The cDNA and protein sequences of human lactate dehydrogenase B.

Human lactate dehydrogenase B (LDH-B) cDNA was isolated and sequenced. The LDH-B cDNA insert consists of the protein-coding sequence (999 bp), the 5' (54 bp) and 3' (203 bp) non-coding regions, and the poly(A) tail (50 bp). The predicted sequence of 333 amino acid residues was confirmed by amino acid composition and/or sequence analyses of a total of 185 (56%) residues from tryptic peptides of human LDH-B protein. The nucleotide and amino acid sequences of the human LDH-B coding region show 68% and 75% homologies respectively with those of the human LDH-A. The peptide map and amino acid composition data have been deposited as Supplementary Publication SUP 50139 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment [see Biochem. J. (1987) 241, 5].     

Nucleotide sequence and genomic organization of a human T lymphocyte serine protease gene

We have determined the nucleotide sequence of 4508 base pairs of human genomic DNA which contain the human serine esterase gene from cytotoxic T lymphocytes (SECT) (equivalent to the 1-3E cDNA clone) and include 879 bp of 5' flanking DNA and 393 bp of 3' flanking DNA. The gene consists of five exons of 88, 148, 136, 261, and 257 nucleotides separated by four introns of 1043, 455, 205, and 643 nucleotides. The location of introns with respect to protein coding sequences in the SECT gene is identical to that of the human cathepsin G and murine granzyme B genes. Comparison of SECT gene exonic sequences to murine granzyme B-F cDNA sequences indicates similarities of 75 and 72% for granzymes B and C and 61, 59, and 61% for granzymes D, E, and F, respectively. The 5' flanking sequence of the SECT gene showed similarity only to the 5' flanking sequence of the murine granzyme B gene, indicating that these genes are homologous. Comparison of the SECT gene sequence to the human cathepsin G sequence indicated no similarity in the 5' flanking DNA although the exonic sequences show 64% sequence similarity overall and 45% sequence similarity in the respective 3' untranslated regions. These similarities suggest that the SECT and cathepsin G genes are members of the same family of serine protease genes. Evidence from high and low stringency Southern transfer analysis of human genomic DNA indicates the presence of another gene of at least 85% sequence similarity to the SECT gene.     

Sequence analysis, tissue distribution, and expression of rat cathepsin S.

Cysteine proteases are involved in many diverse cellular processes ranging from processing of precursor proteins to intracellular degradation. In an effort to identify novel cysteine proteases, we used the polymerase chain reaction and primers directed against the catalytic sites of previously cloned cysteine proteases. From rat brain mRNA, a 600-base pair band was amplified; cloning and partial sequence analysis of this band resulted in the identification of cathepsins B and L and five novel sequences. The novel cDNAs contained a number of residues conserved in lysosomal cysteine proteases, including the active site residue His159 (papain numbering). In addition, the amino acid homology between the novel sequences and either cathepsins B, L, or H, ranged from 63 to 32%. The insert with highest homology was used to screen a rat brain cDNA library; a 1334-base pair cDNA was isolated and the nucleotide sequence determined. This sequence encodes an open reading frame of 330 amino acids which is 82% homologous to human cathepsin S, suggesting that this sequence represents rat cathepsin S. Northern blot analysis for rat cathepsin S revealed tissue-specific expression distinct from the distribution of cathepsin B and L. The regulation of expression of rat cathepsin S mRNA in response to thyroid-stimulating hormone was studied in a rat thyroid cell line FRTL-5. The level of cathepsin S mRNA was substantially increased in response to thyroid-stimulating hormone, whereas cathepsin B and cathepsin L mRNA levels were not altered by this treatment. A portion of cDNA encoding the predicted mature protein of rat cathepsin S was expressed as a glutathione S-transferase-fusion protein. The affinity-purified protein exhibited proteolytic activity with properties similar to bovine cathepsin S. Taken together, these results imply highly specific functions for cathepsin S.     

Cloning of the C alpha catalytic subunit of the bovine cAMP-dependent protein kinase.

