids not carryingD. simulans X, and (3) temperature-sensitive pupal lethality in hybrids carryingD. simulans X. The same lethal phases are also observed when either of the two other sibling species,D. mauritiana orD. sechellia, is employed for hybridization withD. melanogaster. Here, we describe genetic analyses of each hybrid lethality, and demonstrate that these three types of lethality are independent phenomena. We then propose two models to interpret the mechanisms of each hybrid lethality. The first model is a modification of the conventional X/autosome imbalance hypothesis assuming a lethal gene and a suppressor gene are involved in the larval lethality, while the second model is for embryonic lethality assuming an interaction between a maternal-effect lethal gene and a suppressor gene.     

A Genomic Comparison of Faster-Sex, Faster-X, and Faster-Male Evolution Between Drosophila melanogaster and Drosophila pseudoobscura

A genomic comparison of Drosophila melanogaster and Drosophila pseudoobscura provides a unique opportunity to investigate factors involved in sequence divergence. The chromosomal arrangements of these species include an autosomal segment in D. melanogaster which is homologous to part of the X chromosome in D. pseudoobscura. Using orthologues to calculate rates of nonsynonymous (d_N) substitutions, we found genes on the X chromosome to be significantly more diverged than those on the autosomes, but it is not true for segment 3L-XR which is autosomal in D. melanogaster (3L) and X-linked in D. pseudoobscura (XR). We also found that the median d_N values for genes having reproductive functions in either the male, the female, or both sexes are higher than those for sequences without reproductive function and even higher for sequences involved in male-specific function. These estimates of divergence for male sex-related sequences are most likely underestimates, as the very rapidly evolving reproductive genes would tend to lose homology sooner and thus not be included in the comparison of orthologues. We also noticed a high proportion of male reproductive genes among the othologous genes with the highest rates of d_N. Reproductive genes with and without an orthologue in D. pseudoobscura were compared among D. melanogaster, D. simulans, and D. yakuba and it was found that there were in fact higher rates of divergence in the group without a D. pseudoobscura orthologue. These results, from widely separated taxa, bolster the thesis that sexual system genes experience accelerated rates of change in comparison to nonsexual genes in evolution and speciation. [Reviewing Editor: Dr. Willie J. Swanson]     

Organization and expression of mouse Hox3 cluster genes

Summary The three yolk protein genes (yp) of Drosophila melanogaster are transcribed in a sex- and tissue-limited fashion. We have searched for cis-regulatory sequences in regions flanking yp1 and yp2 to identify the elements that confer female-specific expression in the fat body. One such 127 by element has previously been identified in this region. We show here the existence of two additional regions which confer female fat body-specific expression on an Adh reporter gene and on the native yp2 gene, respectively. This suggests some redundancy in the regulation of expression of the yp genes. Computer searches for putative binding sites for the DSX protein, which regulates sex-specific expression of the yp genes, revealed several such sites in our constructs. However, the significance of these is unclear since many such sites also occur in genes which one would not expect to be regulated in a sex-specific manner (e.g. Adh, Actin 5C). We suggest that DSX acts in concert with other proteins to mediate sex- and tissue-specific expression of the yp genes.     

The recent origin of the Sdic gene cluster in the melanogaster subgroup

Gene families are composed of closely related genes and are an important part of eukaryotic genomes. In the proximal region of the X chromosome of Drosophila melanogaster there is a cluster of four tandem Sdic genes, located between the gene Cdic and the gene AnnX. Sdic is a chimeric gene that encodes a novel protein with sperm-specific expression. It had been hypothesized that the Sdic gene cluster was formed after the split of D. melanogaster and D. simulans. To study the evolution of this cluster, the sequence of this region was studied in several Drosophilidae species. In all species analyzed, Sdic genes are absent and AnnX and Cdic are adjacent to each other. The results allowed the inference of the ancestral situation and the reconstruction of the evolution of the cluster, and confirm that the Sdic cluster was indeed formed in the lineage that gave rise to D. melanogaster, being one of the youngest gene clusters known. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Investigation of cis-acting sequences regulating expression of the gene encoding yolk protein 3 in Drosophila melanogaster.

