Vitellogenesis in Drosophila: sequestration of a yolk polypeptide/invertase fusion protein into developing oocytes

The mechanism of yolk deposition into developing oocytes of Drosophila was investigated by following the fate of a reporter protein fused to a vitellogenin, or yolk polypeptide (YP). Embryos were transformed with a hybrid gene consisting of the promotor and amino terminal 430 codons of the Yp2 gene fused to the cytoplasmic form of the invertase gene from the yeast Saccharomyces cerevisiae. RNA hybridization experiments with established lines of transformed flies showed that the hybrid gene was expressed in female fat bodies and ovaries but not in any male cells. Immunoblotting and endoglycosidase digestion showed that the hybrid protein was secreted from fat body cells via the secretory pathway, transported in hemolymph, and sequestered into developing oocytes. Transfusion experiments with hemolymph and pure invertase showed that sequestration of invertase depended on its attachment to YP. Immunocytochemistry demonstrated that the hybrid protein became localized in yolk granules as oocytes developed. Females homozygous for the fusion gene are generally sterile; their eggs containing the hybrid protein often collapse and their embryos fail to develop, suggesting that the structure of the yolk polypeptides is important for embryonic development. These experiments show that YP2 carries structural information sufficient to direct a reporter protein from fat body cells, through the hemolymph, and into the yolk granules of developing oocytes. This work provides a means of identifying the features of yolk polypeptides that are responsible for their deposition into yolk during oogenesis.     

The nucleotide sequence of the gene coding for Drosophila melanogaster yolk protein 3

The entire sequence of the Drosophila melanogaster yolk protein 3 (YP3) gene (yp3), including 1822 nucleotides (nt) of 5'- and 834 nt of 3'-flanking DNA, has been determined. In addition, the 5' and 3' ends of the mRNA and the two introns have been mapped. The predicted amino acid sequence of YP3 has considerable homology (43%) to the other two yolk proteins of D. melanogaster. The nucleotide sequence of yp3 was compared to the other two yolk protein genes which have the same developmental pattern of expression. In addition to extensive homology between the protein coding regions, we found two small regions of homology between yp3 flanking sequences and a segment of DNA required for normal expression of the yolk protein 1 gene in adult female fat bodies.     

CDNA cloning and characterization of S6 kinase and its effect on yolk protein gene expression in the oriental fruit fly Bactrocera dorsalis (Hendel)

p70 S6 kinase (S6K), a serine/threonine protein kinase, is a downstream target of target of rapamycin (TOR) gene and an important regulator of protein synthesis responsible for cell growth and reproduction. In this study, a S6K gene, named BdS6K (GenBank Accession No. GQ203802), was isolated from the oriental fruit fly Bactrocera dorsalis (Hendel). Quantitative RT-PCR showed that BdS6K mRNA is expressed at a higher level in egg than in other developmental stages, as well as in ovary than in fat body. Downregulation of BdS6K activity by rapamycin treatment in larval stage resulted in the developmental defects of larvae, pupae, and adults, with a reduced yolk protein (YP) expression in the fat body throughout the first reproductive cycle with a substantial reduction in ovary size, and also repressed the egg development in female fruit fly. Knockdown of BdS6K gene by RNA interference in the adult significantly decreased the YP expression. These observations support the involvement of BdS6K signaling in the regulation of the YP synthesis and egg development in B. dorsalis.     

