Human platelet cGI-PDE: expression in yeast and localization of the catalytic domain by deletion mutagenesis

Cyclic adenosine monophosphate (cAMP) is an important modulator of platelet responses to agonists. Cyclic nucleotide phosphodiesterase (PDE) controls intracellular cAMP concentrations by hydrolyzing it to AMP. The major PDE activity in platelets is PDE3A (cyclic guanosine monophosphate [cGMP]-inhibited PDE). To obtain structural information on platelet PDE3A, we cloned the enzyme cDNA from a human erythroleukemia cell (HEL) library since the cell line expresses many platelet proteins. This clone consists of 87% of the full-length human myocardial PDE3A cDNA, spanning from nucleotides 456 to 4606, and is identical in sequence. The nucleotide coding for the N terminal 179 amino acid sequence (nt 1-536) as well as four other cDNAs (nt 1459- 1632, nt 1765-1986, nt 2152-2538, and nt 2978-3375) obtained by RT-PCR of platelet RNA are also identical to the myocardial sequences, indicating that the HEL, myocardial, and platelet PDE3As are the same. Northern blot analysis of HEL cell RNA detected two mRNAs of 7.5 and 4.4 kb. Four new deletion mutants are reported. PDE 3A delta 1 and PDE 3A delta 2, encoding amino acids 665 to 1141 and amino acids 679 to 1141, respectively, were expressed in a PDE-deficient yeast. They displayed PDE activities of 172 and 79 pmol/mg/min, respectively. PDE 3A delta 3 and PDE 3A delta 4, encoding amino acids 686 to 1141 and 700 to 1141, had no detectable PDE activity. All mutant proteins were expressed as determined by Western blot analysis. These findings localize the PDE3A catalytic domain to within amino acid residues 679 to 1141.     

Cloning of cDNA coding for connective tissue activating peptide III from a human platelet-derived lambda gt11 expression library

We report here the cloning of the cDNA coding for platelet connective tissue-activating peptide-III (CTAP-III) from a lambda gt11 expression library prepared using messenger RNA (mRNA) isolated from human platelets. The open reading frame of the clone coded for a protein with 128 amino acid residues. Since the precursor of CTAP-III, platelet basic protein (PBP is 94 amino acids long, the 5'-translated region of the cDNA codes for a leader sequence 34 amino acids long. This leader sequence, like the sequence of mature CTAP-III, shows significant homology to the sequence of platelet factor 4 (PF4), the only other platelet specific alpha-granule protein cloned until now, from a human erythroleukemic (HEL) cell line-derived cDNA library. These leader sequences are probably critical for targeting such proteins to the alpha-granule. Northern blot hybridization with platelet and megakaryocyte mRNA shows a single species mRNA of approximately 0.8 kb, suggesting that the corresponding cDNA is full length. The cloning of platelet specific CTAP-III provides additional evidence for the platelet specificity of the cDNA library used.     

Isolation and characterization of cDNA clones for human apolipoprotein A-I.

We have isolated cDNA clones encoding human apolipoprotein (apo) A-I. Twenty putative apo A-I cDNA clones were selected by screening 10,000 clones of an adult human liver cDNA library with an oligonucleotide probe. The probe was a mixture of synthetic 14-base-long DNA oligomers constructed to correspond to the codons for apo A-I amino acids 105-109. Four of these clones were examined further and showed 600- to 800-base-pair (bp) inserts. Preliminary restriction mapping and partial DNA sequence analysis indicated that the shorter inserts were a subset of the longer DNA inserts. DNA sequence analysis of the clone with an insert of approximately equal to 600 bp, designated pAI-113, revealed that it contained a DNA sequence corresponding to apo A-I amino acids 94-243. The DNA base sequence of this clone also contained a standard termination codon, polyadenylylation signal, and poly(A) tail. Partial DNA sequence of a second clone that contained an 800-bp insert, designated pAI-107, showed that it corresponded to apo A-I amino acids 18-243 and also included the 3' untranslated region. Isolation of these cDNA clones will facilitate molecular analyses of apolipoproteins in normal and disease states.     

