Yolk synthesis in ovarian follicles ofDrosophila

Summary The autonomous synthesis of yolk proteins in ovarian follicles ofDrosophila melanogaster was analyzed. Vitellogenic follicles were labelled with³⁵S-methionine in vitro and the newly synthesized yolk proteins were separated by SDS-polyacrylamide gel electrophoresis. Possible contamination of the follicle preparations caused by adhering fat body cells could be excluded by culturing follicles in males prior to labelling in vitro. When labelled follicles were cut at the nurse cell/oocyte border the three yolk proteins (YP1, YP2, YP3) were found only in posterior fragments containing ooplasm and follicle cells, whereas two radioactive protein bands (A and B) were detected in nurse cells (anterior fragments). The yolk proteins of these five bands were characterized by peptide mapping. Band A protein, migrating a little more slowly than YP2, is closely related to both YP1 and YP2 while band B contains a yolk protein which is very similar to YP3. Hence, the nurse cells have been identified as a site of vitellogenin synthesis within the ovary ofDrosophila.Supported by the Deutsche Forschungsgemeinschaft, SFB 46     

Vitellogenin-derived yolk proteins in a hybrid sturgeon, bester (Huso huso x Acipencer ruthenus): identification, characterization and course of proteolysis during embryogenesis

Vitellogenin (Vg) and its corresponding yolk protein (YP) products, YP1, YP2 and YP3, were isolated from serum of estrogen-treated hybrid sturgeon (bester; Huso huso X Acipencer ruthenus) and eggs from untreated fish, respectively. Vitellogenin had an apparent molecular mass of 580 kDa and appeared as two major bands corresponding to 180 kDa and 120 kDa after SDS-PAGE. Apparent molecular weights of YP1, YP2 and YP3 were 370 kDa, 88 kDa and 19 kDa, respectively. After SDS-PAGE, YP1 appeared as a main band of 110 kDa, while YP2 was resolved as a single band of 94 kDa and 29 kDa band under non-reducing and reducing conditions, respectively. Yolk protein 3 appeared as a diffuse band corresponding to 16 kDa and two faint bands below 14.4 kDa after SDS-PAGE. However, the 16 kDa band alone was observed after dephosphorylation with alkaline phosphatase. The course of cleavage of yolk proteins in bester embryos and alevins was observed by SDS-PAGE and Western blotting from fertilization onward. After hatching, the main 110 kDa band of YP1 was degraded into smaller peptides during development, while YP2 hardly showed any such structural changes. The amino acid compositions of purified yolk proteins indicated that YP1, YP2 and YP3 were bester lipovitellin, beta-component, and phosvitin, respectively.     

Vitellogenin-derived yolk proteins of white perch,Morone americana: purification,characterization, and vitellogenin-receptor binding

The objectives of this study were to 1) purify and characterize vitellogenin-derived yolk proteins of white perch (Morone americana), 2) develop a nonisotopic receptor binding assay for vitellogenin, and 3) identify the yolk protein domains of vitellogenin recognized by the ovarian vitellogenin receptor. Four yolk proteins derived from vitellogenin (YP1, YP2 monomer [YP2m] and dimer [YP2d], and YP3) were isolated from ovaries of vitellogenic perch by selective precipitation, ion exchange chromatography, and gel filtration. The apparent molecular masses of purified YP1, YP2m, and YP2d after gel filtration were 310 kDa, 17 kDa, and 27 kDa, respectively. YP3 appeared in SDS-PAGE as a approximately 20-kDa band plus some diffuse smaller bands that could be visualized by staining for phosphoprotein with Coomassie Brilliant Blue complexed with aluminum nitrate. Immunological and biochemical characteristics of YP1, YP2s, and YP3 identified them as white perch lipovitellin, beta'-components, and phosvitin, respectively. A novel receptor-binding assay for vitellogenin was developed based on digoxigenin (DIG)-labeled vitellogenin tracer binding to ovarian membrane proteins immobilized in 96-well plates. Lipovitellin from white perch and vitellogenin from perch and other teleosts effectively displaced specifically bound DIG-vitellogenin in the assay, but phosvitin and the beta'-component could not, demonstrating for the first time that the lipovitellin domain of teleost vitellogenin mediates its binding to the oocyte receptor. Lipovitellin was less effective than vitellogenin in this regard, suggesting that the remaining yolk protein domains of vitellogenin may interact with its lipovitellin domain to facilitate binding of vitellogenin to its receptor.     

