cDNA cloning and sequence analysis of the Xenopus laevis egg envelope glycoprotein gp43

The glycoproteins of the Xenopus laevis egg envelope function in fertilization and development. As the unfertilizable coelomic egg transits the pars recta region of the oviduct, it is converted to a fertilizable egg by limited proteolysis of the envelope glycoprotein gp43 to gp41. This conversion is caused by an oviductally secreted serine active site protease, oviductin. We cloned a cDNA for gp43 from an oocyte cDNA library. The cDNA encoded a 454 amino acid protein homologous to the ZPC family of glycoproteins previously shown to be present in mammalian and fish egg envelopes. Conserved ZPC domains and motifs present in the Xenopus sequence included a signal peptide sequence, an N-linked glycosylation site, and 12 aligned Cys residues. In mammalian and Xenopus sequences, a furin-like (convertase) site and a C-terminal transmembrane domain were present reflecting the biosynthesis of ZPC in these species via the secretory glycoprotein pathway. However, fish envelope glycoproteins lack these sequences since they are synthesized via a different route (in the liver, transported to the ovary, and assembled into the egg envelope surrounding the oocyte). Consensus amino acid residues were identified by sequence comparisons of seven ZPC family members; 19% of the amino acid residues were invariant and 48% of the residues were identical in at least four of the seven sequences. The consensus sequence was used to make structure-fertilization function predictions for this phylogenetically conserved family of glycoproteins.     

cDNA cloning of the developmentally regulated lamin LIII of Xenopus laevis.

Lamins are nucleoskeletal proteins which form intermediate type filaments in close association with the inner nuclear envelope membrane. Based on molecular and biochemical properties the lamins were grouped as type-A and type-B lamins, respectively. I have cloned the cDNA encoding lamin LIII of Xenopus which is the lamin protein present in oocyte nuclei and in cleavage nuclei. The data presented here indicate that a pool of maternal lamin LIII RNA is synthesized very early in oogenesis and that it continues to be present until gastrulation when the vast majority of the LIII RNA is degraded. Despite the similarities shared by all lamin proteins, the lamin LIII sequence neither possesses the features diagnostic for either type-A or type-B lamins nor does it show greater sequence similarity to one of the lamin types than to the other and thus it may represent a third type of lamin protein which may reflect its special function in oogenesis and early development.     

Molecular cloning of the cDNA coding for Xenopus laevis prion protein.

Isolation and characterization of the cDNA coding for the 216-residue Xenopus laevis prion protein is reported. Existence of this protein in amphibians was suggested by an EST fragment (accession number BG813008), while a conclusive demonstration is presented here. This protein exhibits a higher identity level to avian and turtle prion (more than 44%) than to mammalian prion (about 28%). Although most of the structural motifs common to known prion proteins are conserved in X. laevis, the lack of repeats represents a substantial difference. Other features worth noting are the presence of not perfectly conserved hydrophobic stretch, which is considered the prion signature, as well as the complete absence of histidine residues.     

Xenopus laevis contains two nonallelic preproinsulin genes. cDNA cloning and evolutionary perspective

We undertook the cloning of preproinsulin cDNAs from the South African clawed toad, Xenopus laevis, in order to study the role of insulin during embryogenesis in this species. We found that X. laevis contains two different preproinsulin cDNAs, both of which code for peptides containing 106 amino acids of typical structure but which differ by eight amino acids: one in the signal peptide, two in the B-chain, four in the C-peptide, and one in the A-chain. Southern blot analysis indicates that the two preproinsulin cDNAs identified correspond to two different nonallelic genes which we believe arose through a recent gene duplication within the amphibian radiation possibly during the development of tetraploidy in this species. Both genes are expressed, since we have recently identified the two corresponding insulins in pancreatic extracts of adult toads (Shuldiner, A.R., Bennett, C., Robinson, E.A., and Roth, J. (1989) Endocrinology, in press). These cDNAs represent the first amphibian preproinsulin sequences to be elucidated.     

cDNA cloning and developmental expression of fibroblast growth factor receptors from Xenopus laevis.