Summary The regulatory sequences leading to the ovarian and fat body expression of yolk proteins 1 and 2 (YP1 and 2) of Drosophila melanogaster have been characterised in some detail. These genes (yp1 and yp2) share many enhancer elements, and some important regulatory sequences lie within the coding regions. We have begun to investigate the cis-regulation of the gene encoding yolk protein 3 (yp3). We describe a system for P element transformation using the complete and unaltered yp3 gene rather than reporter genes and describe sequences conferring correct expression in the ovary and carcass.     

Evolution of the LINE-like I element in the Drosophila melanogaster species subgroup

LINE-like retrotransposons, the so-called I elements, control the system of I-R (inducer-reactive) hybrid dysgenesis in Drosophila melanogaster. I elements are present in many Drosophila species. It has been suggested that active, complete I elements, located at different sites on the chromosomes, invaded natural populations of D. melanogaster recently (1920–1970). But old strains lacking active I elements have only defective I elements located in the chromocenter. We have cloned I elements from D. melanogaster and the melanogaster subgroup. In D. melanogaster, the nucleotide sequences of chromocentral I elements differed from those on chromosome arms by as much as 7%. All the I elements of D. mauritiana and D. sechellia are more closely related to the chromosomal I elements of D. melanogaster than to the chromocentral I elements in any species. No sequence difference was observed in the surveyed region between two chromosomal I elements isolated from D. melanogaster and one from D. simulans. These findings strongly support the idea that the defective chromocentral I elements of D. melanogaster originated before the species diverged and the chromosomal I elements were eliminated. The chromosomal I elements reinvaded natural populations of D. melanogaster recently, and were possibly introduced from D. simulans by horizontal transmission.     

Mutant fs(1) 1163 of Drosophila melanogaster alters yolk protein secretion from the fat body

Summary The mutant fs(1) 1163 of Drosophila melanogaster, which was isolated by Gans et al. (1975) is a recessive homozygous female sterile at 18°C and a dominant female — sterile at 29°C. We reported previously that there are reduced quantities of the largest of the three yolk polypeptides in Drosophila melanogaster in the haemolymph and eggs of this mutant at 29°C (Bownes and Hames 1978 a). In this paper we show that the yolk protein defect maps within approximately 2.5 recombination units of the female sterility at 21±2.5 map units on the X-chromosome. The temperature-sensitive period of the yolk protein defect is after emergence. In vitro labelling of fs(1) 1163 ovaries and fat bodies showed that they were able to synthesise yolk polypeptide 1. Interestingly, studies on the proteins present in the various tissues indicate that the fat body tends to accumulate all three yolk polypeptides in the mutant. This phenotype is partially co-dominant in that an effect is seen in heterozygotes as well as homozygotes and is enhanced by increased temperature. This mutant could therefore have a defect (a) in the structural gene for yolk polypeptide 1, (b) in the processing and secretion enzyme systems; (c) in the fat body or all tissues leading to altered secretion properties.Mutants like fs(1) 1163 which alter specific steps in vitellogenesis should be of value for analysing the genetic and biochemical control of the synthesis, transport and sequestering of the yolk polypeptides during oogenesis.     

Drosophila melanogaster genes encoding three troponin-C isoforms and a calmodulin-related protein

Using low-stringency hybridization and polymerase chain reaction (PCR)-based DNA amplification, we have isolated threeDrosophila melanogaster genes that encode troponin-C isoforms and one specifying a protein that is closely related to calmodulin. Two of the troponin-C genes, located within the 47D and 73F subdivisions of chromosomes 2 and 3, respectively, encode very closely related isoforms. That specified by the 47D gene accumulates almost exclusively in larval muscles, while that encoded by the 73F gene is present in both larvae and adults. The third gene, located within the 41C subdivision of chromosome 2, encodes a more distantly related troponin-C isoform that accumulates only within adults. The gene that encodes the calmodulin-related protein is located within the 97A subdivision of chromosome three. The protein encoded by this gene has a different primary sequence from that of conventional calmodulin, which is specified by a gene located within the 49A subdivision of chromosome 2. Our report is the first to describe insect troponin-C isoforms and further avails genetic methods for investigating thein vivo functions of the troponin-C/myosin light-chain/calmodulin protein superfamily.This work was supported by grants from the NIH and Muscular Dystrophy Association to E. F.Sequences described herein have been filed in the EMBL and GenBank databases under Accession Numbers X76042, X76043, X76044, and X76045.     

