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Authentic cDNAs encoding the activator protein for acid beta-glucosidase (EC3.2.1.45), co-beta-glucosidase, were cloned from the pCD and lambda gt11 human cDNA libraries. Initial screening with oligonucleotide mixtures encoding amino acid sequences of co-beta-glucosidase identified partial cDNAs which were used to obtain a potentially full-length cDNA from the lambda gt11 library. This clone (2767 bp), EGTISI, contained 5' (38 bp) and 3' (1157 bp) noncoding sequences, a translation initiation site, and an open reading frame encoding 524 amino acids which included a typical hydrophobic signal sequence (16 amino acids). Computer analyses identified three regions of high similarity to co-beta-glucosidase encoded by tandem sequences in EGTISI. Searches revealed that two of these regions encoded peptides of known function; SAP1 (sphingolipid activator protein 1) and protein C (a new sphingolipid activator protein) were encoded by EGTISI sequences 5' and 3', respectively, to those for co-beta-glucosidase. The third region of similarity, encoding a theoretical peptide (undefined function), was located most 5' in the cDNA. EGTISI and its encoded polypeptide had high similarity (77% nucleotide identity and about 80% amino acid similarity) to a rat Sertoli cell cDNA and its encoded sulfated glycoprotein-1. These results indicate that a single highly conserved gene encodes the precursor for four potential sphingolipid activator proteins in rat and man.  相似文献   

3.
Structure of a precursor to human pancreatic polypeptide   总被引:3,自引:0,他引:3  
We have isolated mRNA from a human pancreatic islet cell tumor and have identified among the cell-free translation products a precursor of pancreatic polypeptide with an approximate Mr = 11,000. Recombinant DNA molecules encoding this precursor were selected from a cDNA library prepared from the islet tumor mRNA. From the nucleotide sequences of cDNAs encoding the precursor, we have deduced the complete amino acid sequence of pre-propancreatic polypeptide. These sequences encode a protein consisting of 95 amino acid residues with a Mr = 10,432. The sequence of human pancreatic polypeptide occurs in the middle of the precursor and is flanked at its carboxyl terminus by a 27-amino acid sequence which is similar to a peptide previously isolated from canine pancreatic islets. At the amino terminus of the precursor is a probable leader sequence which is rich in hydrophobic residues. A smaller pancreatic polypeptide-related protein was generated in cell-free translations of mRNA supplemented with microsomal membranes. Sequential Edman degradations of this smaller peptide indicate that the sequence of pancreatic polypeptide is located at the amino terminus of the prohormone.  相似文献   

4.
从广西产眼镜王蛇(Ophiophagus hannah)毒腺中抽提总RNA,经mRNA纯化后构建眼镜王蛇毒腺cDNA文库。从所构建的cDNA文库中,随机筛选200个克隆测序,得到两个在进化上高度保守的基因:泛素融合蛋白基因(GenBank登录号为AF297036)和核糖体蛋白L30基因(GenBank登录号是AF297033)。前者cDNA的开放阅读框为387bp,后者为348bp。前者编码128个氨基酸残基组成的泛素融合蛋白前体;后者编码115个氨基酸残基组成的核糖体蛋白L30前体。由cDNA序列推导出的氨基酸序列分析表明,泛素融合蛋白前体包括N-末端的泛素结构域(76个氨基酸残基)和C-末端的核糖体蛋白L40结构域(52个氨基酸残基)。该蛋白为一高碱性蛋白,C末端含有一个“锌指”模式结构。与16个物种比较的结果表明,眼镜王蛇与脊椎动物的泛素融合蛋白氨基酸序列相似度较高,具有高度的保守性。  相似文献   