Tissue-Specific Expression Phenotypes of Hawaiian Drosophila Adh Genes in Drosophila Melanogaster Transformants

Interspecific differences in the tissue-specific patterns of expression displayed by the alcohol dehydrogenase (Adh) genes within the Hawaiian picture-winged Drosophila represent a rich source of evolutionary variation in gene regulation. Study of the cis-acting elements responsible for regulatory differences between Adh genes from various species is greatly facilitated by analyzing the behavior of the different Adh genes in a homogeneous background. Accordingly, the Adh gene from Drosophila grimshawi was introduced into the germ line of Drosophila melanogaster by means of P element-mediated transformation, and transformants carrying this gene were compared to transformants carrying the Adh genes from Drosophila affinidisjuncta and Drosophila hawaiiensis. The results indicate that the D. affinidisjuncta and D. grimshawi genes have relatively higher levels of expression and broader tissue distribution of expression than the D. hawaiiensis gene in larvae. All three genes are expressed at similar overall levels in adults, with differences in tissue distribution of enzyme activity corresponding to the pattern in the donor species. However, certain systematic differences between Adh gene expression in transformants and in the Hawaiian Drosophila are noted along with tissue-specific position effects in some cases. The implications of these findings for the understanding of evolved regulatory variation are discussed.     

Receptor-mediated endocytosis in the Caenorhabditis elegans oocyte

The Caenorhabditis elegans oocyte is a highly amenable system for forward and reverse genetic analysis of receptor-mediated endocytosis. We describe the use of transgenic strains expressing a vitellogenin::green fluorescent protein (YP170::GFP) fusion to monitor yolk endocytosis by the C. elegans oocyte in vivo. This YP170::GFP reporter was used to assay the functions of C. elegans predicted proteins homologous to vertebrate endocytosis factors using RNA-mediated interference. We show that the basic components and pathways of endocytic trafficking are conserved between C. elegans and vertebrates, and that this system can be used to test the endocytic functions of any new gene. We also used the YP170::GFP assay to identify rme (receptor-mediated endocytosis) mutants. We describe a new member of the low-density lipoprotein receptor superfamily, RME-2, identified in our screens for endocytosis defective mutants. We show that RME-2 is the C. elegans yolk receptor.     

Structure, Expression, Chromosomal Location and Product of the Gene Encoding Adh2 in Petunia

In most higher plants the genes encoding alcohol dehydrogenase comprise a small gene family, usually with two members. The Adh1 gene of Petunia has been cloned and analyzed, but a second identifiable gene was not recovered from any of three genomic libraries. We have therefore employed the polymerase chain reaction to obtain the major portion of a second Adh gene. From sequence, mapping and northern data we conclude this gene encodes ADH2, the major anaerobically inducible Adh gene of Petunia. The availability of both Adh1 and Adh2 from Petunia has permitted us to compare their structures and patterns of expression to those of the well-studied Adh genes of maize, of which one is highly expressed developmentally, while both are induced in response to hypoxia. Despite their evolutionary distance, evidenced by deduced amino acid sequence as well as taxonomic classification, the pairs of genes are regulated in strikingly similar ways in maize and Petunia. Our findings suggest a significant biological basis for the regulatory strategy employed by these distant species for differential expression of multiple Adh genes.     

Identification, synthesis, and characterization of the yolk polypeptides of Plodia interpunctella

The mature eggs of Plodia interpunctella were found to contain four major polypeptides. These yolk polypeptides (YPs) were found to have approximate molecular weights of 153,000 daltons (YP1), 69,000 daltons (YP2), 43,000 daltons (YP3), and 33,000 daltons (YP4) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, we found YP1 was resolved by a 5% polyacrylamide gel into two separate polypeptides of 153,000 and 147,000 daltons. All of the YPs could be labeled in vivo or in vitro with [35S]-methionine. Yolk peptide 1 and YP3 were synthesized by fat body of pharate adult and adult females and secreted into the hemolymph. Yolk peptide 2 and YP4 were synthesized and secreted into incubation medium by ovaries that contained vitellogenic oocytes, but these polypeptides were not found in the hemolymph. Fat bodies of males synthesized and secreted an immunoprecipitable polypeptide similar to YP3 as well as immunoprecipitable polypeptides larger than 200,000 daltons that had no counterparts in the oocytes. Peptide mapping by protease digestion showed each YP to be cleaved into unique fragments, suggesting that no precursor-product relationship exists between the YPs. Ion exchange chromatography and gel permeation chromatography separated that yolk proteins into two groups with approximate molecular weights of 462,000 and 264,000 daltons. By resolving these peaks on SDS-PAGE, it was found that YP1 and YP3 formed the 462,000-dalton yolk protein and YP2 and YP4 formed the 264,000-dalton yolk protein.     

