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.     

从云南烟草上检测到的黄瓜花叶病毒亚组Ⅱ分离物*

在对云南省烟草病毒病的研究中,分离到一种直径约２６￣３０ｎｍ的球形病毒。提纯病毒进行的ＳＤＳ－ＰＡＧＥ发现一条５５ｋＤ蛋白带。５５ｋＤ蛋白Ｎ－端１０个氨基酸与ＣＭＶ亚组Ⅱ的Ｑ株系外壳蛋白Ｎ－氨基酸同源性为１００％。以ＣＭＶ－Ｑ抗血清对５５ｋＤ蛋白进行了Ｗｅｓｔｅｒｎ ｂｌｏｔ检测,发现５５ｋＤ蛋白与ＣＭＶ Ｑ株系抗血清有血清学反应。根据已报道的ＣＭＶ亚组Ⅱ外壳蛋白基因序列合成引物,采用ＲＴ－ＰＣＲ     

Molecular cloning and characterization of a catalase gene from Zhikong scallop Chlamys farreri

Catalase is one of the central enzymes involved in scavenging the high level of reactive oxygen species (ROS) by degradation of hydrogen peroxide to oxygen and water. The full-length catalase cDNA of Zhikong scallop Chlamys farreri (denoted as CfCAT) was identified from hemocytes by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The nucleotide sequence of CfCAT cDNA consisted of 3146bp with a 5' UTR of 103bp, an unusually long 3' UTR of 1519bp with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1521bp encoding a polypeptide of 507 amino acids with predicted molecular weight of 57.5kDa. The deduced amino acid sequence of CfCAT has significant homology to catalases from animals, plants and bacteria. Several highly conserved motifs including the proximal heme-ligand signature sequence RLFSYNDTH, the proximal active site signature FNRERIPERVVHAKGGGA, and the three catalytic amino acid residues of His(72), Asn(145) and Tyr(355) were identified in the deduced amino acid sequence of CfCAT. The CfCAT was demonstrated to be a peroxisomal glycoprotein with two potential glycosylation sites and a peroxisome targeting signal of ANL that was consistent with human, mouse and rat catalases. The time-course expression of CfCAT in hemocytes was measured by quantitative real-time PCR. The expression of CfCAT increased gradually and reached the highest point at 12h post-Vibrio infection, then recovered to the original level at 24h. All these results indicate that CfCAT, a constitutive and inducible protein, is a member of the catalase family and is involved in the process against ROS in scallop.     

桑泛素延伸蛋白基因片段的克隆及序列分析

目的:利用3’RACE技术克隆植物泛素基因,是进一步研究其功能的基础。方法:本研究从桑树(丰驰桑)(Morus bomby-cis)幼叶中提取总RNA,反转录成cDNA,根据已报道的泛素基因序列设计1条正向引物,利用3’RACE(Rapid Amplification of cDNAEnd)技术进行扩增。结果:扩增出1条690 bp的泛素基因片段。该片段5’端为编码156个氨基酸残基的阅读框,3’末端有219bp的非翻译区。结论:同源分析表明,此cDNA序列为泛素延伸蛋白基因(Genebank登录号为DQ839403)。用Genedoc软件对该片段编码的氨基酸序列进行同源性分析的结果表明:桑树泛素延伸蛋白与马铃薯、烟草、陆地棉、黄瓜的泛素延伸蛋白以及苜蓿的核糖体S27A蛋白的同源性都在96%以上。     

Molecular cloning and immune responsive expression of a novel C-type lectin gene from bay scallop Argopecten irradians

C-type lectins are Ca(2+)-dependent carbohydrate-recognition proteins that play crucial roles in innate immunity. The cDNA of C-type lectin (AiCTL1) in the bay scallop Argopecten irradians was cloned by expressed sequence tag (EST) and RACE techniques. The full-length cDNA of AiCTL1 was 660 bp, consisting of a 5'-terminal untranslated region (UTR) of 30 bp and a 3' UTR of 132 bp with a polyadenylation signal sequence AATAAA and a poly(A) tail. The AiCTL1 cDNA encoded a polypeptide of 166 amino acids with a putative signal peptide of 20 amino acid residues and a mature protein of 146 amino acids. The deduced amino acid sequence of AiCTL1 was highly similar to those of the C-type lectins from other animals and contained a typical carbohydrate-recognition domain (CRD) of 121 residues, which has four conserved disulfide-bonded cysteine residues that define the CRD and two additional cysteine residues at the amino terminus. AiCTL1 mRNA was dominantly expressed in the hemocytes of the bay scallop. The temporal expression of AiCTL1 mRNA in hemocytes was increased by 5.7- and 4.9-fold at 6h after injury and 8h after injection of bacteria, respectively. The structural features, high similarity and expression pattern of AiCTL1 indicate that the gene may be involved in injury healing and the immune response in A. irradians.     