The bovine C alpha type catalytic subunit of the cAMP-dependent protein kinase was cloned. A partial cDNA was isolated from a bovine heart cDNA library. This clone contained 120 bp of the coding sequence and the entire 3' untranslated region of 1431 bp. The complete coding region was cloned by PCR amplification from total bovine heart and skeletal muscle RNA. The sequence of the 3' oligonucleotide was taken from the partial cDNA clone whereas the 5' oligonucleotide was chosen by comparison of sequences of published C alpha subunits from other species. In the deduced amino acid sequence there is one deviation from the published bovine C alpha protein sequence, aspartic acid 286 is exchanged by an asparagine. The C alpha mRNA was found to be expressed differentially in various bovine tissues.     

Molecular cloning of cDNA for rat cathepsin C. Cathepsin C, a cysteine proteinase with an extremely long propeptide

A cDNA for rat cathepsin C (dipeptidylaminopeptidase I) was isolated. The deduced amino acid sequence of cathepsin C comprises 462 amino acid residues: 28 NH2-terminal residues corresponding to the signal peptide, 201 residues corresponding to the propeptide, and 233 COOH-terminal residues corresponding to the mature enzyme region. Four potential glycosylation sites were found, three located in the propeptide region, and one in the mature enzyme region. The amino acid sequence of mature cathepsin C has 39.5% identity to that of cathepsin H, 35.1% to that of cathepsin L, 30.1% to that of cathepsin B, and 33.3% to that of papain. Cathepsin C, therefore, is a member of the papain family, although its propeptide region is much longer than those of other cysteine proteinases and shows no significant amino acid sequence similarity to any other cysteine proteinase.     

Investigation of diversity and isotypes of the beta-tubulin cDNA in several small strongyle (Cyathostominae) species

The diversity of the beta-tubulin cDNAs of the cyathostominae and the occurrence of further isotypes were examined in adult worms isolated from an anthelmintic-na?ve horse. cDNAs encoding beta-tubulin from Cyathostomum catinatum, Cylicocyclus nassatus, Cylicocyclus insigne, Cylicocyclus radiatus, Cylicocyclus elongatus, Cyathostomum coronatum, and Cyathostomum pateratum were characterized using specific primers developed from the cDNA sequence of Cc. nassatus. The cDNA sequences span 1,429 bp and show identities ranging from 95.6 to 100%. The deduced protein sequences span 448 amino acids and were 98-100% identical. The amino acid sequences of the 7 species varied within and between species at 10 positions. A 3' Rapid Amplification of cDNA ends using a degenerate forward primer was carried out with cDNA from Cy. pateratum, Cy. coronatum, Cy. catinatum, and Cc. nassatus to investigate the occurrence of further beta-tubulin isotypes. The expected polymerase chain reaction (PCR) product of 400 bp, including 306 bp of coding sequence, was amplified, as was an additional fragment of 600 nucleotides in the case of Cy. pateratum, Cy. coronatum, and Cy. catinatum. Sequencing of the PCR products revealed no evidence for the existence of a second beta-tubulin isotype in cyathostomes. The variation in size was caused by a length polymorphism within the 3' untranslated region, and 2 functional mRNAs seem to be transcribed from the same gene.     

孔石莼(Ulva pertusa)质体蓝素基因的克隆及基因特征分析(英文)

采用cDNA末端快速扩增的办法,从孔石莼(Ulva pertusa)中克隆获得质体蓝素基因。该基因完整的cDNA为787bp,包括40 bp 5’端非编码区和327 bp的3’端非编码区,以及一个420 bp的开放阅读框架,编码139个氨基酸的蛋白质。该基因编码质体蓝素的前体肽,其N端41个氨基酸残基为信号肽,后面为98个氨基酸残基的成熟肽。从Genbank中选择了13个质体蓝素的前体肽基因进行序列比对分析和构建进化树。孔石莼质体蓝素基因与其它质体蓝素基因的同源性为48.2%至78.8%。该进化树将来源于6种藻类植物的7个质体蓝素基因聚类在一起,显示出它们较近的进化关系。同样,也表现出11种生物的分子进化关系。序列比对结果显示,在质体蓝素的基因序列中存在两个高度保守的基序,它编码质体蓝素蛋白的铜结合活性位点。     

Purification of juvenile hormone esterase and molecular cloning of the cDNA from Manduca sexta.