An evolutionary model for the duplication and divergence of esterase genes in Drosophila

Summary The esterase 5 (Est-5 = gene, EST 5 = protein) enzyme in Drosophila pseudoobscura is encoded by one of three paralogous genes, Est-5A, Est5B, and Est-5C, that are tightly clustered on the right arm of the X chromosome. The homologous Est-6 locus in Drosophila melanogaster has only one paralogous neighbor, Est-P. Comparisons of coding and flanking DNA sequences among the three D. pseudoobscura and two D. melanogaster genes suggest that two paralogous genes were present before the divergence of D. pseudoobscura from D. melanogaster and that, later, a second duplication occurred in D. pseudoobscura. Nucleotide sequences of the coding regions of the three D. pseudoobscura genes showed 78–85% similarity in pairwise comparisons, whereas the relatedness between Est-6 and Est-P was only 67%. The higher degree of conservation in D. pseudoobscura likely results from the comparatively recent divergence of Est-5B and Est-5C and from possible gene conversion events between Est-5A and Est-5B. Analyses of silent and replacement site differences in the two exons of the paralogous and orthologous genes in each species indicate that common selective forces are acting on all five loci. Further evidence for common purifying selective constraints comes from the conservation of hydropathy profiles and proposed catalytic residues. However, different levels of amino acid substitution between the paralogous genes in D. melanogaster relative to those in D. pseudoobscura suggest that interspecific differences in selection also exist.Offprint requests to: R.C. Richmond     

The sex-lethal gene homologue in Chrysomya rufifacies is highly conserved in sequence and exon-intron organization

A great variety of sex determination mechanisms exists in insect species. In Drosophila melanogaster sex is determined by the ratio between X chromosomes and autosomes, while in the blowfly Chrysomya rufifacies it is maternally determined. A cascade of genes which are involved in sex determination has been identified in D. melanogaster with the Sex-lethal gene (Sxl) as the key gene. We screened genomic libraries of C. rufifacies with a probe of the Sxl gene from D. melanogaster and isolated a genomic region that included most of the homologous gene. DNA- and protein-sequence comparison showed a high percent identity between the Chrysomya and the Drosophila gene. Up to 90% identity of the amino acid sequences was found in the region that contained the RNA-binding domains. The degree of identity is much lower outside of this functionally important region (18% identity). cDNA analysis showed a highly conserved exon-intron structure between the two species, although sex-specific splicing as used in D. melanogaster for the regulation of Sxl activity, could not be detected in C. rufifacies.     

Nucleotide and repeat length variation at the nonA gene of the Drosophila virilis group species and its effects on male courtship song

Genes found to affect male courtship song characters in Drosophila melanogaster are good candidates when tracing genes responsible for species-specific songs in other Drosophila species. It has previously been shown that Thr–Gly repeat length variation at the period gene affects song traits in D. melanogaster, which gives the repetitive regions a special interest. In this work, we have characterised the patterns of nucleotide variation for gene regions containing two Gly and one Gln–Ala repeat in another D. melanogaster song gene, no-on-transient A, in D. virilis group species. The levels of nucleotide variability in D. virilis nonA were similar to those found for other genes of the species, and the gene sequences showed no signs of deviation from neutrality. The Gly 2 repeat preceding the central domain of the gene exhibited length variation, which did not, however, correlate with song variation either within D. virilis or between the species of D. virilis group. The Gly 3 repeat located on the other side of the central domain showed amino acid divergence parallel to the consensus phylogeny of the D. virilis group species. The species of the virilis subgroup having Asn after the first three glycines in this repeat have simple songs with no species-specificity, while the species of the montana subgroup having two Gly or Asn–Ser in this site have unique courtship songs. Amino acid differences between the species in this repeat may, however, reflect species phylogeny rather than have an effect on song divergence per se.     

The evolutionary genetics of thehobo transposable element in theDrosophila melanogaster complex

Hobo elements are a family of transposable elements found inDrosophila melanogaster and its three sibling species:D. simulans, D. mauritiana andD. sechellia. Studies inD. melanogaster have shown thathobo may be mobilized, and that the genetic effects of such mobilizations included the general features of hybrid dysgenesis: mutations, chromosomal rearrangements and gonadal dysgenis in F1 individuals. At the evolutionary level somehobo-hybridizing sequences have also been found in the other members of themelanogaster subgroup and in many members of the relatedmontium subgroup. Surveys of older collected strains ofD. melanogaster suggest that completehobo elements were absent prior to 50 years ago and that they have recently been introduced into this species by horizontal transfer. In this paper we review our findings and those of others, in order to precisely describe the geographical distribution and the evolutionary history ofhobo in theD. melanogaster complex. Studies of the DNA sequences reveal a different level of divergence between the groupD. melanogaster, D. simulans andD. mauritiana and the fourth speciesD. sechellia. The hypothesis of multiple transfers in the recent past into theD. melanogaster complex from a common outside source is discussed.     