The heparin-binding growth factors constitute a family of homologous polypeptides including basic and acidic fibroblast growth factors (FGFs). These factors participate in a variety of processes, including wound healing, angiogenesis, neuronal survival, and inductive events in the early amphibian embryo. We have isolated three closely related species of cDNA clones for Xenopus FGF receptors. One of these, designated XFGFR-A1, encodes an open reading frame of 814 amino acids. A second class encodes an identical amino acid sequence with the exception of an 88-amino-acid deletion near the 5' end. This species probably arises through alternative splicing. A third class of cDNA corresponding to the shorter form of XFGFR-A1 was isolated and shown to be 95% homologous and is designated XFGFR-A2. Xenopus FGF receptors are similar to FGF receptors from other species in that they contain a transmembrane domain, a tyrosine kinase domain split by a 14-amino-acid insertion, and a unique conserved stretch of eight acidic residues in the extracellular domain. Overexpression of Xenopus FGF receptor protein by transfection of COS1 cells with the corresponding cDNA in a transient expression vector leads to the appearance of new FGF binding sites on transfected cells, consistent with these cDNAs encoding for FGF receptors. RNA gel blot analysis demonstrates that Xenopus FGF receptor mRNA is a maternal message and is expressed throughout early development. When blastula-stage ectoderm is cultured in control amphibian salt solutions, Xenopus FGF receptor mRNA declines to undetectable levels by late neurula stages. However, when cultured in the presence of FGF of XTC mesoderm-inducing factor, Xenopus FGF receptor RNA expression is maintained.     

稻瘟菌Ⅰ型烯醇化酶基因全长cDNA的电子克隆

利用电子克隆技术从稻瘟菌中克隆到一个新的Ⅰ型烯醇化酶全长cDNA,暂命为MgEno-1。MgEno-1全长1571核薯酸,其预测的ORF为1317核苷酸,共编码438个氨基酸。起始密码子ATG位于第53位,终止密码子TAA位于第1369位。序列分析表明该烯醇化酶与丝状真菌中已报道的其它烯醇化酶高度同源,且长度一致,这暗示烯醇化酶基因进化上高度保守,甚至有可能像18SrRNA一样可作为进化尺度。这将是第一个用电子克隆技术从稻瘟菌中克隆到的基因。     

Nuclear pore complex glycoprotein p62 of Xenopus laevis and mouse: cDNA cloning and identification of its glycosylated region

cDNA clones for nuclear pore complex glycoprotein p62 of two distantly related species, mouse and Xenopus laevis, were isolated. Antibodies raised against recombinant murine p62 react on protein blots with p62 of both species and decorate pore complexes. Analysis of the predicted protein sequence indicates that vertebrate p62 is organized into two structurally different regions. The entire carboxy-terminal half (86.7% identical amino acids) and the amino-terminal 56 amino acids (62.5% identity) have been highly conserved during evolution. The amino-terminal half contains several penta amino acid repeats and is able to form beta-sheets, whereas the carboxy-terminal half is predominantly organized in alpha-helical structures in part with heptad repeats typical for intermediate filament proteins. p62 of mouse and Xenopus is glycosylated by N-acetylglucosamine additions in the amino-terminal half. The region containing these potential glycosylation sites has been identified.     

cDNA cloning and expression in Xenopus laevis oocytes of pig renal dipeptidase, a glycosyl-phosphatidylinositol-anchored ectoenzyme.