Isolation and identification of yolk proteins in Indian major carp,Labeo rohita

Two yolk proteins (YP1 and YP2) from the ovaries of Indian major carp, Labeo rohita were isolated by gel filtration and partially characterized by the use of hydroxyapatite ultrogel column in conjunction with native PAGE. On native PAGE YP1 gave a single protein band, whereas YP2 of gel filtration revealed the contamination of YP1, which was removed by adsorption chromatography on hydroxyapatite ultrogel and then the YP2 was the purified one as judged by electrophoresis. Both YP1 and YP2 also stained for lipid and contained alkalilabile phosphorus. Therefore, both yolk proteins were lipophosphoprotein. The molecular weights of YP1 and YP2 were 620 kDa and 225 kDa respectively as determined by gel filtration on Sepharose 4B. When YP1 and YP2 were compared in relation to some physicochemical characteristics with yolk proteins of other oviparous vertebrates including fish, they were lipovitellin like. Antiserum to YP2 crossreacted with YP2 and vitellogenin suggesting that YP2 was the cleaved product of vitellogenin. Anti-YP2 antiserum was not crossreacted with native YP1, whereas reduced and/or denatured YP1 was crossreacted indicating the presence of antigenic determinants in the inner core region of YP1 polypeptide.     

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.     

Egg yolk proteins in gray mullet (Mugil cephalus): purification and classification of multiple lipovitellins and other vitellogenin-derived yolk proteins and molecular cloning of the parent vitellogenin genes

Seven yolk proteins (YPs), four large lipoproteins (YPs1-4) and three minor yolk components (YPs5-7) including one phosphoprotein (YP7), were purified from extracts of vitellogenic ovaries of grey mullet (Mugil cephalus) by combinations of hydroxylapatite, ion exchange, immunoadsorbent, and gel filtration chromatography. The molecular masses of native YP1, YP2, YP3, and YP4 were estimated to be 330, 325, 335, and 570 kDa, respectively. The tertiary structures of YP1, YP2, and YP3 revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis were typical of teleost lipovitellins (Lvs), consisting of a heavy chain ( approximately 110, approximately 99, and approximately 97 kDa, respectively) and a light chain ( approximately 30, approximately 29, and approximately 21.5 kDa, respectively), while YP4 exhibited a heavy chain ( approximately 110 kDa) and two more polypeptide bands ( approximately 70 and approximately 54 kDa). Mapping of N-terminal peptide sequences of the purified YPs to the primary structure of multiple mullet vitellogenins (Vgs) deduced from their respective complete cDNAs, which were cloned and sequenced, conclusively identified YP1, YP2, and YP3 as Lvs derived from mullet VgA, VgB, and VgC, respectively. The fourth YP (YP4) appeared to be a proteolytic variant consisting of Lv and phosvitin components of VgA. Two other YPs (YP5 and YP6) were identified as beta'-components derived from VgA and VgB based on their structures and common, but not identical, antigenicity to salmonid beta'-component, while purified YP7, a phosphoprotein with a high content of serine residues, was identified as a phosvitin derived from VgB. This is the first report, of which we are aware, on purification and molecular classification of three distinct forms of Lv from any oviparous vertebrate.     

The Regulation of yolk polypeptide synthesis in Drosophila ovaries and fat body by 20-hydroxyecdysone and a juvenile hormone analog