Molecular characterization of the PK-LR gene in sixteen pyruvate kinase-deficient patients

We studied the PK-LR gene in 16 unrelated patients with congenital haemolytic anaemia associated with erythrocyte pyruvate kinase deficiency. Fifteen different mutations were detected among the 28 mutated alleles identified: two deletions (del 1010G, del 1042--1044); one four nucleotide duplication (nt 1515--1518, GGTC); one splice site [IVS6(-2)t]; nine missense (991A, 1003A, 1151T, 1160G, 1181T, 1181A, 1456T, 1483A, 1529A); and two nonsense (721T, 1675T) mutations. Eight of them [del 1010G, del 1042--1044, dupl 1515--1518, IVS6(-2)t, 1003A, 1160G, 1181T, 1181A] were novel. The deletion 1042-1044 causes the loss of Lys 348. Deletion 1010G and duplication 1515-1518 determine a frameshift and the creation of a stop codon at nucleotides 1019 and 1554 respectively. Mutation IVS6(-2)t leads to an alteration of the 5' and 3' splice site consensus sequence; the cDNA analysis shows a 67-bp deletion in the first part of exon 11 (del 1437--1503). All the four new missense mutations involve highly conserved amino acids. The most frequent mutation in Italy would appear to be 1456T. Correlation was made between mutations, biochemical characteristics of the enzyme and clinical course of the disease.     

Complete cDNA and derived amino acid sequence of human factor V.

cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A) tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approximately equal to 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approximately 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approximately 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues.     

中华按蚊防御素基因 cDNA序列和基因组序列的克隆及鉴定

目的 克隆中华按蚊防御素基因全长cDNA序列及基因组序列,并对其进行鉴定和生物信息学分析。方法 根据已发表的埃及伊蚊和冈比亚按蚊等的防御素基因序列设计引物,提取中华按蚊总RNA并构建其基因组文库,分别进行多轮RT-PCR和巢式PCR扩增,将所得片段进行克隆、测序,并应用相关生物信息学软件对序列进行鉴定和分析。结果 从中华按蚊基因组文库中扩增出完整的防御素基因组序列(由两个外显子和一个内含子组成)以及5′端和3′端的非编码序列(UTR)片段,总长度为2 256 bp;从中华按蚊总RNA中扩增出大小为324 bp的cDNA片段,经测序证实为中华按蚊防御素基因全长cDNA序列,其开放阅读框共编码107个氨基酸,成熟肽部分具有40个氨基酸残基。结论 首次克隆出中华按蚊防御素基因全长cDNA序列及基因组序列,为进一步的功能研究奠定了基础。     

Human platelet glycoprotein IX: an adhesive prototype of leucine-rich glycoproteins with flank-center-flank structures.

The glycoprotein (GP) Ib-IX complex on the surface of human platelets functions as the von Willebrand factor receptor and mediates von Willebrand factor-dependent platelet adhesion to blood vessels. GPIX is a relatively small (Mr, 17,000) protein that may provide for membrane insertion and orientation of the larger component of the complex, GPIb (Mr, 165,000). Using antibody screening, we cloned a cDNA encoding GPIX from a human erythroleukemia cell cDNA library constructed in phage lambda gt11. Lacking a 5' untranslated region and start codon, the cDNA sequence includes 604 nucleotides, beginning with 495 bases at the 5' end coding for 165 amino acids, followed by a stop codon and 106 noncoding bases at the 3' end. By Northern blot analysis, the GPIX cDNA hybridizes with a single 1.0-kilobase species of platelet poly(A)+ RNA. Translation of the cDNA sequence gives a predicted protein sequence beginning with a truncated putative signal sequence of 5 amino acid followed by a sequence of 17 amino acids matching that determined directly by Edman degradation of intact GPIX. The predicted amino acid sequence of mature GPIX includes an NH2-terminal extracytoplasmic domain of 134 residues, a transmembrane domain of 20 residues, 6 intracytoplasmic residues, and 1 N-linked glycosylation site. GPIX contains a leucine-rich glycoprotein (LRG) sequence of 24 amino acids similar to conserved LRG sequences in GPIb and other proteins from humans, Drosophila, and yeast. "Flanking" sequences of approximately 22 amino acids are present at the NH2 and/or COOH sides of the "central" LRG sequence(s) in GPIX, GPIb, and the other human and Drosophila members of the LRG family. The role of the flank-LRG center-flank structure in the evolution and function of the LRG proteins remains to be defined.     

A 13-mer peptide straddling the leucine33/proline33 polymorphism in glycoprotein IIIa does not define the PLA1 epitope.

