Molecular characterization of a cDNA encoding vitellogenin in the banana shrimp,Penaeus (Litopenaeus)merguiensis and sites of vitellogenin mRNA expression (original) (raw)
Related papers
Gene, 2003
Vitellogenin is the major egg yolk protein synthesized in female shrimp during gonad maturation. Although there are several reports for the cloning of vitellogenin complementary DNA (cDNA) in different crustaceans, little is known of the gene organization of this protein. This study reports the first cloning and characterization of a full-length gene encoding the vitellogenin precursor from the shrimp Metapenaeus ensis. By genomic DNA library screening, six different lambda clones were isolated using shrimp partial gene sequence as probe. Initial DNA sequence determination revealed that these clones are derived from different genes with coding sequence similar to other crustacean vitellogenins. Two of these clones were used for further analysis. One of the lambda clones (l3.3) carries most of the coding sequence that correspond to the M. ensis vitellogenin gene (MeVg1) and the other clone (l8.3) carries a smaller portion of the coding sequence of a different vitellogenin gene (MeVg2). The l3.3 clone was chosen for further characterization. To clone the remaining 5 0 end upstream promoter region, 5 0 untranslated region and the remaining coding sequence of MeVg1, a polymerase chain reaction (PCR)-based gene walking approach was used. Subsequently, a PCR clone with overlapping sequence identical to the genomic clone was obtained and the organization of MeVg1 gene was constructed. The MeVg1 gene consists of 15 exons and 14 introns spanning approximately 10 kb. Several potential cleavage sites were identified from the deduced vitellogenin precursor. Cleaving of the precursor in these sites would result in the production of several vitellogenin subunits. To clone the cDNA for the vitellogenin, 5 0 and 3 0 rapid amplification of cDNA ends was performed using ovary cDNA of the shrimp. A 4.4 kb 5 0 cDNA clone and a 4 kb 3 0 end cDNA clone were isolated. The size of the reconstructed cDNA for M. ensis Vg is 7.97 kb and consists of the longest open reading frame of 7776 bp. Unlike the vitellogenin precursor of most insects and vertebrates, the deduced vitellogenin precursor lacks the polyserine domain important for receptor-mediated endocytosis. Phylogenetic analysis revealed a closer relationship of the MeVg1 with other crustacean vitellogenins but distantly related to other invertebrate and vertebrate vitellogenins. By reverse transcription-PCR, we have demonstrated that the shrimp MeVg1 gene is expressed only in the ovary and hepatopancreas while the MeVg2 gene is expressed exclusively in the hepatopancreas. In conclusion, the shrimp ovary also contribute significantly in the production of vitellogenin at transcription level and the gene organization of the shrimp protein may provide an insight in the evolution of this group of important proteins. q
Journal of Experimental Zoology, 2004
In order to determine the primary structure of vitellogenin in a protandric species, the coonstriped shrimp Pandalus hypsinotus, we previously purified four vitellin components (designated as VnA, VnB, VnC, and VnD, respectively), and chemically analyzed their partial amino acid sequences. In this study, we subsequently cloned a cDNA encoding vitellogenin in this species based on the N-terminal and internal amino acid sequences of VnA, as well as the N-terminal sequence of VnC. The open reading frame of this cDNA encoded a pro-vitellogenin in which vitellins were arranged as follows: NH 2 -VnA-VnB-VnC/D-COOH. The deduced amino acid sequence possessed a single consensus cleavage sequence, R-X-K/R-R, along the lines of vitellogenins reported in other crustaceans and insects, and the N-terminal sequence of VnB was immediately preceded by this sequence. The comparison of primary structures revealed the existence of a basic and characteristic structure for the vitellogenin molecule in decapod crustacean species, and phylogenetic analysis reflected the current taxonomic classifications of Crustacea. An approximately 8 kb-long transcript of the vitellogenin gene was detected in the hepatopancreas of female shrimps having a gonadosomatic index higher than 1.0 by Northern blot analysis, but was not observed in the hepatopancreas and gonads of male shrimps and the hepatopancreas of female shrimps having a gonadosomatic index lower than 1.0. These results indicate that the hepatopancreas is responsible for vitellogenin synthesis.
