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1.
Similarly to epidermal growth factor (EGF) and EGF-like repeats, the "P-domain" represents a cysteine-rich module that has been detected in the past in a variety of polypeptides, as well as in high molecular weight proteins. Here, a precursor for a secretory polypeptide (xP2) is characterized that consists of two P-domains. xP2 has been discovered in Xenopus laevis with the help of the polymerase chain reaction. In contrast to all other P-domain peptides, it is synthesized in the skin but not in the stomach or the pancreas. By this and other criteria, it cannot be considered simply as the X. laevis homologue of recently described P-domain peptides, viz. the spasmolytic polypeptides (PSP/hSP/mSP). Furthermore, a polyclonal antiserum was generated against the deduced C-terminal end of xP2. Due to its immunoreactivity with granular glands, as well as with the epidermis, the possibility of a growth factor activity for xP2 in the germinal layer is discussed. 相似文献
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3.
TFF-peptides (formerly P-domain peptides, trefoil factors) represent major secretory products of the mammalian gastrointestinal tract. A molecular cloning approach revealed the existence of two TFF-peptides, xP1 and xP4, also in the stomach of Xenopus laevis. Here, the localization of these two peptides by Western blot analysis as well as immunohistochemistry is presented. xP1 is found predominantly in the surface mucous cells of the stomach, whereas xP4 is mainly localized to a specific population of goblet cells in the esophagus, to mucous neck cells of the stomach, and to closely resembling cells in antral glands. xP4 in the esophagus and in the stomach differ by their N-glycosylation patterns. Compared to mammalian TFF-peptides, xP1 obviously represents the frog homologue of human TFF1 (formerly pS2) and xP4 seems to be the amphibian equivalent of human TFF2 (formerly hSP). 相似文献
4.
The amphibian gastrointestinal tract is remodeled from a larval-type to an adult-type during metamorphosis. In the present study, we examined the products of subtractive hybridization between tadpole and frog stomach cDNAs of Xenopus laevis in order to identify genes expressed specifically in the larval stomach epithelium. A new gene homologous to xP1 was obtained and named xP1-L. In the genome database of Silurana tropicalis, we found a homologue of xP1-L and named it stP1-L. RT-PCR showed that the expression of xP1-L was detected in stage 41/42 tadpoles. In addition, in situ hybridization showed that xP1-L was localized to surface mucous cells of the larval stomach. The H(+)/K(+)-ATPase beta subunit, a marker gene for manicotto gland cells in the tadpole stomach, was also detected at the same time. However, adult marker genes such as xP1 for surface mucous cells and pepsinogen C (PgC) for oxynticopeptic cells were not expressed in the tadpole stages. The expression of xP1-L gradually decreased towards the metamorphic climax and disappeared after stage 61 when larval-type gastric epithelium is replaced by adult-type. We found that xP1-L was never expressed in surface mucous cells of the adult-type stomach, and xP1, instead of xP1-L, was expressed. During T3-induced metamorphosis, xP1-L expression decreased in the same manner as during natural metamorphosis. Thus, xP1-L is a useful marker for larval surface mucous cells in tadpole stomach. This is the first demonstration of a marker gene specific for the surface mucous cells of the larval stomach. 相似文献
5.
《Gene expression patterns : GEP》2008,8(1):12-18
The amphibian gastrointestinal tract is remodeled from a larval-type to an adult-type during metamorphosis. In the present study, we examined the products of subtractive hybridization between tadpole and frog stomach cDNAs of Xenopus laevis in order to identify genes expressed specifically in the larval stomach epithelium. A new gene homologous to xP1 was obtained and named xP1-L. In the genome database of Silurana tropicalis, we found a homologue of xP1-L and named it stP1-L. RT-PCR showed that the expression of xP1-L was detected in stage 41/42 tadpoles. In addition, in situ hybridization showed that xP1-L was localized to surface mucous cells of the larval stomach. The H+/K+-ATPase β subunit, a marker gene for manicotto gland cells in the tadpole stomach, was also detected at the same time. However, adult marker genes such as xP1 for surface mucous cells and pepsinogen C (PgC) for oxynticopeptic cells were not expressed in the tadpole stages. The expression of xP1-L gradually decreased towards the metamorphic climax and disappeared after stage 61 when larval-type gastric epithelium is replaced by adult-type. We found that xP1-L was never expressed in surface mucous cells of the adult-type stomach, and xP1, instead of xP1-L, was expressed. During T3-induced metamorphosis, xP1-L expression decreased in the same manner as during natural metamorphosis. Thus, xP1-L is a useful marker for larval surface mucous cells in tadpole stomach. This is the first demonstration of a marker gene specific for the surface mucous cells of the larval stomach. 相似文献
6.