In adult female Drosophila melanogaster an increase in the synthesis and secretion of three yolk polypeptides (YPs) occurs during the first 24 hr after eclosion. During organ culture, these same polypeptides are synthesized and secreted into the medium by both fat body and ovaries. Two hormones, 20-hydroxyecdysone (20-HE) and a juvenile hormone analog (ZR-515) stimulate synthesis and secretion of YPs into the hemolymph of isolated female abdomens. The present experiments were undertaken to compare synthesis of YPs in normal females with YP synthesis in preparations deprived of anterior endocrine glands, and to find which hormone stimulates synthesis in the different organs. Separation of hemolymph proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that at eclosion incorporation of [³⁵S]methionine into YP1 and YP2 was low and was barely detectable in YP3. Over the next 24 hr the rate of label incorporation increased for all the YPs. Isolation of female abdomens at eclosion prevented this increase in label incorporation but did not entirely abolish YP synthesis. Application of either ZR-515 or 20-HE to isolated abdomens stimulated up to ninefold label incorporation into three polypeptides which comigrated with YPs from normal vitellogenic females. The response of isolated abdomens to ZR-515 or 20-HE was first detectable between 90 and 135 min after hormone application. The stimulated bands were confirmed to be YPs by a comparison of peptide digests of each of the three labeled polypeptides with those of the yolk polypeptides from intact vitellogenic females. The hypothesis that the two hormones might act on different organs was tested by treating isolated female abdomens with various concentrations of either ZR-515 or 20-HE and then culturing the stimulated organ in vitro with [³⁵S]methionine. The fat body responded to both hormones by synthesizing and secreting into the culture medium polypeptides which comigrated with the YPs found in hemolymph, whereas the ovary produced similar polypeptides only after ZR-515. These secreted polypeptides were confirmed to be YPs by repeating the experiment using organs from heterozygotes for both YP2 and YP3 electrophoretic variants. Such organs synthesized five polypeptides which comigrated with the corresponding yolk polypeptides. These findings are discussed in relation to a hypothesis for the action of the two hormones.     

Comparison of Yolk Production in Seven Pyralid Moth Species

Summary

The yolk proteins of six pyralid moths were analyzed and compared with the yolk proteins of Plodia interpunctella (Hübner). When cross-reacted in an Ouchterlony double immunodiffusion with antiserum raised to either total yolk proteins or purified vitellin from P. tnterpunctella, the yolk proteins of Anagasta kuehniella (Zeller), Cadra cautella (Walker), C. figulilella (Gregson), and Ephestia elutella (Hübner), closely related members of the subfamily Phycitinae, showed strong precipitation lines that consisted of four major yolk polypeptides (YPs). The yolk proteins from Amyelois transitella (Walker) were only weakly reactive, whereas yolk proteins from Galleria mel-lonella (L.) were not precipitated by either antiserum. Abdominal body walls (containing primarily fat body) from late pharate adult females were incubated in vitro and they secreted two major polypeptides that had molecular masses similar to the vitellogenins (YP1 and YP3) from P. interpunctella. In addition, ovarioles from late pharate adult females were incubated in vitro, and they secreted two major polypeptides that had molecular masses similar to YP2 and YP4 from P. interpunctella. When late pharate adult females were injected with ³⁵S-Met, the hemolymph of all species contained vitellogins that were secreted by their respective body walls in vitro. Ovarioles from injected females contained many labeled polypeptides, but there were four major bands that corresponded consistently to the vitellogenins secreted from the fat body and the two major polypeptides secreted from the ovarioles. These data show that the production of the major YPs in these closely related pyralid species is very similar, and that there is considerable conservation of immunological characters of yolk proteins in the subfamily Phycitinae.     

Chromosome polymorphism in the yeast Saccharomyces

The variability of chromosomal band patterns was determined by pulse electrophoresis. The natural strains differed by the quantity and electrophoretic mobility of chromosomal DNA bands. The strains of independent genetic stocks originated from the XII race of Saccharomyces cerevisiae showed less significant difference in band patterns than the strains of different species of the Saccharomyces genus. The progeny of among strains with different karyotypes hybrid showed non-regular segregation of parental bands, the occurrence of new bands and the bands with altered mobility. Reverse crosses of hybrid progeny with strains of Peterhoff genetic stocks of S. cerevisiae led to decrease in chromosomal polymorphism. Homozygotization for ski5 allele and selection for increasing the copy number of killer plasmids was accompanied with repeated splash of polymorphism in 1-2 generations of intratetrad and intrafamily crossed hybrid progeny. Subsequent stabilization of electrophoretic karyotype took place, excluding the mendelian dimorphism of chromosome III, with was a stable trait of the last 6 generations of that progeny.     