Use of recombination techniques to examine the structure of the csg locus of Myxococcus xanthus

Summary The technique of chromosome walking was used to isolate approximately 60 kb of DNA from the region containing the complementation group uncoordinated of Drosophila melanogaster, located in that part of the X chromosome which spans the euchromatin-heterochromatin junction. The cloned DNA can be divided into two distinct regions. The first contains sequences that are low copy number or unique and are largely conserved between strains. The second region is characterized by units repeated in tandem arrays and is polymorphic within, and between, strains. Each repetitive unit is separated by a member of an abundant sequence family, part of which is homologous to the ribosomal type 1 insertion sequence of D. melanogaster. The molecular organization of the cloned DNA was compared with that of sequences isolated from regions of intercalary heterochromatin and also with genes which have been characterized from more conventional euchromatic regions.     

Structure,regulation and evolution of the crystal-Stellate system of Drosophila

The crystal–Stellate system is one of the most known example of interaction between heterochromatin and euchromatin: a heterochromatic locus on the Y chromosome (crystal) 'represses a euchromatic locus (Stellate) on the X chromosome in Drosophila melanogaster. The molecular mechanism regulating this interaction is not completely understood. It is becoming clear that an RNA interference (RNAi) mechanism could be responsible for the silencing carried out by crystal on the Stellate sequences. Here, a detailed structural analysis of all the sequences involved in the system is reported, demonstrating a their 'puzzling structure. In addition three autosomal mutations: sting, scratch and sirio are described that interfere with the system. All of them are male sterile mutations and exhibit crystals made by the STELLATE protein in their primary spermatocytes. They are requested during oogenesis and early in embryogenesis as well. Hypothesis on the involvement of these genes in activating the Stellate sequences are discussed.     

Evolutionary changes in the organization of the major LCP gene cluster during sex chromosomal differentiation in the sibling speciesDrosophila persimilis,D. pseudoobscura andD. miranda

The major larval cuticle protein (LCP) genes I–IV ofDrosophila melanogaster are clustered on the right arm of the second chromosome. By cross-hybridization we cloned the corresponding genes from three different members of theobscura group:D. persimilis, D. pseudoobscura andD. miranda. InD. pseudoobscura andD. persimilis the gene cluster maps to autosome3. In contrast, inD. miranda it was found on theX2 andY sex chromosome. Hence, this exceptional karyotypic situation offers a unique opportunity to analyse the molecular processes underlying the phenomenon of chromosome degeneration. Comparison of LCP genes I–IV in theX2 andY chromosomal region inD. miranda revealed extensive DNA rearrangements at the latter. TheY chromosomal LCP cluster is characterized by DNA insertions which are absent in the correspondingX2 chromosomal DNA, suggesting that these DNA sequences must have invaded this area. In addition, part of the analysedY chromosomal region is duplicated.     

Chromosomal Evolution of Sibling Species of the Drosophila willistoni Group. I. Chromosomal Arm IIR (Muller’s Element B)

The phylogenetic relationships among nine entities of Drosophila belonging to the D. willistoni subgroup were investigated by establishing the homologous chromosomal segments of IIR chromosome, Muller’s element B (equivalent to chromosome 2L of D. melanogaster). The sibling species of the D. willistoni group investigated include D. willistoni, D. tropicalis tropicalis, D. tropicalis cubana, D. equinoxialis, D. insularis and four semispecies of the D. paulistorum complex. The phylogenetic relationships were based on the existence of segments in different triads of species, which could only be produced by overlapping inversions. Polytene banding similarity maps and break points of inversions between species are presented. The implications of the chromosomal data for the phylogeny of the species and comparisons with molecular data are discussed. The aim of this study is to produce phylogenetic trees depicting accurately the sequence of natural events that have occurred in the evolution of these sibling species. Claudia Rohde, Ana Cristina Lauer Garcia: These authors contributed equally to this work