Immunological relationship between oocyte nuclear proteins of Xenopus laevis and X. borealis 总被引:1,自引:0,他引:1
Monoclonal antibodies (mABs) have been raised against oocyte nuclear proteins of Xenopus laevis and X. borealis and have been screened for species specificity. Although about 40% of all germinal vesicle polypeptides differ between the two species as judged by two-dimensional gel analysis, most mABs react with polypeptides of both species. Biochemical analysis of the antigens by immunoblotting revealed that a homologue of each antigen of one species could be detected in the other species, despite frequent differences in molecular structure. Nevertheless, five strictly species-specific mABs have been identified. All five are directed against the same acidic polypeptide B3 of X. borealis, which is a structurally altered homologue of the protein N1, previously described in X. laevis germinal vesicles. In oocyte nuclei of hybrids between X. laevis females and X. borealis males, polypeptide of both species appear to be accumulated equivalently. Exceptions to this rule are most easily explained by differences between individuals and by the loss of certain alleles resulting from the cross. 相似文献
7.
Sinorhizobium fredii USDA257, a soybean symbiont, exports several nodulation outer proteins (Nops) into the rhizosphere. These proteins, which are exported by a type III secretion system (TTSS), have a pivotal role in host-specific nodulation. The entire TTSS of S. fredii lies within a 31-kb region that includes conserved genes that code for secretion machinery proteins, Nops, and several open reading frames (ORF) of unknown function. Identifying the functions of these ORF is essential to understand fully the role of TTSS in nodulation. Here, we report the characterization of y4xP, an ORF of previously unknown function. Southern blot analysis revealed that USDA257 contains two copies of y4xP, while a sibling, USDA191, contains a single copy. The amino acid sequence of Y4XP is homologous to both eukaryotic and prokaryotic cysteine synthase, a key enzyme in sulfur assimilation. The coding region of USDA257 y4xP under control of T7 promoter was expressed in Escherichia coli, and the recombinant protein was purified by nickel-affinity chromatography. Antibodies generated against soybean cysteine synthase cross-reacted with the recombinant protein. A nonpolar mutant of y4xP of USDA191 showed a marked reduction in cysteine synthase activity. Enzyme activity was completely restored when the mutant was complemented with a plasmid containing the y4xP sequence. Cysteine synthase activity was confined to the cell cytosol. Extracellular protein fraction from genistein-induced USDA191 showed no cysteine synthase activity. This observation indicates that cysteine synthase, which is located in the TTSS locus, is not a type III secreted protein. A nonpolar cysteine synthase mutant was able to export all the Nops to the rhizosphere, albeit in reduced amounts compared with the wild-type USDA191. Interestingly, USDA191 cysteine synthase mutant was able to initiate nodules on 'McCall' soybean more efficiently than the wild-type. Our results demonstrate that y4xP encodes a cysteine synthase and inactivation of this gene enhances the ability of USDA191 to form nodules on 'McCall' soybean by regulating Nops production. 相似文献
8.
Biosynthetic pathways of two polypeptide subunits of the light-
harvesting chlorophyll a/b protein complex 总被引:20,自引:8,他引:12
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《The Journal of cell biology》1981,91(2):468-478
We have used an in vitro reconstitution system, consisting of cell-free translation products and intact chloroplasts, to investigate the pathway from synthesis to assembly of two polypeptide subunits of the light-harvesting chlorophyll-protein complex. These polypeptides, designated 15 and 16, are integral components of the thylakoid membranes, but they are products of cytoplasmic protein synthesis. Double immunodiffusion experiments reveal that the two polypeptides share common antigenic determinants and therefore are structurally related. Nevertheless, they are synthesized in vitro from distinct mRNAs to yield separate precursors, p15 and p16, each of which is 4,000 to 5,000 daltons larger than its mature form. In contrast to the hydrophobic mature polypeptides, the precursors are soluble in aqueous solutions. Along with other cytoplasmically synthesized precursors, p15 and p16 are imported into purified intact chloroplasts by a post- translational mechanism. The imported precursors are processed to the mature membrane polypeptides which are recovered exclusively in the thylakoids. The newly imported polypeptides are assembled correctly in the thylakoid lipid bilayer and they bind chlorophylls. Thus, these soluble membrane polypeptide precursors must move from the cytoplasm through the two chloroplast envelope membranes, the stroma, and finally insert into the thylakoid membranes, where they assemble with chlorophyll to form the light-harvesting chlorophyll protein complex. 相似文献
9.