Primary structure of a lipoxygenase from barley grain as deduced from its cDNA sequence

A full length cDNA sequence for a barley grain lipoxygenase was obtained. It includes a 5′ untranslated region of 69 nucleotides, an open reading frame of 2586 nucleotides encoding a protein of 862 amino acid residues and a 3′ untranslated region of 142 nucleotides. The molecular mass of the encoded polypeptide was calculated to be 96.392. Its amino acid sequence shows a high homology with that of other plant lipoxygenases identified to date.     

cDNA-derived amino acid sequence of rat mitochondrial 3-oxoacyl-CoA thiolase with no transient presequence: structural relationship with peroxisomal isozyme.

The sorting of homologous proteins between two separate intracellular organelles is a major unsolved problem. 3-Oxoacyl-CoA thiolase is localized in mitochondria and peroxisomes, and provides a good system for the study on the problem. Unlike most mitochondrial matrix proteins, mitochondrial 3-oxoacyl-CoA thiolase in rats is synthesized with no transient presequence and possess information for mitochondrial targeting and import in the mature protein. Two overlapping cDNA clones contained an open reading frame encoding a polypeptide of 397 amino acid residues (predicted Mr = 41,868), a 5' untranslated sequence of 164 bp, a 3' untranslated sequence of 264 bp and a poly(A) tract. The amino acid sequence of the mitochondrial thiolase is 37% identical with that of the mature portion of rat peroxisomal 3-oxoacyl-CoA thiolase precursor. These results suggest that the two thiolases have a common origin and obtained information for targeting to respective organelles during evolution. Two portions in the mitochondrial thiolase that may serve as a mitochondrial targeting signal are presented.     

Isolation and characterization of a cDNA coding for human myeloperoxidase

A cDNA encoding the carboxyl-terminal fragment of the human myeloperoxidase heavy chain was isolated and characterized. It was then used to determine the locations of the myeloperoxidase light and heavy chains in the polypeptide precursor. A cDNA library from poly(A)+ RNA from human leukemia HL-60 cells was constructed in pBR322 and screened by differential hybridization with enriched and depleted cDNA probes and then by hybridization with an oligonucleotide probe. A cDNA clone containing 1278 bp with an open reading frame of 474 bp and a 3' noncoding region of 804 bp was isolated. The amino acid sequence deduced from the nucleotide sequence consisted of 158 residues including a sequence of 14 amino acids known to be present in the heavy chain of the molecule. The cDNA also included a stop codon of TAG followed by a noncoding sequence that included a potential recognition site for polyadenylylation and a poly(A) tail. RNA transfer blot analysis with the cDNA probe indicated that myeloperoxidase mRNA was approximately 3.3 kb in length. In vitro translation of the mRNA selected by cDNA hybridization revealed preferential synthesis of a 74,000-Da polypeptide precursor that could be precipitated with anti-myeloperoxidase IgG. Antibodies specific for the heavy and light chains of myeloperoxidase were isolated from antiserum by affinity chromatography employing Sepharose columns covalently bound to the heavy or light chains. Antibodies specific for the light chain or the heavy chain readily precipitated the 74,000-Da precursor polypeptide. These results indicated that myeloperoxidase is synthesized as a single chain which undergoes processing into a light and heavy chain. Furthermore, the heavy chain of myeloperoxidase originates from the carboxyl terminus of the precursor polypeptide.     

Nucleotide sequence of mouse L19 ribosomal protein cDNA isolated in screening with tre oncogene probes.

A cDNA library was prepared from cytoplasmic poly(A)RNA from mouse NIH-3T3 cells carrying a transfected human tre oncogene. Screening with tre gene probes identified a tre cDNA clone 11-4 and a co-purifying weakly hybridizing cDNA clone 11-5. The 11-5-specific RNA was expressed in both nontransfected and tre-transfected NIH-3T3 cells, showing it is of mouse rather than tre gene origin. Its nucleotide sequence was 717 bp long and contained, starting from the first nucleotide, an open reading frame of 588 bp followed by a 3' noncoding region and 26 A residues at the 3' terminus. Comparison with the GenBank data base revealed 93.7% homology with cDNA encoding the rat L19 ribosomal protein. Furthermore, the 196-amino-acid polypeptide deduced from 11-5 was of the same length and contained only one amino acid difference compared with the rat L19 protein. Comparison with the weakly hybridizing tre gene probe showed stretches of homology that were, however, too short to be taken into consideration. We conclude that the 11-5 sequence encodes the mouse L19 ribosomal protein.     