We confirm the recent report (J Clin Invest 83:1778, 1989) of a polymorphism at amino acid 33 of platelet GPIIIa associated with the PLA1/PLA2 phenotype by using the polymerase chain reaction on cDNA derived from platelet RNA, using the base-pair primers 105-129 and 452-428. Platelet cDNA from three PLA2-homozygous individuals, when digested with Nci I, gave two bands of 256 bp and 91 bp, whereas eight PLA1 cDNAs gave a single band of 347 bp. Two 13-mer amino acid peptides straddling the amino acid polymorphism: SDEALP (L/P) GSPRCD were synthesized for epitope studies. Two mouse polyclonal antibodies were raised: one against the PLA1-associated peptide, the other against the PLA2 peptide. Both antibodies react with either peptide, as well as with both PLA1 and PLA2 platelets. The PLA1 peptide did not block the binding of two different human anti-PLA1 antibodies to the 100-Kd GPIIIa band on immunoblot of platelet extracts; neither did it block the binding of the same antibodies to PLA1-platelet extracts in an enzyme-linked immunosorbent assay. Further studies were performed on the PLA1 epitope following subtilisin digestion of purified GPIIIa. A 55-Kd fragment was obtained that retained the PLA1 epitope as well as the first 13 N-terminal amino acids of GPIIIa. Reduction of the 55-Kd fragment resulted in loss of the PLA1 epitope with production of a 67-Kd, 21-Kd, and 10-Kd band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 55-Kd band does not react with LK-2, a monoclonal antibody versus GPIIIa that inhibits adenosine diphosphate, collagen, epinephrine, and thrombin-induced aggregation. Thus, the PLA1 epitope is conformation-induced, resides on an N-terminal 55-Kd fragment composed of two or more peptides held together by -SH bonds, and is not required for platelet aggregation.     

日本血吸虫(中国大陆株)20.2 kDa分子编码基因(Sj20.2)的克隆及序列分析

目的：克隆日本血吸虫中国大陆株20.2kDa分子的编码基因，方法：根据筛选到的日本血吸虫肝期童虫cDNA文库阳性克隆之一的插入片段序列，设计合成1对引物，通过PCR技术对其最大开放阅读框进行扩增，然后克隆人pGEM-T载体，并对克隆产物进行核苷酸序列测定和分析，利用在线软件推测其编码氨基酸的序列，结果：利用PCR方法从上述插入片段中扩增出1条单一的DNA条带，大小介于515bp与697bp之间，将此产物克隆入pGEM-T载体，双酶切和PCR反应结果都出现与前述结果一致的目的片段，核苷酸序列测定结果表明此开放阅读框全长564bp，在线软件分析结果表明，此片段编码187个氨基酸，编码肽段分子量大小约为20．0dDa，结论：本次实验成功扩增和克隆出目的基因片段，为下一步研究奠定了基础。     

Isolation and sequence of a human apolipoprotein CII cDNA clone and its use to isolate and map to human chromosome 19 the gene for apolipoprotein CII.

cDNA clones encoding human apolipoprotein CII (apo CII) were identified by screening an adult human liver cDNA library with a mixed oligonucleotide probe corresponding to all possible codons for apo CII amino acid 6-10. One clone with an approximately equal to 500-base-pair (bp) insert, designated pCII -711, was selected for DNA sequence analysis. This clone contained a DNA sequence that corresponded with the previously reported amino acid sequence of apo CII with only minor differences. The DNA sequence specified a polypeptide of 79 amino acids, compared to the 78 amino acids previously reported. The pCII -711 clone contains a 36-bp DNA sequence upstream from that specifying the NH2-terminal threonine which, when read in frame, specifies the amino acid sequence Leu-Val-Leu-Leu-Val-Leu-Gly-Phe-Glu-Val-Gln-Gly and may be part of an apo CII signal peptide. The pCII -711 clone also contains a 144-bp region that corresponds to the 3' untranslated region of apo CII mRNA as well as a portion of the poly(A) tail. Clone pCII -711 was used to isolate and characterize by restriction endonuclease digestion the gene for apo CII from a human genomic library. In addition, through Southern blot analysis of DNA from human-rodent somatic cell hybrids, clone pCII -711 also was used to provisionally map the gene for apo CII to human chromosome 19.     