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2001
The site of yolk protein synthesis in crustaceans has long been a subject of controversy. The vitellogenin gene structure was partially reported only very recently in Macrobrachium rosenbergii, after which the hepatopancreas was confirmed as the extraovarian site of vitellogenin synthesis in that species. Ovaries are the most frequently reported as the site of yolk protein synthesis in penaeid shrimp. Using cDNA reversed-transcribed from mRNA isolated from the hepatopancreas of vitellogenic female shrimp, Penaeus monodon, we found that its deduced amino acid sequence had high identity of 48% with that from M. rosenbergii vitellogenin. A similar location of the intron in the sequenced region of genomic DNA was also found between these two species. We therefore concluded that the hepatopancreas the extraovarian site of vitellogenin synthesis in P. monodon in vivo. The partial structure of vitellogenin gene is presented in this study. ᮊ
Characterization of vitellin from the ovaries of the banana shrimp Litopenaeus merguiensis
Comparative Biochemistry and …, 2006
Vitellin (Vt) was purified from ovary extracts of mature females of the banana shrimp Litopenaeus merguiensis using DEAE-Sephacel and Superdex 200 columns. Native Vt had an apparent molecular mass of 398 kDa as determined by native PAGE and by gel filtration chromatography. Under reducing and denaturing conditions (SDS-PAGE), Vt is composed of two major subunits of 87 and 78 kDa, although some faint bands were also detected. The N-terminal 10 amino acids sequence of the 78 kDa subunit is identical to that of Litopenaeus vannamei Vt and very similar to that of Litopenaeus japonicus vitellogenin (Vg) as well as Litopenaeus semisulcatus Vt, with an identity of 89%. Anti-Vt polyclonal antibody raised against purified Vt shows a high specificity with only ovarian Vt and hemolymph Vg of vitellogenic shrimps in double immunodiffusion and Western blot assays. Vg and Vt concentrations in hemolymph, hepatopancreas and ovaries were measured by ELISA. Vg concentrations increased in the hemolymph in the early stages of ovarian development and declined in the maturation stages. As there were undetectable concentrations of Vg in the hepatopancreas while an elevation of Vg levels occurred in the hemolymph, during the time that Vt was accumulating in the ovaries during oogenesis, this would suggest that the contribution of Vg synthesized by the hepatopancreas only might be not sufficient for adequate development of the oocytes in the banana shrimp L. merguiensis during vitellogenesis. D
Molecular Reproduction and Development, 2007
An open reading frame (ORF) of vitellogenin (Vg) cDNA was amplified from the ovaries of the banana shrimp, Penaeus merguiensis. An examination of Vg-deduced amino acid sequence revealed the presence of cleavage sites at a consensus motif for subtilisin-like endoproteases prior to the N-terminal sequences of purified vitellin (Vt) subunits. A comparison of the primary structures of Vg molecules in decapod crustacean species revealed the existence of a common characteristic structure, and phylogenetic analysis reflected the current taxonomic classifications of crustaceans. A PCR product of 1.1 kb encoding the 3'-end of Vg cDNA was cloned from the hepatopancreas. Although its sequence was almost identical to that of the same region of the ovarian Vg, with only 18 nucleotide differences, analysis suggests that they have been subjected to natural selection, indicating that there may be two different, tissue-specific Vg genes in P. merguiensis. This is consistent with the different expression patterns of Vg mRNA, as determined by real-time PCR. Vg mRNA levels were maintained at low levels during the previtellogenic stage and they increased as vitellogenesis progressed to reach a peak at the early vitellogenic stage in the ovary or at the vitellogenic stage in the hepatopancreas, and thereafter, levels decreased. Expression of Vg mRNA was much higher in the ovary compared to the hepatopancreas at all stages of ovarian development, implying that the ovary is mainly responsible for Vt synthesis. These indicate that penaeids constitute a unique model for vitellogenesis, showing intraovarian gene expression and synthesis of yolk protein.