Ikuzawa M Yasumasu S Kobayashi K Inokuchi T Iuchi I 《Journal of experimental zoology. Part A, Comparative experimental biology》2004,301(12):992-1002
Through subtractive hybridization, H+/K+-ATPase beta subunit mRNA, highly expressed in the larval stomach of Xenopus laevis, was isolated. In situ hybridization demonstrated that the H+/K+-ATPase beta subunit mRNA was exclusively expressed in manicotto gland cells of the larval stomach, not in any other cell. Northern blot analysis showed that metamorphosis-associated changes of the H+/K+-ATPase beta subunit mRNA expression in the stomach were characterized by high expression in tadpoles, a considerably lower expression in metamorphosing tadpoles, and a re-increase of expression in froglets. Further in situ hybridization showed that the decrease of expression correlated with the degeneration of larval type epithelium in the manicotto gland, while the re-increase correlated with the differentiation of oxynticopeptic cells of the adult type stomach. Moreover, the H+/K+-ATPase beta subunit mRNA was expressed in adult epithelial primordia. Such changes were found in thyroid hormone-induced precocious metamorphosis. Based on studies using this ATPase as well as xP1 and PgC (pepsinogen C) as molecular markers, this study discusses a probable cell lineage involved in metamorphosis-associated stomach remodeling. The pH of luminal contents of the larval stomach was found to be lower than 2. In addition, the pH of an isolated stomach changed from 7.2 to lower than 4 after incubation in Ringer's solution, suggesting acid production from the larval stomach. This is the first demonstration of the H+/K+-ATPase-mediated acid production and secretion in the larval stomach of Xenopus laevis. 相似文献
10.
Efficient transformation of the yellow fever mosquito Aedes aegypti using the piggyBac transposable element vector pBac[3xP3-EGFP afm 总被引:6,自引:0,他引:6
Kokoza V Ahmed A Wimmer EA Raikhel AS 《Insect biochemistry and molecular biology》2001,31(12):275-1143
We report efficient germ-line transformation in the yellow fever mosquito Aedes aegypti accomplished using the piggyBac transposable element vector pBac[3xP3-EGFP afm]. Two transgenic lines were established and characterized; each contained the Vg-Defensin A transgene with strong eye-specific expression of the enhanced green fluorescent protein (EGFP) marker gene regulated by the artificial 3xP3 promoter. Southern blot hybridization and inverse PCR analyses of genomic DNA demonstrated a precise piggyBac-mediated, single copy insertion of the pBac[3xP3-EGFP afm,Vg-DefA] transposon in each transgenic line. For each line, genetic analysis confirmed stability and integrity of the entire transposon construct in the mosquito genome through the G2–G6 generations. Successful establishment of homozygous transgenic lines indicated that in both cases a non-lethal integration of the transposon into the mosquito genome had occurred. The 3xP3-EGFP marker was tested in mosquitoes with different genetic backgrounds. In white-eyed transgenic mosquitoes, the strong eye-specific expression of GFP was observed throughout all stages of development, starting from newly hatched first instar larvae to adults. A similar level and pattern of fluorescence was observed in red-eyed mosquitoes that were generated by crossing the 3xP3-EGFP transformants with the khw white-eye mosquitoes transformed with the Drosophila cinnabar gene. Importantly, the utility of the 3xP3-EGFP, as marker gene for transformation of wild type mosquitoes, was demonstrated by strong eye-specific GFP expression in larval and pupal stages of black-eyed hybrids of the 3xP3-EGFP white-eye transformants and the wild type Rockefeller/UGAL strain. Finally, analysis of the Vg-DefA transgene expression in transformants from two established lines demonstrated strong blood-meal activation and fat-body-specific expression regulated by the Vg 1.8-kb 5′ upstream region. 相似文献
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12.