5.
Signal peptide of Bacillus subtilis alpha-amylase   总被引:4,自引:0,他引:4  
Mature alpha-amylase of Bacillus subtilis is known to be formed from its precursor by the removal of the NH2-terminal 41 amino acid sequence (41 amino acid leader sequence). DNA fragments coding for short sequences consisting of 28 (Pro as the COOH terminus) 29 (Ala), 31 (Ala), and 33 (Ala) amino acids from the translation initiator, Met, in the leader sequence were prepared and fused in frame to the DNA encoding the mature alpha-amylase. The secretion activity of the 33 amino acid sequence was nearly twice as high as that of the parental 41 amino acid sequence, whereas the activity of the 31 amino acid sequence was 75% of that of the parent. In contrast, almost no secretion activity was observed with the 28 and 29 amino acid sequences. The signal peptide cleavage site of the precursor expressed from the plasmid encoding the 33 amino acid sequence was located between Ala and Leu at positions 33 and 34 and that from the 31 amino acid sequence between Thr and Ala at positions 33 and 34. The NH2-terminal amino acid from the latter corresponded to the 3rd amino acid of the mature enzyme. These results indicated that the functional signal peptide of the B. subtilis beta-amylase consists of the first 33 amino acids from the initiator, Met.  相似文献   

6.
Complementary DNA sequences and structural genes encoding the atrial natriuretic peptide precursor (prepro-ANP) have been cloned. Analysis of DNA sequences, complementary to rat atrial prepro-ANP mRNA, has revealed that the various natriuretic peptides isolated from rat atrium reside at the carboxy terminus of a 152-amino-acid precursor protein. The human gene, comprised of three exons and two intervening sequences, encodes a protein of 151 amino acids highly homologous to the rat precursor. Although putative proteolytic processing sites can be identified throughout the prepro-ANP amino acid sequence, the natural form of the mature ANP has not been identified. Therefore, the sites and mechanisms of prepro-ANP processing to mature peptides forms are unknown. However, the successful cloning of the prepro-ANP gene and corresponding cDNAs provide the necessary molecular tools to address these fundamental questions relating to the regulation of ANP synthesis and processing in atrial and extraatrial tissues.  相似文献   

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Carassius RFamide (C-RFa) is a peptide, isolated originally from the brain of Japanese crucian carp and sharing homologies with mammalian prolactin-releasing peptides. From the physiological aspect, it is known that C-RFa has contraction-promoting action on fish intestines, but its localization in peripheral tissues is unknown. We observed the localization of C-RFa in teleost guts using an immunohistochemical technique. C-RFa-like immunoreactive (irC-RFa) sites were observed in not only the smooth muscle cells in the longitudinal muscle layer, but also in both Auerbach's and Meissner's nerve plexus in the stomach, pyloric ceca and intestine. In epithelial mucous cells, irC-RFa sites were observed in the surface mucous cells in the stomach in freshwater fish (FW), and in the goblet cells of the apical sites in the villi of the pyloric ceca and intestine in all fish. In the stomach, irC-RFa sites were found in the fundic glands of the body regions in seawater (SW) and brackish water (BW) fish, but not in FW fish. This study confirmed that one of the functions of C-RFa is the smooth muscle contraction of the longitudinal muscle layer in digestive organs. We suggest that C-RFa may have functional roles in both central and peripheral neurotransmission. In addition, it appears that the difference in C-RFa localization of SW, BW, and FW fish reflects the adaptation of the stomach function to different salinity habitats.  相似文献   

9.
P Koski  L Hirvas  M Vaara 《Gene》1990,88(1):117-120
The complete nucleotide sequence of the ompH gene encoding the 16-kDa basic outer membrane protein of Salmonella typhimurium was determined. The OmpH protein is synthesized in a precursor form with additional 20 amino acid residues in the N terminus of the protein. This peptide has common characteristics of signal sequences. The promoter region has strong homology to consensus sequences of Escherichia coli. The expression of ompH was detected in minicells.  相似文献   

10.
The cDNAs corresponding to the mRNA encoding a polypeptide which is immunoreactive with the antisera specific to carcinoembryonic antigen (CEA) (1) are cloned. The amino acid sequences deduced from the nucleotide sequences of the cDNAs show that it is synthesized as a precursor with a signal peptide followed by 668 amino acids of the putative mature CEA peptide, whose N-terminal 24 amino acids and amino acids 286 to 295 exactly coincide with those known for N-terminal sequences of CEA (2) and NFA-1 (3), respectively. The first 108 N-terminal residues are followed by three very homologous repetitive domains of 178 residues each and then by 26 mostly hydrophobic residues which probably comprise a membrane anchor. Each repetitive domains contains 4 cysteines at precisely the same positions and as many as 28 possible N-glycosylation sites are found in the CEA peptide region agreeing with high carbohydrate content of purified CEA.  相似文献   