Clones expressing renal dipeptidase (EC 3.4.13.11) have been isolated from a pig kidney cortex cDNA library after employing the polymerase chain reaction technique to amplify a region of the dipeptidase cDNA. The complete primary sequence of the enzyme has been deduced from a full length cDNA clone. This predicts a protein of 409 amino acids, a cleavable N-terminal signal sequence of 16 residues and two N-linked glycosylation sites. At the C-terminus of the predicted sequence is a stretch of mainly hydrophobic amino acids which is presumed to direct the attachment of the glycosyl-phosphatidylinositol membrane anchor. Expression of the mRNA for pig renal dipeptidase in Xenopus laevis oocytes led to the production of a disulphide-linked dimeric protein of subunit Mr 48,600 which was recognized by a polyclonal antiserum raised to renal dipeptidase purified from pig kidney cortex. Bacterial phosphatidylinositol-specific phospholipase C released renal dipeptidase from the surface of the oocytes and converted the amphipathic detergent-solubilized form of the dipeptidase to a hydrophilic form, indicating that Xenopus laevis oocytes can process expressed proteins to their glycosyl-phosphatidylinositol anchored form.     

In vitro translation of B-cell stimulatory factor-1 in Xenopus laevis oocytes

B-cell stimulatory factor-1 (BSF-1) can be translated in vitro in Xenopus laevis oocytes. This activity is blocked by an antibody to BSF-1. The RNA species coding for BSF-1 activity sediments of approximately 8-9S and is separable from RNA coding for interleukin-2 activity which sediments at approximately 11.5S. Finally, the fact that BSF-1 can be translated in vitro confirms that functions attributed to BSF-1 do not depend on contamination with other biologically active molecules such as phorbol myristate acetate.     

Purification, cDNA cloning, and expression profiles of the cyclobutane pyrimidine dimer photolyase of Xenopus laevis

Photolyase is a light-dependent enzyme that repairs pyrimidine dimers in DNA. Two types of photolyases have been found in frog Xenopus laevis, one for repairing cyclobutane pyrimidine dimers (CPD photolyase) and the other for pyrimidine-pyrimidone (6-4)photoproduct [(6-4)photolyase]. However, little is known about the former type of the Xenopus photolyases. To characterize this enzyme and its expression profiles, we isolated the entire coding region of a putative CPD photolyase cDNA by extending an EST (expressed sequence tag) sequence obtained from the Xenopus database. Nucleotide sequence analysis of the cDNA revealed a protein of 557 amino acids with close similarity to CPD photolyase of rat kangaroo. The identity of this cDNA was further established by the molecular mass (65 kDa) and the partial amino acid sequences of the major CPD photolyase that we purified from Xenopus ovaries. The gene of this enzyme is expressed in various tissues of Xenopus. Even internal organs like heart express relatively high levels of mRNA. A much smaller amount was found in skin, although UV damage is thought to occur most frequently in this tissue. Such expression profiles suggest that CPD photolyase may have roles in addition to the photorepair function.     

Comparative analysis of cloned larval and adult globin cDNA sequences of Xenopus laevis

cDNA clones complementary to 9 S poly(A)⁺ RNA from erythroblasts of anemic larvae and adults of Xenopus laevis have been prepared. Clones, containing at least 400 bp of cDNA, have been analyzed by cross-hybridization and restriction mapping. They were found to comprise four unrelated main groups of sequences (two larval and two adult) and each main group contained two related subgroups. Partial sequence analysis and comparison of restriction data to previously published maps allowed the four main groups to be identified as α- or β-globin sequences. The sequence divergence between the subgroups was determined by melting curves of homo- and heteroduplexes. We found that the larval sequences have diverged twice as far as the adult ones. To account for this result, different hypotheses on globin gene evolution are proposed.     

Cloning and expression of Xenopus laevis xSox12 cDNA

A family of SRY-related genes has been termed SOX. We have isolatedand sequenced a cDNA encoding xSox12 from Xenopus laevis ovary. The cDNA contained an open reading frame (ORF) coding for 470 amino acids encompassing an HMG box characteristic of the SOX family, a leucine zipper motif and glutamine-rich segments. The size of the xSox12 mRNA was determined to be 3.0 knt by Northern analysis. The ovary was the most prominent in the expression of the Sox mRNA among the various tissues of adult frog as far as examined.