A monoclonal antibody against nuclear lamina proteins reveals cell type-specificity in Xenopus laevis 总被引:15,自引:0,他引:15
Immunofluorescence microscopy shows that the monoclonal murine antibody PKB8 stains the nuclear lamina of various somatic cells from vertebrates as diverse as mammals, birds and amphibia. It also decorates the nuclear periphery of oocytes from rat and chicken but does not react with spermatocytes, spermatids and spermatozoa. Immunoblotting experiments demonstrate reaction with lamina polypeptides A, B and C of rat, with lamina polypeptide A of chicken, and with lamina polypeptides LI and LII of erythrocytes of the frog, Xenopus laevis. Antibody PKB8 does, however, not bind, on blotted polypeptides and on sections through ovaries, to the pore complex-lamina polypeptide of Mr 68000 present in Xenopus oocytes. These results reveal the existence of a common antigenic determinant in all three lamina polypeptides of mammals, in one lamina polypeptide of chicken and in two amphibian lamina polypeptides. The immunological data also indicate that, in Xenopus laevis, pore complex-lamina polypeptides of somatic cells and oocytes are distinct. The Mr 68000 protein of Xenopus oocytes is also different from polypeptides LI and LII of somatic Xenopus cells by tryptic peptide mapping. The results suggest that nuclear pore complex-lamina polypeptides represent a family of related polypeptides containing regions highly conserved during evolution and that these polypeptides can be differentially expressed in cells of at least one species, Xenopus laevis. 相似文献
13.
From skin of Xenopus laevis, cDNA libraries were constructed and clones coding for the precursors of caerulein were isolated and sequenced. Using restriction endonuclease digestions, three different types of preprocaerulein cDNAs could be discerned. These were termed types I, III, and IV in accordance with the number of caerulein copies present in the sequence, the type III being the most abundant one. An incomplete copy of a fourth variant, termed type I', was also found. Besides deletions/insertions encompassing one or two caerulein sequences, these types also differ from each other by several point mutations. In the homologous precursor polypeptides deduced from the nucleotide sequence of these cloned cDNAs, the caerulein copies are flanked by complex processing sequences. These are Arg-Arg-Phe-Ala-Asp-Gly or Arg-Arg-Asp-Gly at the amino-terminal side and Gly-Arg-Arg at the carboxyl end. Between caerulein copies, highly homologous segments are present both at the polypeptide and cDNA level. This homology is evident both within a given precursor, where up to three such segments are present, and between the different types of precursors. We conclude that preprocaerulein cDNAs in the skin of X. laevis represent a small family, at least part of which is derived from different genes rather than being formed by alternative splicing of pre-mRNAs. 相似文献
14.
Three intermediates in adenovirus assembly have been defined; nuclear intermediates, young virions, and mature virions. The nuclear intermediates are fragile and heterogenous in size (550S-670S) and withstand separation on ficoll gradients but fall apart upon CsCl gradient centrifugation unless prefixed with glutaraldehyde. They contain both capsid and core structures, and the core structures are preferentially released during purification in CsCl. The precursor polypeptides pVI and pVII are present in the intermediates without any corresponding mature polypeptide. The young virions (Ishibashi and Maizel, 1974) are stable and preferentially confined to the nuclei after cell fractionation. They contain both uncleaved precursor polypeptides and their cleavage products. The mature virions accumulate in the cytoplasm during cell fractionation and contain the final mature polypeptides. Pulse-chase labeling kinetics, focusing on the precursor polypeptides, suggest that these three classes participate in assembly of adenovirus. Tryptic peptide maps establish that polypeptide pVI is the precursor of polypeptide VI, but only a small fraction of polypeptide 26K can in vivo account for polypeptide VIII. 相似文献
15.
The precursors of the legumin-like storage protein from developing white lupin seeds (35 days after flowering) are trimers composed of protomers of M(r) 72,000 or 67,000. Some subunits of these oligomers contain processed precursor polypeptides, namely alpha polypeptides of either 52,000 or 44,000 linked through disulphide bonds to a beta polypeptide of 21,000, typical of the mature legumin. The prolegumin is glycosylated. Legumin oligomers purified from the same seeds are both trimers and hexamers; some of their subunits are still made of precursor polypeptides. The hexamer contains less precursor polypeptide than the trimer. A low level or absence of precursor appears to be a condition of hexamer assembly. The heterogenous prolegumin and legumin oligomers represent intermediates in the processing of the prolegumin to mature legumin. Hydrophobic-interaction chromatography on TSK-phenyl-5PW and titration with the hydrophobic probe 8-anilino-1-naphthalenesulphonate indicate that the legumin is less hydrophobic than the prolegumin. This is attributed to structural rearrangements at processing of the propolypeptide, made evident by the behaviour in CD and by the second-derivative ultraviolet spectra of the two proteins. The total protein extract of developing cotyledons at 40 days after flowering contains endopeptidases, similar to those existing in the resting seeds, which cause a limited cascade degradation of the prolegumin and legumin. 相似文献
16.