Biosynthesis of Drosophila yolk polypeptides

The three yolk polypeptides (YPs) of Drosophila are synthesized and secreted by female fat body and ovarian follicle cells, sequestered by pinocytosis into oocytes, and finally deposited into yolk granules. The biosynthesis of the YPs was studied using two-dimensional gels. Labeling the YPs with [³⁵S]-cysteine, an amino acid found only near the amino terminus of YP1 and YP2, showed that an amino terminal peptide is removed from YP1 and YP2 shortly after or during translation. Intermediates in YP biosynthesis corresponding in electrophoretic mobility to pancreatic membrane-processed primary translation products were also detected in a 5-min pulse label with [³⁵S]-methionine. Genetic variants that alter YP structure were used to identify which YP precursor comes from which Yp gene. Pulse labeling with [³⁵S]-methionine revealed that all three YPs becomes more negatively charged, that YP1 and YP2 become heterogeneously charged, and that YP1 gains in apparent molecular weight within 15 min after translation. Injecting female flies with radioiabeled sugars or orthophosphate revealed that the YPs are glycosylated and phosphorylated. Treating hemolymph proteins with phosphatase showed that phosphorylation is responsible for much of the change in charge and increase in molecular weight of the maturing YPs. These experiments with wild-type flies provide a basis for the analysis of mutations at the Yp genes which alter the structure of individual YPs.     

A yolk protein mutant leads to defects in the secretion machinery of Drosophila melanogaster.

The three yolk proteins of Drosophila melanogaster are synthesized in the fat body and ovarian follicle cells. A mutation in yolk protein 3, YP3S1, has been described in which the leader sequence is not cleaved from the protein. We describe here ultrastructural and molecular studies on the YP3S1 mutant and show that the mutant protein enters the secretory pathway and forms precipitates, often as electron dense material in excessive elaborations of the plasma membrane. Females homozygous for YP3S1 lay fewer eggs than wild type flies and these embryos are less viable. The abnormal ultrastructure of the yolk spheres observed suggests that whilst YP3 is not completely essential for viability, it is required for normal yolk sphere morphogenesis.     

Detection of relationships among microsporidan isolates by electrophoretic analysis: hydrophilic extracts

Hydrophilic spore proteins were extracted from Nosema sp. and Nosema trichoplusiae. These proteins were subjected to electrophoretic analysis. The resulting electrophoretic spectra were found to be unstable when (1) two genera of hosts were used for spore propagation, (2) hosts were reared at a variety of temperatures, (3) protein was extracted from spores stored for different periods of time, or (4) spore incubation period was varied. Comparison of the major bands obtained from spore protein of the isolates indicated no overlap in relative migration values. Although variation in spectra was observed, the use of major band patterns indicate electrophoretic analysis of hydrophilic spore protein can provide characters useful in the separation and identification of microsporidan isolates. Nosema sp. and Nosema trichoplusiae are not considered to be closely related phylogenetically.     

Protein intensity changes in the hemoglobin and plasma electrophoretic patterns of Oreochromis niloticus in response to single and combined Zn and Cd exposure

The purpose of this study was to evaluate the effects of metals on the electrophoretic patterns of hemoglobin and blood plasma proteins of Oreochromis niloticus. Fish were exposed to 0.5 and 5.0 mg/L Zn, 0.1 and 1.0 mg/L Cd, and 0.5 mg/L Zn + 0.1 mg/L Cd, and 5.0 mg/L Zn + 1.0 mg/L Cd mixtures for 7 and 28 days. In all concentrations tested, electrophoretic pattern of hemoglobin and plasma proteins by cellulose acetate electrophoresis consist of three and eight bands, respectively. The three bands for hemoglobin are one cathodic (Hb1) and two anodic (Hb2 and Hb3) bands. The protein intensity in hemoglobins of fish following Zn, Cd, and Zn + Cd exposures decreased in Hb1, whereas it increased in Hb3. The eight bands for plasma proteins are 60, 78, 87, and 94 kDA high molecular weight proteins (HMP) for four bands and 120, 132, 176, and 273 kDA very high molecular weight proteins (VHMP) for four bands. The level of 60, 78, and 94 kDA HMP and 120, 132, and 176 kDA VHMP increased in response to single and combined Zn and Cd exposure. Also, there was increasing level of the metals in the whole blood with increasing concentrations of metals in the exposure medium and with increasing duration of exposure.     

