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.     

Identification and comparisons of the yolk polypeptides and the genes which code for them in D. melanogaster sibling species

Summary The yolk proteins stored in Drosophila, oocytes for utilisation during embryogenesis are an ideal system for studying the regulation of gene expression during development. The 3 major polypeptides found in yolk in D. melanogaster are synthesised in the fat body and ovarian follicle cells and selectively accumulated by the oocyte during vitellogenesis. In order to understand more about their regulation and the mechanism of uptake, studies on other species are necessary.Three yolk polypeptides have previously been identified in the D. melanogaster sibling species (D. melanogaster, D. simulans, D. mauritiana, D. erecta, D. teissieri, D. orena and D. yakuba). In D. melanogaster three genes located on the X chromosome are known to code for these yolk polypeptides. in this study genomic Southern transfers and in situ hybridisation experiments were carried out on the sibling species. Using the three cloned yolk protein genes from D. melanogaster, homologous sequences could be detected in the sibling species. It is suggested that three yolk protein genes occur in each of these species, all being located on the X chromosome, and that two of the genes are very closely linked in these same species. Yolk protein gene-homologous DNA sequences have also been identified in two more distantly related species D. funebris and D. virilis.     

Isolation and partial chemical characterization of the three major yolk polypeptides from Drosophila melanogaster.

The three major yolk polypeptides of Drosophila melanogaster have been isolated from yolk spheres of early embryos. Their molecular weights, as determined by SDS-polyacrylamide electrophoresis, are 44,000, 45,000, and 46,000. A number of approaches have been used to show that each of these yolk polypeptides are different. They have different isoelectric points, they have different digestion products upon peptide mapping by limited proteolysis, and they show three different antigen-antibody systems when each polypeptide is reacted with an antisera made to a mixture of all three. Both the digestion with chymotrypsin and the immunoelectrophoresis studies indicate similarities between two of the polypeptides while the third appears unique. This is the first example of multiple yolk polypeptides of similar molecular weight.     

Roles of young serine-endopeptidase genes in survival and reproduction revealed rapid evolution of phenotypic effects at adult stages

Our recent study found that 30% of young genes were essential for viability that determines development through stages from embryo to pupae in Drosophila melanogaster, revealing rapidly evolving genetic components involved in the evolution of development. Meanwhile, many young genes did not produce complete lethal phenotype upon constitutive knockdown, suggesting that they may not be essential for viability. These genes, nevertheless, were fixed by natural selection, and might play an important functional role in their adult stage. Here we present a detailed demonstration that a newly duplicated serine-type endopeptidase gene that originated in the common ancestor in the D. melanogaster subgroup 6~11 million years ago, named Slfc, revealing a strong effect in post-eclosion. Although animals survived constitutive knockdown of Slfc to adult stage, however, their life span reduced significantly by two-thirds compared to wildtype. Furthermore, the Slfc-RNAi males dropped their fertility to less than 10% of the wildtype level, with over 80% of these males being sterile. The Slfc-RNAi females, on the other hand, showed a slight reduction in fertility. This case study demonstrates that a young gene can contribute to fitness on the three important traits of life history in adults, including the life expectancy, male fertility and female fertility, suggesting that new genes can quickly evolve and impact multiple phenotypes.     

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.     

Analysis of the Drosophila female sterile mutation fs(1) 1304 by pole cell transplantation experiments

The Drosophila melanogaster mutant fs(1)1304 is an ovary autonomous female sterile mutant that causes abnormal morphology of the egg. Vitellogenesis proceeds at an abnormally slow rate in homozygous females. We have used pole cell transplantation to construct germ line mosaics in order to determine whether the 1304 defect depends upon the genotype of the germ line cells (oocyte or nurse cells) or the somatic line (follicle cells). We have found that the germ line is the primary target tissue where the mutant gene is expressed.     

Ovarian yolk-protein synthesis in Drosophila melanogaster

The ovaries and fat bodies of Drosophila melanogaster adult females both synthesize yolk polypeptides. In a series of experiments it has been shown that the ovaries become competent to mature in an adult male host, where only ovarian synthesis occurs, very early in metamorphosis and synthesis of yolk polypeptides begins in a time-dependent sequence related to the age of the ovary when transplanted. Maturation of ovaries occurs prior to eclosion when they are transplanted to an earlier developmental stage showing that neither the event of eclosion not the adult environment is essential in triggering yolk-polypeptide synthesis by the ovary. When metamorphosing ovaries are transplanted to a female host they take up host yolk polypeptides from the haemolymph, but this does not lead to the implanted ovary developing substantially better than in a male host where only synthesis by the ovary can occur. The regulation of ovarian yolk-polypeptide synthesis therefore appears to be autonomous to the ovary itself. There may be a trigger early in metamorphosis which induces competence in the ovary so that it subsequently initiates yolk-polypeptide gene expression at eclosion.     