Isolation and characterisation of a cDNA encoding the precursor for human apolipoprotein AII

cDNA clones encoding human apolipoprotein AII have been isolated from an adult liver cDNA library. Apo AII mRNA was shown to be approximately 600 bases in length by RNA blot hybridisation. The intracellular precursor of apo AII was inferred from the cDNA sequence to be a 100 amino acid polypeptide consisting of the 77 residue mature protein and an additional 23 amino terminal residues. The amino terminal extension, divisible into an 18 residue signal peptide and a 5 residue propeptide, is separated from the first amino acid of mature apo AII by dibasic residues. The 5' untranslated region of the message is 61 bases in length and the 3' untranslated region 113 bases. A polyadenylation signal is situated 14 bases 3' of the poly(A) tail.     

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].     

Molecular cloning of a rhoptry protein (ROP6) secreted from Toxoplasma gondii

Monoclonal antibody (mAb) Tg786 against Toxoplasma gondii has been found to detect a 42-kDa rhoptry protein (ROP6) which showed protease activity and host cell binding characteristics after secretion. Using the mAb, a colony containing a 3o-UTR was probed in a T. gondii cDNA expression library. A full length cDNA sequence of the rhoptry protein was completed after 5o-RACE, which consisted of 1,908 bp with a 1,443 bp ORF. The deduced amino acid sequence of ROP6 consisted of a polypeptide of 480 amino acids without significant homology to any other known proteins. This sequence contains an amino terminal stop transfer sequence downstream of a short neutral sequence, hydrophilic middle sequence, and hydrophobic carboxy terminus. It is suggested that the ROP6 is inserted into the rhoptry membrane with both N- and C-termini.     

Isolation and sequence analysis of a full-length cDNA for human M creatine kinase

A full length cDNA for human M creatine kinase has been isolated and sequenced. The cDNA contains 77 bp of 5' untranslated, 338 bp of 3' untranslated sequence and the entire coding region (1146 bp) for human M creatine kinase. The M creatine kinases from different species share considerable sequence homology within the coding region (77-91%) and in amino acid sequence (82-97%). Little or no sequence homology is observed in the 3' untranslated sequence of the mammalian M creatine kinases, although canine and human creatine kinase share overall 80% sequence homology in 5' untranslated sequence. A unique 8 bp sequence was identified in the 5' untranslated regions of mammalian M creatine kinase but is not present in B creatine kinase cDNA. The degree of sequence conservation observed implies an evolutionary constraint on M creatine kinase structure beyond that which would be expected for the maintenance of enzymatic function.     

Characterization and primary sequence of a human hepatic microsomal estriol UDPglucuronosyltransferase

A human liver microsomal UDP glucuronosyltransferase (UDPGT) that demonstrates reactivity with estriol (pI 7.4 UDPGT) has been purified to homogeneity and characterized further. No activity toward morphine, 4-hydroxybiphenyl, bilirubin, or tripelennamine was observed. The estriol UDPGT shows immunoreactivity with antibodies raised against rat hepatic microsomal 3 alpha- and 17 beta-hydroxysteroid UDPGTs but not with antibodies raised against rat hepatic microsomal p-nitrophenol UDPGT. The NH2-terminal sequence of the purified protein was determined and found to correspond to an identical sequence in the deduced amino acid sequence of a cDNA obtained from a human liver library in lambda gt11 (HLUG4). Sequence analysis revealed that HLUG4 is 2094 bp in length and encodes a protein of 523 amino acids which has a 16 amino acid leader sequence, followed by an untranslated 3' region of 525 bp. Three potential N-glycosylation sites were identified in the predicted sequence. The deduced amino acid sequence of estriol UDPGT showed 82% identity with the deduced amino acid sequence of another human hepatic cDNA (HLUG25), which has been expressed as a UDPGT capable of 6 alpha-hydroxyglucuronidation of hyodeoxycholic acid, strongly suggesting that these proteins are members of the same gene subfamily.     

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.     

Human lysosomal acid phosphatase: cloning, expression and chromosomal assignment.

A 2112-bp cDNA clone (lambda CT29) encoding the entire sequence of the human lysosomal acid phosphatase (EC 3.1.3.2) was isolated from a lambda gt11 human placenta cDNA library. The cDNA hybridized with a 2.3-kb mRNA from human liver and HL-60 promyelocytes. The gene for lysosomal acid phosphatase was localized to human chromosome 11. The cDNA includes a 12-bp 5' non-coding region, an open reading frame of 1269 bp and an 831-bp 3' non-coding region with a putative polyadenylation signal 25 bp upstream of a 3' poly(A) tract. The deduced amino acid sequence reveals a putative signal sequence of 30 amino acids followed by a sequence of 393 amino acids that contains eight potential glycosylation sites and a hydrophobic region, which could function as a transmembrane domain. A 60% homology between the known 23 N-terminal amino acid residues of human prostatic acid phosphatase and the N-terminal sequence of lysosomal acid phosphatase suggests an evolutionary link between these two phosphatases. Insertion of the cDNA into the expression vector pSVL yielded a construct that encoded enzymatically active acid phosphatase in transfected monkey COS cells.