Aquaculture, 2006
Although the gene fragment and partial cDNA for the vitellogenin (Vg) of Penaeus monodon have been reported for several years, the complete gene sequence and full-length cDNA of this Vg has not been reported. Here we report the gene organization and cloning of the full-length cDNA of P. monodon Vg. Similar to the MeVg1 of M. ensis, the Vg gene of P. monodon (PmVg1) is confirmed to consist of 14 introns and 15 exons. Additionally, genomic DNAs that show high sequence identity to PmVg1 were isolated from a single shrimp. The result confirmed the existence of multiple Vg genes in P. monodon. PmVg cDNA is 7.8 kb in size and the deduced precursor is most similar to the Vg of Penaeus merguenesis (86% identity). Contrary to the previous report, similar levels of PmVg1 transcripts can be detected from the hepatopancreas and ovary suggesting that the ovary also plays a major role in the contribution of PmVg1 transcript during vitellogenesis. In shrimp at early vitellogenesis (i.e., GSI b 3%), the expression of PmVg1 in the ovary and hepatopancreas is low; the expression reached the maximum level in shrimp with GSI 4-8% and decreased in shrimp with GSI N 9%. Farnesoic acid and 20-hydroecdysone can cause a significant stimulation of PmVg1 expression in an in vitro hepatopancreas explant culture. In conclusion, vitellogenesis in P. monodon is most likely the result of the expression of multiple vitellogenin genes and the hepatopancreas and ovary contribute equally to the production of PmVg1 transcripts.
Journal of Crustacean Biology, 2013
A complete cDNA sequence of vitellogenin (Vg-H) was cloned from the hepatopancreas of Fenneropenaeus merguiensis (De Man, 1888). The full-length Vg-H gene consists of 7958 bp and contains an ORF of 7761 bp. It encodes a polypeptide of 2587 amino acids, comprising a predicted molecular mass of 283 kD and the theoretical pI is 6.5. The amino acid sequences of the mature vitellogenin (Vg) composed of a signal peptide, three possible O-glycosylation sites, three phosphorylation sites and putative possessing sites (R-X-K/R-R) recognized by subtilisin-like endoproteases. Cleavage at residues 725 to 728 will produce two Vg subunits (78 and 200 kD), which the N-terminal amino acid sequence of 78 kD subunit is identical to that of vitellin purified from the ovary. These properties suggest that Vg protein encoding by Vg-H undergoes processing at its synthetic sites prior to being transported to developing oöcytes. The deduced primary structure of Vg-H showed the highest identity (98.8%) to the Vg-O previously cloned from the ovary of the same species. It is more related to the penaeid Vg sequences than that of nonpenaeids. In situ hybridization revealed that mRNA encoding Vg was expressed both in follicle cells in the ovary and parenchyma cells in the hepatopancreas. In the developing ovary, highest levels were detected during the early vitellogenic stage 2, declining thereafter. In the hepatopancreas, Vg mRNA levels reached a maximum at stage 3 of ovarian development. Profiles of Vg mRNA expression in the ovary and hepatopancreas suggest that the contribution of these tissues to vitellogenesis is inversely related during gonad maturation. Thus, Vg gene sequences expressed in the ovary and hepatopancreas are most likely identical and both tissues are responded for yolk protein synthesis in F. merguiensis.