Genetically modified yolk proteins precipitate in the adult Drosophila fat body

Ultrastructural and genetic studies were carried out on the fat body of a female sterile mutant fs(1)1163 to ascertain why yolk protein 1 (YP1) is not secreted from this tissue. Earlier molecular studies demonstrated that (a) normally yolk protein is synthesized in the fat body, secreted into the hemolymph and taken up by the ovary, (b) the 1163 mutation causes a single amino acid substitution in YP1, and (c) females homozygous for the mutation, or heterozygous females raised at 29 degrees C, retain YP1 in the fat body. Ultrastructural analysis in this paper shows that the fat body of these females contains masses of electron-dense material deposited in the subbasement membrane space. This subbasement membrane material (SBMM), which occasionally has a crystalline-like, fibrous component, is found in females whose genotypes include at least one copy of the mutant 1163 gene. These strains include a deletion strain that is hemizygous for the 1163 gene and two strains that are transgenic for the mutant gene. Immunogold studies indicate that SBMM contains yolk protein. We propose that the mutant protein is secreted into the subbasement membrane space, but because of the amino acid substitution in YP1, the oligomers containing YP1 condense into SBMM, which cannot penetrate the basement membrane. The similarity of SBMM and deoxyhemoglobin S fibers is discussed.     

Differential control of yolk protein gene expression in fat bodies and gonads by the sex-determining gene tra-2 of Drosophila.

We studied the regulation of the yolk protein (YP) genes in the somatic cells of the gonads, using temperature sensitive mutations (tra-2ts) of transformer-2, a gene required for female sexual differentiation. XX;tra-2ts mutant animals were raised at the permissive temperature so that they developed as females and were then shifted to the restrictive male-determining temperature either 1-2 days before or 0-2 h after eclosion. These animals formed vitellogenic ovaries. Likewise, mutant gonads transplanted into either normal female hosts or normal male hosts, kept at the restrictive temperature, underwent vitellogenesis. Thus, the ovarian follicle cells can mature and express their YP genes in the absence of a functional product of the tra-2 gene. Although the gonadal somatic cells of ovary and testis may derive from the same progenitor cells, the testicular cells of XX;tra-2ts pseudomales did not express their YP genes nor take up YP from the haemolymph at the permissive female-determining temperature. We conclude that in the somatic cells of the gonad, the YP genes are no longer under direct control of the sex-determining genes, but instead are regulated by tissue specific factors present in the follicle cells. It is the formation of follicle cells which requires the activity of tra-2.     

Differential expression of protein S genes during Myxococcus xanthus development

Protein S, the most abundant protein synthesized during development of the fruiting bacterium Myxococcus xanthus, is coded by two highly homologous genes called protein S gene 1 (ops) and protein S gene 2 (tps). The expression of these genes was studied with fusions of the protein S genes to the lacZ gene of Escherichia coli. The gene fusions were constructed so that expression of beta-galactosidase activity was dependent on protein S gene regulatory sequences. Both the gene 1-lacZ fusion and the gene 2-lacZ fusion were expressed exclusively during fruiting body formation (development) in M. xanthus. However, distinct patterns of induction of fusion protein activity were observed for the two genes. Gene 2 fusion activity was detected early during development on an agar surface and could also be observed during nutritional downshift in dispersed liquid culture. Gene 1 fusion activity was not detected until much later in development and was not observed after downshift in liquid culture. The time of induction of gene 1 fusion activity was correlated with the onset of sporulation, and most of the activity was spore associated. This gene fusion was expressed during glycerol-induced sporulation when gene 2 fusion activity could not be detected. The protein S genes appear to be members of distinct regulatory classes of developmental genes in M. xanthus.