细粒棘球蚴内蒙株FABP基因cDNA的克隆与核酸疫苗的构建

【提要】 根据GenBank中细粒棘球蚴脂肪酸结合蛋白(FABP)基因cDNA序列设计引物, 并在起始密码子前加上Kozak 序列(CCACC),提取细粒棘球蚴(内蒙株)的原头蚴总RNA,RT-PCR扩增目的基因。回收其纯化的产物克隆到 pMD19-T载体后进行序列分析。克隆到的FABP基因cDNA序列长402 bp,开放阅读框(ORF)编码133个氨基酸。FABP基因cDNA亚克隆到pcDNA3.1(+)中,构建核酸疫苗pcDNA3.1-FABP-NM, 经测序验证, 结果正确。     

Identification of an additional member of the protein-tyrosine-phosphatase family: evidence for alternative splicing in the tyrosine phosphatase domain.

Protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.13.48) have been implicated in the regulation of cell growth; however, to date few tyrosine phosphatases have been characterized. To identify additional family members, the cDNA for the human tyrosine phosphatase leukocyte common antigen (LCA; CD45) was used to screen, under low stringency, a mouse pre-B-cell cDNA library. Two cDNA clones were isolated and sequence analysis predicts a protein sequence of 793 amino acids. We have named the molecule LRP (LCA-related phosphatase). RNA transfer analysis indicates that the cDNAs were derived from a 3.2-kilobase mRNA. The LRP mRNA is transcribed in a wide variety of tissues. The predicted protein structure can be divided into the following structural features: a short 19-amino acid leader sequence, an exterior domain of 123 amino acids that is predicted to be highly glycosylated, a 24-amino acid membrane-spanning region, and a 627-amino acid cytoplasmic region. The cytoplasmic region contains two approximately 260-amino acid domains, each with homology to the tyrosine phosphatase family. One of the cDNA clones differed in that it had a 108-base-pair insertion that, while preserving the reading frame, would disrupt the first protein-tyrosine-phosphatase domain. Analysis of genomic DNA indicates that the insertion is due to an alternatively spliced exon. LRP appears to be evolutionarily conserved as a putative homologue has been identified in the invertebrate Styela plicata.     

Molecular cloning, characterization, and functional expression of rat oxidosqualene cyclase cDNA.

A cDNA encoding rat oxidosqualene lanosterol-cyclase [lanosterol synthase; (S)-2,3-epoxysqualene mutase (cyclizing, lanosterol-forming), EC 5.4.99.7] was cloned and sequenced by a combination of PCR amplification, using primers based on internal amino acid sequence of the purified enzyme, and cDNA library screening by oligonucleotide hybridization. An open reading frame of 2199 bp encodes a M(r) 83,321 protein with 733 amino acids. The deduced amino acid sequence of the rat enzyme showed significant homology to the known oxidosqualene cyclases (OSCs) from yeast and plant (39-44% identity) and still retained 17-26% identity to two bacterial squalene cyclases (EC 5.4.99.-). Like other cyclases, the rat enzyme is rich in aromatic amino acids and contains five so-called QW motifs, highly conserved regions with a repetitive beta-strand turn motif. The binding site sequence for the 29-methylidene-2,3-oxidosqualene (29-MOS), a mechanism-based irreversible inhibitor specific for the vertebrate cyclase, is well-conserved in all known OSCs. The hydropathy plot revealed a rather hydrophilic N-terminal region and the absence of a hydrophobic signal peptide. Unexpectedly, this microsomal membrane-associated enzyme showed no clearly delineated transmembrane domain. A full-length cDNA was constructed and subcloned into a pYEUra3 plasmid, selected in Escherichia coli cells, and used to transform the OSC-deficient uracil-auxotrophic SGL9 strain of Saccharomyces cerevisiae. The recombinant rat OSC expressed was efficiently labeled by the mechanism-based inhibitor [3H]29-MOS.     

Isolation and heterologous expression of a cDNA encoding bovine inositol polyphosphate 1-phosphatase.