Juvenile hormone esterase (JHE) is a highly specific enzyme important for regulating the onset of metamorphosis in lepidopteran insects. After affinity chromatography of the hemolymph proteins of Manduca sexta, the pure JHE protein was digested with Lys-C and the resultant peptides were purified by microbore HPLC. Two peptides were selected for sequencing. Based upon these amino acid sequences, degenerate RT-PCR was performed in order to amplify a partial cDNA sequence from mRNA from the fat body of M. sexta. A 1512bp partial cDNA was generated and found to be highly homologous to the JHE from Heliothis virescens. 5' and 3' RACE were performed to obtain the full length cDNA sequence. The cDNA has a total length of 2220bp, with a 1749bp coding region. The deduced protein sequence contains 573 amino acids.     

cDNA sequence coding for a rat glia-derived nexin and its homology to members of the serpin superfamily

Rat glial cells release a neurite-promoting factor with serine protease inhibitory activity. By using a rat glioma cDNA clone as a probe, it was possible to isolate rat cDNAs containing the entire sequence coding for this neurite-promoting factor. The largest rat cDNA (approximately 2100 bp) was characterized by DNA sequencing. It contained the entire coding region, 135 bp of the 5' nontranslated region, and about 750 bp of the 3' nontranslated region. The open reading frame coded for 397 amino acids including a putative signal peptide of 19 amino acids. The correct identity of the coding sequence was substantiated by the fact that the sequence of tryptic peptides, derived from the purified rat factor, matched exactly with the deduced amino acid sequence. The rat protein sequence had 84% homology with the corresponding protein from human glioma cells. Both amino acid sequences indicated that the proteins belong to the protease nexins [Baker, B.J., Low, D. A., Simmer, R. L., & Cunningham, D.D. (1980) Cell (Cambridge, Mass.) 21, 37-45] and therefore can be defined as glia-derived nexins (GDNs). Further analysis showed that both rat and human GDN belong to the serpin superfamily and share 41%, 32%, and 25% homology with human endothelial-cell-type plasminogen activator inhibitor, antithrombin III, and alpha-1 proteinase inhibitor, respectively.     

Complementary DNA and derived amino acid sequence of the alpha subunit of human complement protein C8: evidence for the existence of a separate alpha subunit messenger RNA

The entire amino acid sequence of the alpha subunit (Mr 64,000) of the eighth component of complement (C8) was determined by characterizing cDNA clones isolated from a human liver cDNA library. Two clones with overlapping inserts of net length 2.44 kilobases (kb) were isolated and found to contain the entire alpha coding region [1659 base pairs (bp)]. The 5' end consists of an untranslated region and a leader sequence of 30 amino acids. This sequence contains an apparent initiation Met, signal peptide, and propeptide which ends with an arginine-rich sequence that is characteristic of proteolytic processing sites found in the pro form of protein precursors. The 3' untranslated region contains two polyadenylation signals and a poly(A) sequence. RNA blot analysis of total cellular RNA from the human hepatoma cell line HepG2 revealed a message size of approximately 2.5 kb. Features of the 5' and 3' sequences and the message size suggest that a separate mRNA codes for alpha and argues against the occurrence of a single-chain precursor form of the disulfide-linked alpha-gamma subunit found in mature C8. Analysis of the derived amino acid sequence revealed several membrane surface seeking domains and a possible transmembrane domain. These occur in a cysteine-free region of the subunit and may constitute the structural basis for alpha interaction with target membranes. Analysis of the carbohydrate composition indicates 1 or 2 asparagine-linked but no O-linked oligosaccharide chains, a result consistent with predictions from the amino acid sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

三疣梭子蟹NF-κB家族基因Relish和Dorsal的克隆及表达特征

为研究Relish和Dorsal在三疣梭子蟹免疫过程中所起到的作用, 研究利用RACE技术克隆获得三疣梭子蟹Relish(Pt-Rel)、Dorsal基因(Pt-Dor) cDNA全长, 并通过实时荧光定量PCR技术分析了Pt-Rel和Pt-Dor基因在健康蟹不同组织及其原代培养的血淋巴细胞在感染不同微生物后的表达情况。结果显示, Pt-Rel cDNA长3254 bp, ORF长2949 bp, 编码983个氨基酸, Pt-Dor cDNA长2348 bp, ORF长1911 bp, 编码637个氨基酸; 蛋白结构预测分析发现Pt-Rel和Pt-Dor均包含RHD (Rel homology domain)及IPT (Immunoglobulin-like fold, Plexins, TranscriPtion factors)Rel/NF-κB家族蛋白经典结构域。Pt-Rel和Pt-Dor与其他节肢动物Relish、Dorsal氨基酸序列具有很高的相似性; 在系统进化分析中Pt-Rel和Pt-Dor分别与中华绒螯蟹等甲壳动物的Relish、Dorsal聚在一支, 而昆虫类聚在另一支。Pt-Rel和Pt-Dor在检测的6种组织中均有表达, 且2个基因均在血淋巴细胞中表达量最高。蟹血淋巴细胞体外感染实验结果表明, 不同病原微生物对2个基因表达的影响非常相似, 假丝酵母在2h明显诱导了Pt-Rel和Pt-Dor基因的表达, 而金黄色葡萄球菌及溶藻弧菌在感染4h后使Pt-Rel和Pt-Dor基因的表达显著上调。上述研究结果表明Pt-Rel和Pt-Dor基因很有可能参与了三疣梭子蟹的抗感染免疫过程。     