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Carassius RFamide (C-RFa) is a novel peptide found in the brain of the Japanese crucian carp. It has been demonstrated that mRNA of C-RFa is present in the telencephalon, optic tectum, medulla oblongata, and proximal half of the eyeball in abundance. Immunohistochemical methods were employed to elucidate the distribution of the peptide in the brain of the goldfish (Carassius auratus) in detail. C-RFaimmunoreactive perikarya were observed in the olfactory bulb, the area ventralis telencephali pars dorsalis and lateralis, nucleus preopticus, nucleus preopticus periventricularis, nucleus lateralis tuberis pars posterioris, nucleus posterioris periventricularis, nucleus ventromedialis thalami, nucleus posterioris thalami, nucleus anterior tuberis, the oculomotor nucleus, nucleus reticularis superior and inferior, facial lobe, and vagal lobe. C-RFa immunoreactive fibers and nerve endings were present in the olfactory bulb, olfactory tract, area dorsalis telencephali pars centralis and medialis, area ventralis telencephali, midbrain tegmentum, diencephalon, medulla oblongata and pituitary. However, in the optic tectum the immunopositive perikarya and fibers were less abundant. Based on these results, some possible functions of C-RFa in the nervous system were discussed.  相似文献   

13.
Two maize genes and cDNAs encoding the mitochondrial adenine nucleotide translocator (ANT), a nuclear-encoded inner mitochondrial membrane carrier protein, have previously been isolated in this laboratory. Sequence analysis revealed the existence of much longer open reading frames than the corresponding fungal and mammalian ANT genes. Potato ANT cDNAs have subsequently been isolated and sequenced and alignment of the deduced plant amino acid sequences with the equivalent fungal and mammalian polypeptides indicated that the plant proteins contain N-terminal extensions. When the plant cDNA clones are expressed in vitro they direct the synthesis of precursor proteins that are specifically processed at the N-terminus upon import into isolated mitochondria. N-terminal amino acid sequence data obtained from the native proteins purified from both maize and potato mitochondria has allowed identification of the putative processing sites. Further import analysis has shown that two distinct regions of the maize precursor protein contain targeting information, the 97 amino acids at the N-terminus and the 267 C-terminal amino acids. This is the first report that provides experimental evidence that the adenine nucleotide translocator of higher plants is synthesized as a large precursor protein that is specifically cleaved upon import into mitochondria. Import of ANT into higher plant mitochondria therefore appears to be different to the corresponding process in fungal and mammalian systems where targeting of ANT to mitochondria is mediated by internal signals and there is no N-terminal processing.  相似文献   

14.
Organization, structure, and polymorphisms of the human profilaggrin gene   总被引:8,自引:0,他引:8  
Profilaggrin is a major protein component of the keratohyalin granules of mammalian epidermis. It is initially expressed as a large polyprotein precursor and is subsequently proteolytically processed into individual functional filaggrin molecules. We have isolated genomic DNA and cDNA clones encoding the 5'- and 3'-ends of the human gene and mRNA. The data reveal the presence of likely "CAT" and "TATA" sequences, an intron in the 5'-untranslated region, and several potential regulatory sequences. While all repeats are of the same length (972 bp, 324 amino acids), sequences display considerable variation (10-15%) between repeats on the same clone and between different clones. Most variations are attributable to single-base changes, but many also involve changes in charge. Thus, human filaggrin consists of a heterogeneous population of molecules of different sizes, charges, and sequences. However, amino acid sequences encoding the amino and carboxyl termini are more conserved, as are the 5' and 3' DNA sequences flanking the coding portions of the gene. The presence of unique restriction enzyme sites in these conserved flanking sequences has enabled calculations on the size of the full-length gene and the numbers of repeats in it: depending on the source of genomic DNA, the gene contains 10, 11, or 12 filaggrin repeats that segregate in kindred families by normal Mendelian genetic mechanisms. This means that the human profilaggrin gene system is also polymorphic with respect to size due to simple allelic differences between different individuals. The amino- and carboxyl-terminal sequences of profilaggrin contain partial or truncated repeats with unusual un-filaggrin-like sequences on the termini.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