Inositol polyphosphate 1-phosphatase, an enzyme of the phosphatidylinositol signaling pathway, catalyzes the hydrolysis of the 1-position phosphate from inositol 1,3,4-trisphosphate and inositol 1,4-bisphosphate. The protein was isolated from calf brain and digested with trypsin or CNBr, and the amino acid sequence of several peptides was determined. Degenerate oligonucleotide primers were designed from amino acid sequence and used to synthesize an 80-base-pair (bp) fragment by the polymerase chain reaction. This product was used to isolate a 1.6-kbp cDNA with an open reading frame of 400 amino acids, 185 bp of 5' untranslated region, and 171 bp of 3' untranslated region followed by a putative poly(A) tail. The coding region of the cDNA was inserted into an expression vector that was used to obtain the recombinant protein from Escherichia coli cells. The recombinant enzyme (44 kDa) had a specific activity and other properties similar to those of native bovine brain inositol polyphosphate 1-phosphatase. It hydrolyzed both inositol phosphate substrates and was inhibited by lithium ions. The enzyme shows minimal sequence similarity to inositol monophosphate phosphatase, the other enzyme inhibited by lithium ions in the signaling pathway.     

The alpha and beta chains of human platelet glycoprotein Ib are both transmembrane proteins containing a leucine-rich amino acid sequence.

The primary structure of the beta chain of human glycoprotein Ib (GPIb), the platelet receptor for von Willebrand factor, has been established by a combination of cDNA cloning and amino acid sequence analysis. A lambda phage cDNA expression library prepared from human erythroleukemia cells (HEL cells) was screened with a radiolabeled affinity-purified rabbit polyclonal antibody to the beta chain of GPIb. Eighteen positive clones were isolated and plaque-purified and the nucleotide sequences of three were determined. The composite sequence spanned 968 nucleotides and included a 5' untranslated region of 22 nucleotides, an open reading frame of 618 nucleotides encoding a signal peptide of 28 amino acids and a mature protein of 181 amino acids, a stop codon, and a 3' noncoding region of 307 nucleotides. The 3' noncoding sequence also contained a polyadenylylation signal (AATAAA) 14 nucleotides upstream from the poly(A) tail of 18 nucleotides. Edman degradation of the intact beta chain and of peptides produced by chemical cleavage yielded amino acid sequences spanning 76 residues that were identical to those predicted from the cDNA. The amino-terminal region of the beta chain contains a leucine-rich sequence of 24 amino acids that is similar to a sequence that occurs as seven tandem repeats in the alpha chain of GPIb and nine tandem repeats in leucine-rich alpha 2-glycoprotein. The leucine-rich sequence in the beta chain of GPIb is flanked on both sides by amino acid sequences that are similar to those flanking the leucine-rich tandem repeats of the alpha chain of GPIb and leucine-rich alpha 2-glycoprotein. The amino-terminal region of the beta chain of GPIb is followed by a transmembrane segment of 25 amino acids and an intracellular segment of 34 amino acids at the carboxyl terminus of the protein. The intracellular segment contains an unpaired cysteine and two potential sites for phosphorylation by cAMP-dependent protein kinase.     

Cloning of the alpha chain of human platelet glycoprotein Ib: a transmembrane protein with homology to leucine-rich alpha 2-glycoprotein.

Glycoprotein Ib is a surface membrane glycoprotein of platelets that functions as a receptor for von Willebrand factor. It is a heterodimer composed of an alpha and a beta chain linked by a disulfide bond(s). A phage lambda gt11 cDNA expression library prepared from mRNA from a human erythroleukemia cell line, HEL, was screened using an affinity-purified antibody to the glycocalicin portion of the alpha chain of glycoprotein Ib. Eleven positive clones were isolated and plaque-purified. The largest cDNA insert was 2420 nucleotides in length and coded for a leader sequence of 16 amino acids, a mature protein of 610 amino acids, and a stop codon. It also contained 42 nucleotides of 5' noncoding sequence and 497 nucleotides of 3' noncoding sequence, including a poly(A) tail. The amino acid sequence of the alpha chain of GPIb predicted from the cDNA agreed completely with the sequence of 156 amino acids that was determined by Edman degradation of peptides isolated from human platelet glycocalicin after digestion with trypsin or Staphylococcus aureus V8 protease. The extracytoplasmic domain of the alpha subunit of GPIb contains several noteworthy structural features, including a region of seven tandem repeats of 24 amino acids that are homologous with those present in leucine-rich alpha 2-glycoprotein. The extracytoplasmic domain also contains two hydrophilic regions, one rich in charged amino acids and a second rich in serine and threonine residues. The region rich in serine and threonine includes five repeats of nine amino acids as well as the majority of the O-linked carbohydrate sites present in the molecule. The extracytoplasmic domain is followed by a potential transmembrane segment of approximately 29 amino acids and a potential intracellular domain of approximately 100 amino acids located at the carboxyl end of the molecule.