Transfer of molecules from ejaculate to females in Drosophila melanogaster and Drosophila pseudoobscura

Methionine in the form of free amino acids, small peptides and several proteins is transferred from ejaculate to females in Drosophila melanogaster and D. pseudoobscura. The male contribution is found mostly in the ovary and the sizes of the polypeptides transferred have been established. Some components of the ejaculate are rapidly transferred to the somatic tissues outside the ovary and reproductive system where they co-migrate with the yolk polypeptides. The incorporation of free methionine into polypeptides in non-reproductive tissues is higher in females that have been deprived of a source of protein prior to mating. The molecules transferred from ejaculate may have roles in regulating reproductive behaviour and may also be used as a nutrient source. We suggest that free amino acids in the ejaculate are rapidly utilised in protein synthesis in the somatic tissues outside the ovary and could provide a boost to yolk protein and hence egg production in females in nature, where nutrients may be in limited supply.     

Evolution of nested genes with special reference to cuticle proteins inDrosophila melanogaster

Summary One of the pupal cuticle protein (PCP) genes has been found within an intron of aDrosophila housekeeping gene (theGart locus) that encodes three enzymes involved in the purine pathway. This intronic gene has been described as a gene within a gene, and the gene is now called a “nested” gene. Because the intronic PCP gene has sequence similarity with the larval cuticle protein (LCP) gene, it may have been derived from one of the LCP genes or their ancestral gene. We have studied possible phylogenetic relationships among these five genes by comparing nucleotide sequences of four LCP genes with that of the PCP gene. The results obtained suggest that the PCP gene may have originated from an ancestral gene before duplication of the LCP genes occurred. Using the number of synonymous (silent) substitutions, we then estimated the divergence time between the PCP gene and the LCP genes to be about 70 million years (Myr). The divergence time estimated is much larger than that for the sibling species ofD. melanogaster (about 2.5 Myr), indicating that the “nested” gene structure can be seen not only inDrosophila melanogaster, but also in other distantly relatedDrosophila species.     

Single gene mutations in Drosophila: what can they tell us about the evolution of sexual behaviour?

The molecular analysis of specific mutant genes that affect the courtship behaviours of Drosophila melanogaster males and females is discussed in the light of the possibility that they may contribute to mate choice. There is clear evidence that some genes can act as a reservoir of species-specific behaviour, particularly for the male actions during courtship. However, to date there has not been a single genetic locus that has been isolated at the molecular level and shown to be associated with a change in female preference. There are some promising avenues of exploration, in that recent genetic analyses suggest that a small number of genes may make major contributions to female preferences. Finally a candidate gene approach is advocated in which orthologous genes from other species of Drosophila are used as natural mutations, and transformed into D. melanogaster hosts to investigate whether they carry species-specific mating information of the donor.     

Roles of young serine-endopeptidase genes in survival and reproduction revealed rapid evolution of phenotypic effects at adult stages

Our recent study found that 30% of young genes were essential for viability that determines development through stages from embryo to pupae in Drosophila melanogaster, revealing rapidly evolving genetic components involved in the evolution of development. Meanwhile, many young genes did not produce complete lethal phenotype upon constitutive knockdown, suggesting that they may not be essential for viability. These genes, nevertheless, were fixed by natural selection, and might play an important functional role in their adult stage. Here we present a detailed demonstration that a newly duplicated serine-type endopeptidase gene that originated in the common ancestor in the D. melanogaster subgroup 6∼11 million years ago, named Slfc, revealing a strong effect in post-eclosion. Although animals survived constitutive knockdown of Slfc to adult stage, however, their life span reduced significantly by two-thirds compared to wild-type. Furthermore, the Slfc-RNAi males dropped their fertility to less than 10% of the wild-type level, with over 80% of these males being sterile. The Slfc-RNAi females, on the other hand, showed a slight reduction in fertility. This case study demonstrates that a young gene can contribute to fitness on the three important traits of life history in adults, including the life expectancy, male fertility and female fertility, suggesting that new genes can quickly evolve and impact multiple phenotypes.     