Molecular cloning and nucleotide sequence of human pancreatic prechymotrypsinogen cDNA

The cDNA clone encoding human prechymotrypsinogen was isolated from a human pancreas cDNA library and its nucleotide sequence was determined. The sequence consists of a 16 bp 5' non-coding region, a 789 bp amino acid coding region and a 60 bp 3' non-coding region. The predicted product consists of 263 amino acids, including 18 amino acids for a signal peptide and 15 amino acids possible for an activation peptide. Southern blot analyses using the cloned cDNA as a probe revealed that human genomic DNA carries at least two genes that are related to chymotrypsinogen.     

Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

A tissue-specific cDNA library was constructed using polyA⁺ RNA from pituitary glands of the Indian catfishHeteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence ofH. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.     

The triose phosphate-3-phosphoglycerate-phosphate translocator from spinach chloroplasts: nucleotide sequence of a full-length cDNA clone and import of the in vitro synthesized precursor protein into chloroplasts.

The nucleotide sequence of several cDNA clones coding for the phosphate translocator from spinach chloroplasts has been determined. The cDNA clones were selected from a lambda gt10 library prepared from poly(A)+ mRNA of spinach leaves using oligonucleotide probes modeled from amino acid sequences of tryptic peptides prepared from the isolated translocator protein. A 1439 bp insert of one of the clones codes for the entire 404 amino acid residues of the precursor protein corresponding to a mol. wt of 44,234. The full-length clone includes 21 bp at the transcribed non-coding 5' region with the ribosome initiation sequence ACAATGG, a 1212 bp coding region and 199 bp at the non-coding 3' region excluding the poly(A) tail which starts 17 bp downstream from a putative polyadenylation signal, AATAAT. According to secondary structure predictions the mature part of the chloroplast phosphate translocator exhibits high hydrophobicity and consists of at least seven membrane-spanning segments. Using plasmid-programmed wheat germ lysate the precursor protein was synthesized in vitro and could be imported into spinach chloroplasts where it is inserted into the inner envelope membrane.     

The 44-kDa Regulatory Subunit of the Paramecium cAMP-Dependent Protein Kinase Lacks a Dimerization Domain and May Have a Unique Autophosphorylation Site Sequence

ABSTRACT. The 44-kDa regulatory subunit (R₄₄) of one form of cAMP-dependent protein kinase of Paramecium was purified, and two partial internal amino acid sequences from it were used to clone the corresponding cDNA. This R₄₄ cDNA clone was 1022-bp long, including 978 bp of coding sequence and 7 bp and 37 bp of 5' and 3' untranslated sequences, respectively. A 1.1-kb mRNA was labeled on a Northern blot. The deduced R₄₄ amino acid sequence had 31%–38% positional identity to the sequences of other cloned cAMP-dependent protein kinase regulatory subunits. R₄₄ sequence showed equal sequence similarity to mammalian types I and II regulatory subunits. The N -terminal sequence encoding the regulatory subunit dimerization domain found in most regulatory subunits is not present in the R₄₄ clone, confirming the lack of regulatory subunit dimer formation previously reported for the Paramecium cAMP-dependent protein kinase. The putative autophosphorylation site of R₄₄ contains the amino acid sequence TRTS, distinct from the consensus sequence RRXS, where X is any residue, found in other autophosphorylated cAMP-dependent protein kinase regulatory subunits and many cAMP-dependent protein kinase substrates.     

Molecular cloning of a human co-beta-glucosidase cDNA: evidence that four sphingolipid hydrolase activator proteins are encoded by single genes in humans and rats

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.