General and Comparative Endocrinology, 2006
The gene that encodes vitellogenin (Vg), the precursor of the major yolk protein, vitellin, is expressed during vitellogenesis in decapod crustaceans. In this study, we sequenced the full-length cDNA from the Pacific white shrimp Litopenaeus vannamei Vg gene (LvVg). This is the first open thelycum penaeid shrimp Vg cDNA to be sequenced. The transcript encodes a 2587 amino acid polypeptide with up to 85% identity to Vg of different penaeid species. Peptide mass fingerprints (PMFs) of the vitelline polypeptides suggest that the predicted endoprotease cleavage site at amino acids 725-728 does indeed undergo cleavage. Five prominent high-density lipoprotein polypeptides of masses 179, 113, 78, 61, and 42 kDa were isolated from vitellogenic ovary, and their PMFs were determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) spectrometry. It is likely that these polypeptides are all products of the LvVg gene. Removal of the X-organ sinus gland complex (XO-SG), which secretes the neurohormones that control the endocrine system regulating molt and reproduction, can induce both these processes. During the course of a number of molt cycles in induced sub-adult females, periodic ovarian growth and resorption were observed. Ovary growth correlated with LvVg expression in both the hepatopancreas and the ovary. Expression in ovaries of induced intermolt-early premolt females was significantly higher compared to all other sub-groups. Expression in ovaries of induced females was significantly higher compared to hepatopancreas at all molt cycle stages. Periodicity of molt and vitellogenesis in endocrinologically induced sub-adult shrimps may serve as a model to study alternate regulation of gene expression during these two processes.
Identification and characterization of vitellin in a hermophrodite shrimp, Pandalus kessleri
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1989
A female specific protein (FSP, vitellogenin) in hemolymph and its related ovarian protein (vitellin) of Pandalus kessleri were studied by means of electrophoretical and immunological procedures. 2. The vitellin was purified from vitellogenic ovaries using hydroxylapatite, DEAE cellulose and Sepharose 6B columns, consecutively. 3. The vitellin had a molecular weight of approximately 560 kD and was composed of two subunits, 81 and 110 kD, respectively. 4. The vitellogenin concentrations in the hemolymph increased as vitellogenesis in the ovarian oocytes advanced and dropped markedly after the release of mature eggs.
Is There Extraovarian Synthesis of Vitellogenin in Penaeid Shrimp?
The Biological Bulletin, 1992
Extraovarian synthesis of vitellogenin (Vg), has been reported for several crustaceans, mainly in the subepidermal adipose tissue (SAT) or the hepatopancreas (HEP). The precise site(s) of Vg synthesis in penaeid shrimp is hitherto unknown and was investigated in a large local species Pcnaeus semisulcalus de Haan. Protein synthesis was determined in SAT and HEP tissue pieces incubated //; vitro. Incubations were at 25 C for eight hours in an oxygen enriched atmosphere, under sterile conditions in a physiological medium, containing 14 Cleucine. At the end of the incubation period, tissue homogenates and medium samples were analyzed for de novo protein synthesis. Total protein synthesis was determined by trichloroacetic acid precipitation. Specific vitellin (Vt) synthesis was determined by radioimmunoprecipitation with a polyclonal Vt-specific antiserum. Characterization of other de novo synthesized proteins was carried out by fluorography from polyacrylamide gels. Subepidermal adipose tissues removed from females at all stages of ovarian development did not synthesize Vtspecific proteins, in spite of the fact that total protein synthesis levels were high. The major protein synthesized de novo in the SAT of males and females is a protein with an identical electrophoretic mobility as hemocyanin in polyacrylamide gels. In vitro protein synthesis in HEP tissues was low compared to SAT or ovary systems. Vtspecific de novo synthesized protein was identified in HEP's from early vitellogenic females, but constituted less than 15% of total protein synthesis. We have previously shown that ovarian tissues from vitellogenic females incubated in vitro exhibited high levels of protein synthesis, an average of 38% of which is Vt-specific (Browdy et ai. 1990, J. Exp. Zoo/. 255: 205-2 1 5). The calculated Vt syn