Developmental regulation of yolk protein gene expression in Anastrepha suspensa

A partial cDNA clone for the 48,000 dalton yolk polypeptide gene from Anastrepha suspensa was isolated from a cDNA expression library using a yolk polypeptide antibody probe and hybridization to the Drosophila melanogaster yolk protein 1 gene. The sequenced DNA has greatest homology to the yolk protein genes from Ceratitis capitata, D. Melanogaster, and Calliphora erythrocephala and, similar to these genes, shares amino acid sequence domains with those from lipases. RNA hybridization studies indicated that the yolk protein gene expression is completely female-specific and limited to the ovaries, without apparent regulation by 20-hydroxyecdysone or juvenile hormone. This is in contrast to an earlier study which suggested, based on immunological probes, that a very low level of yolk protein synthesis occurred in fat body and was not sex-specific. Arch. Insect Biochem. Physiol. 36:25–35, 1997.Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

In situ study of chorion gene amplification in ovarian follicle cells ofDrosophila nasuta

The temporal and spatial pattern of replication of chorion gene clusters in follicle cells during oogenesis inDrosophila melanogaster andDrosophila nasuta was examined by [^3H thymidine autoradiography and byin situ hybridization with chorion gene probes. When pulse labelled with [³H] thymidine, the follicle cells from stage 10–12 ovarian follicles of bothDrosophila melanogaster and,Drosophila nasuta often showed intense labelling at only one or two sites per nucleus.In situ hybridization of chorion gene probes derived fromDrosophila melanogaster with follicle cell nuclei ofDrosophila melanogaster andDrosophila nasuta revealed these discrete [³H] thymidine labelled sites to correspond to the two amplifying chorion gene clusters. It appears, therefore, that in spite of evolutionary divergence, the organization and programme of selective amplification of chorion genes in ovarian follicle cells have remained generally similar in these two species. The endoreplicated and amplified copies of each chorion gene cluster remain closely associated but the two clusters occupy separate sites in follicle cell nucleus.     

Separate DNA sequences are required for normal female and ecdysone-induced male expression of Drosophila melanogaster yolk protein 1

Summary Drosophila melanogaster flies were transformed with a yp1-Adh fusion gene with 890 bp of yp1 5 flanking sequence. In an Adh ^- background these flies show a stage, tissue and sex-specific pattern of alcohol dehydrogenase (ADH) activity characteristic of yolk protein genes. ADH activity is not present in dsx ^D/dsx pseudomales indicating that this fragment contains sites where the dsx gene product exerts its effect. Transformed male flies do not exhibit ADH activity when injected with 20-hydroxyecdysone while synthesis of native yolk proteins is induced. Thus the hormone inducibility and sex regulation have been separated in this construct.     

Genetic,molecular and developmental analysis of the glutamine synthetase isozymes ofDrosophila melanogaster

The glutamine synthetase isozymes ofDrosophila melanogaster offer an attractive model for the study of the molecular genetics and evolution of a small gene family encoding enzymatic isoforms that evolved to assume a variety of specific and sometimes essential biological functions. InDrosophila melanogaster two GS. isozymes have been described which exhibit different cellular localisation and are coded by a two-member gene family. The mitochondrial GS structural gene resides at the 21B region of the second chromosome, the structural gene for the cytosolic isoform at the 10B region of the X chromosome. cDNA clones corresponding to the two genes have been isolated and sequenced. Evolutionary analysis data are in accord with the hypothesis that the twoDrosophila glutamine synthetase genes are derived from a duplication event that occurred near the time of divergence between Insecta and Vertebrata. Both isoforms catalyse all reactions catalysed by other glutamine synthetases, but the different kinetic parameters and the different cellular compartmentalisation suggest strong functional specialisation. In fact, mutations of the mitochondrial GS gene produce embryo-lethal female sterility, defining a function of the gene product essential for the early stages of embryonic development. Preliminary results show strikingly distinct spatial and temporal patterns of expression of the two isoforms at later stages of development.     

Drosophila melanogaster eggshell development: Localization of the s19 chorion gene

Summary Further IF screening ofDrosophila melanogaster geographic strains has revealed a variant of the s19 major chorion protein. Developmental analysis of F1 hybrids indicates that the source of the variation is found in the structural gene for this protein. The linkage group of the variant gene was determined to be the third, and the gene was localized by several methods of recombination analysis. The s19 gene was found to be tightly linked to thesepia locus, as had been previously found for the s18 gene (Yannoni and Petri 1980). Lack of recombination between the s19 and s18 genes in double heterozygotes suggested that these two genes are within 0.3 map units of each other. Although more precise localization of the s19 gene failed, the s18 gene could be more specifically located to the right ofsepia, betweensepia andhairy. Contrary to our prediction (ibid.), the s19 and s18 genes have been found to be tightly linked in spite of the fact that they display somewhat different developmental stage specificity.