15.
The structures of crustacean hyperglycemic hormones (CHH) were investigated in two crabs, the coastal euryhaline crab Pachygrapsus marmoratus and the fresh water crab Potamon ibericum. The neuropeptide mRNAs were extracted from pericardial and X-organs (PO and XO), and the sequences of the cDNA encoding the hormones' precursors were determined. The X-organ preprohormones are composed of 29 and 28 amino acid signal peptides in P. marmoratus and P. ibericum respectively, followed by 43 and 41 amino acid crustacean hyperglycemic hormone precursor related peptide (CPRP) flanking the 72 amino acid crustacean hyperglycemic hormones. A similar organization is reported for pericardial preprohormones with identical sequences for the signal peptide, the CPRP and the N-terminal sequences of CHH (1-40), but remaining sequences (41-72 and 41-71) differing considerably. In P. marmoratus two CHH cDNAs were characterized from XO and evidences were obtained for the existence of at least two forms in the PO. From our results and by comparison with other known sequences, a consensus pattern for crab pericardial CHH could be pointed out. Analysis of the data presented in this article using phylogenetic methods reveals that the two crab species studied are much closer than previously predicted.  相似文献   

16.
The presence of endothelin (ET)-like immunoreactivity and the cardiovascular effects of mammalian ET-1 in fish have been reported. To identify ET-related peptides in fish, we screened the cDNA library of the salmon (Oncorhynchus keta) stomach by means of rapid amplification of cDNA ends, and we cloned cDNAs encoding an ET-related peptide. The salmon ET-related sequence of 21 amino acids is identical to the trout ET-1 peptide recently purified from kidney specimens of Oncorhynchus mykiss. The deduced amino acid sequence of salmon pre-proET-1 (PPET-1) comprises 244 amino acids, including a putative signal sequence and mature ET-1, as well as big ET-1 and ET-1-like sequences. This precursor, the first reported PPET-1 sequence for Salmoniformes, Teleostei, has low homology with the sequences of human, mouse, frog (Xenopus laevis), and zebrafish (Danio rerio) PPET-1 (26%, 29%, 24%, and 39%, respectively).  相似文献   

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18.
We have cloned a DNA complementary to the messenger RNA encoding the precursor of ornithine transcarbamylase from rat liver. This complementary DNA contains the entire protein coding region of 1062 nucleotides and 86 nucleotides of 5'- and 298 nucleotides of 3'-untranslated sequences. The predicted amino acid sequence has been confirmed by extensive protein sequence data. The mature rat enzyme contains the same number of amino acid residues (322) as the human enzyme and their amino acid sequences are 93% homologous. The rat and human amino-terminal leader sequences of 32 amino acids, on the other hand, are only 69% homologous. The rat leader contains no acidic and seven basic residues compared to four basic residues found in the human leader. There is complete sequence homology (residues 58-62) among the ornithine and aspartate transcarbamylases from E. coli and the rat and human ornithine transcarbamylases at the carbamyl phosphate binding site. Finally, a cysteine containing hexapeptide (residues 268-273), the putative ornithine binding site in Streptococcus faecalis, Streptococcus faecium, and bovine transcarbamylases, is completely conserved among the two E. coli and the two mammalian transcarbamylases.  相似文献   

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Phospholipase A1 (PLA1) is a hydrolytic enzyme that catalyzes removal of the acyl group from position 1 of lecithin to form lysolecithin. The genomic DNA and cDNA encoding PLA1 from Aspergillus oryzae were cloned with the mixed deoxyribonucleotide-primed polymerase chain reaction. The PLA1 gene is composed of 1,056 bp and has four exons and three short introns (63, 54, and 51 bp). The deduced amino acid sequence of PLA1 contained the N-terminal sequence of the mature PLA1 analyzed by Edman degradation. PLA1 cDNA has an open reading frame of 885 bp encoding the PLA1 precursor of 295 amino acid residues. The mature PLA1 is composed of 269 amino acid residues, and a prepro-sequence of 26 amino acid residues is at the N-terminal region of the PLA1 precursor. PLA1 has two possible N-glycosylation sites (Asn27 and Asn55). PLA1 has a consensus pentapeptide (-Gly-His-Ser-Xaa-Gly-), which is conserved in lipases. The amino acid sequence of PLA1 showed 47% identity with that of mono- and diacylglycerol lipase from Penicillium camembertii. The PLA1 cDNA was expressed in Saccharomyces cerevisiae KS58-2D, indicating the cloned gene to be functional.  相似文献   

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