Protein Electrophoretic Analysis of Amyloplast and Cytoplasm Ribosomes from Lotus Cotyledon

Amyloplasts and cytoplasmic ribosomes in cotyledon cells of lotus (Nelvmbo nucifeva Gaertn. ) have been observed on the basis of morphology. Isolation of these ribosomes by centrifugation through 30% to 55% (W/V) sucrose density gradient resulted in three bands of amyloplasts ribosomes and four bands of cytoplasmic ribosomes. The authors used these ribosomes bands for SDS-PAGE electrophoresis to analyse ribosomes of proteins. The patterns of SDS-PAGE between cytoplasmic ribosomes of proteins and amyloplasts ribosomes of proteins were different. The amyloplasts ribosomes of proteins showed 26 kD and 23 kD bands, and the cytoplasmic ribosomes of proteins showed 65 kD band. The analysis of electrophoretic patterns of the cytoplasmic ribosomes of proteins showed that there was a newly synthesized ribosomes protein with 19 kD molecular weight in 18 to 20 days after fertilization.     

曲霉和青霉属内杂种和亲株的蛋白质电泳图谱比较

曲霉属内黑曲霉(Aspergitlus niger)与米曲霉(A.oryzae)具有特征明显不同的可溶性蛋白质电泳图谱,其种间杂种具有双亲的部分或全部电泳带并与黑曲霉相近。来自杂种Ⅰ的多数分离子电泳带与黑曲霉相近,只有一个分离子产生米曲霉的电泳带并具有米曲霉的遗传特性。青霉属内产黄青霉(Penicillium chrysogenum)与展青霉(P.patulum)种间及种内不同菌株间的电泳图谱基本相同,种内或种间杂种具有双亲的电泳带。结果讨论了蛋白质图谱分析的意义。     

Identification of a female-sterile mutation affecting yolk protein 2 in Drosophila melanogaster

Summary The three yolk proteins (YP1, YP2 and YP3) of Drosophila melanogaster are synthesised in the fat body and ovarian follicle cells and selectively accumulated in the developing oocytes to provide a nutrient source for embryogenesis. We have described the phenotype of a temperaturesensitive female-sterile mutant, fs(1) K313, and characterised its yolk proteins. This mutation affects the secretion of YP2 and is the first mutation affecting YP2 to be described. Using genetic and molecular tests we argue that the female-sterile phenotype results, at least in part, from the abnormal secretion of YP2 perturbing the follicle cell secretory pathway in general and thus causing defects in chorion protein secretion. The gene coding for YP2 in fs (1) K313 has been cloned and sequenced. Two amino acid substitutions have been found which probably cause the abnormal secretion of YP2 and the resulting female-sterile phenotype.     

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.     

Inheritance of electrophoretic variants of tuber proteins in Solanum tuberosum haploids

Soluble tuber proteins were separated by discontinuous polyacrylamide gel electrophoresis on vertical slabs. Banding patterns of proteins stained with Coomassie Blue in 7.5% acrylamide gels (pH 4.3) were few and distinctive for haploids (2n = 2x = 24) derived from several cultivars (2n = 4x = 48). Katahdin and Chippewa haploids have only three different banding patterns for the eight fastest moving bands. The haploids have either the parental pattern (all eight bands) or one of two complementary banding patterns (four bands). The frequency of these patterns among the haploids indicates that the eight bands are controlled by one locus which is duplex (A¹A¹A²A²) in the parents. Haploids with the genotype A¹A² have eight bands. A¹A¹ haploids have four bands, and A²A² have the other four bands. Tawa haploids have in equal numbers either the eight (A¹A²) or four (A²A²) band pattern. Thus the genotype of Tawa is A¹A²A²A². The control of four bands by one allele could be explained by assuming that these alleles are involved in posttranslational modification or assembly of one or two protein species. Another explanation is that pseudoalleles or redundant genes produce the groups of protein bands. The eight proteins studied apparently are of similar molecular weight but differ in charge.