Antisera to disrupted Rauscher leukemia virus (RLV) or to the purified Rauscher viral 30,000 dalton polypeptide were used to specifically precipitate newly synthesized intracellular viral polypeptides from extracts of infected NIH Swiss mouse cells (JLS-V16). Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of extracts from cells pulse-labeled for 10–20 min with 35S-methionine showed that immune precipitates contained none of the nonglycosylated internal structural polypeptides of mature viruses. The major viral-specific polypeptides labeled in 10 min included polypeptides of 180,000, 140,000, 110,000, 80,000, and 60,000 daltons with minor polypeptides of 65,000, 50,000, and 40,000 daltons. Labeling the intracellular virus-specific polypeptides with 14C-glucosamine indicated that the 180,000, 110,000, 80,000, and 60,000 dalton polypeptides were glycosylated, and all but the 110,000 dalton polypeptides are contained in the mature virions. Based on pulse-chase experiments, it appears that at least 3 of the large polypeptides (140,000, 65,000, and 50,000 daltons) are precursors to the three major internal structural polypeptides of the mature virions. 相似文献
17.
Connie M. Lee Nancy Osterbauer Shuping Jiao Marvin W. Fawley 《Journal of phycology》1992,28(3):309-311
The biosynthesis of the light-harvesting complex (LHC) polypeptides of the green flagellate Mantoniella squamata (Manton et Parke) Desikachary (Micromonadophyceae, Chlorophyta) was examined by in vivo polypeptide labeling and immunoprecipitation of in vitro translation products. Using protein synthesis inhibitors, the LHC polypeptides were shown to be synthesized on 80S cytoplasmic ribosomes and not in the chloroplasts of cells. Poly (A)+ RNA was isolated and proteins were synthesized by a rabbit reticulocyte lysate system, with antisera raised against M. squamata LHC used for immunoprecipitation from the translation products. One polypeptide 3-5 kDa larger than mature LHC polypeptides was immunoprecipitated. These studies indicate that although the LHC of M. squamata is quite different from the LHC of most green plants, the LHC polypeptides are synthesized as precursors in the cytoplasm of the cell and suggest that the genes encoding these polypeptides are located in the nucleus. 相似文献
18.
K Kuchler K Richter J Trnovsky R Egger G Kreil 《The Journal of biological chemistry》1990,265(20):11731-11733
From the skin of Xenopus laevis, the cloned cDNAs of two precursors of thyrotropin-releasing hormone have been isolated and sequenced. These encode almost identical precursor polypeptides each containing seven copies of the end product flanked by typical prohormones processing signals. Northern blot analysis has corroborated the existence of two thyrotropin-releasing hormone precursor mRNAs of similar size as the cloned cDNA and demonstrated the existence of a third smaller species as well. Two or more mRNAs for this precursor are also present in the brain and eyes of X. laevis adults and tadpoles. 相似文献
19.
The organization of genes encoding the blood group M-specific hemagglutinin (M-agglutinin) of Escherichia coli strain IH11165 was studied with a cloned 6.5-kb DNA segment. This DNA segment contains at least five genes which code for the polypeptides of 12.5, 30, 80, 18.5 and 21 kDa. The 30-, 80- and 21-kDa polypeptides are synthesized as precursors that are approximately 2 kDa larger. The 21-kDa polypeptide was identified as the M-agglutinin subunit by its reactivity with anti-M-agglutinin serum. Nucleotide sequence analysis of the corresponding gene showed that the M-agglutinin precursor had a 24-amino acid (aa) signal sequence, while the mature protein is 146 aa residues long. Although the organization of the M-agglutinin gene cluster resembles those of other E. coli adhesins, there is no significant sequence homology between the M-agglutinin subunit and the subunits of the other potentially related proteins in E. coli. 相似文献