The construction, identification and characterisation of plasmids containing human alpha-lactalbumin cDNA sequences.

We describe the cloning of double-stranded cDNA synthesized from lactating human mammary gland total poly(A)-containing RNA, into the EcoRI site of the plasmid pAT153. Nine recombinants were shown to contain alpha-lactalbumin cDNA sequences as determined by positive hybridisation translation of complementary RNA. Restriction enzyme maps were determined for six of these. Alignment of the restriction map with the known amino acid sequence of human alpha-lactalbumin provided evidence that two plasmids, designated phO-53 and phB-35, contained the complete coding sequence of the primary translation product (pre-alpha-lactalbumin). Hybridisation studies using purified human, monkey and guinea-pig alpha-lactalbumin cDNA demonstrated that greater nucleotide sequence divergence has occurred within the rodents than the primates, and that rodent alpha-lactalbumin mRNAs retain regions of homology with primate alpha-lactalbumin mRNAs.     

Complete nucleotide sequence of ovine alpha-lactalbumin mRNA

The nucleotide sequence of ovine alpha-lactalbumin mRNA has been determined by chemical sequencing of two cDNA recombinant plasmids and a primer extension product. Ovine alpha-lactalbumin mRNA contains 723 nucleotides (excluding the poly(A) tail), with a 5' non-coding region of 26 nucleotides, followed by the 426 nucleotides of the coding region which determines a sequence signal of 19 amino acid residues and the 123 amino acid residues of mature alpha-lactalbumin. The coding region is followed by a 3' untranslated sequence of 271 nucleotides. The derived amino acid sequence of ovine pre-alpha-lactalbumin differs from that of its bovine counterpart by 8 amino acid substitutions, all but one originating from single mutations. Comparison of sequences of guinea pig, rat and human alpha-lactalbumin mRNAs with their ovine and bovine counterparts has revealed that these molecules have rapidly evolved. The highest degree of conservation was observed in the region coding for the mature protein and corresponds essentially to sequences which interact with UDP-galactosyltransferase and Ca2+ ions.     

Structure and evolution of human α-fetoprotein deduced from partial sequence of cloned cDNA

The nucleotide sequence of a recombinant DNA clone, containing a partial mRNA sequence for human α-fetoprotein (AFP) in the plasmid vector pBR322, has been determined. Two regions of the cloned nucleotide sequence were found to agree with published amino acid sequences of two cyanogen bromide peptides derived from human AFP. Examination of the amino acid sequence, deduced from the cloned portion of the mRNA coding region, reveals extensive homology with the third domain of the human serum albumin molecule. A total of 44% ( ) amino acids and 54% ( ) nucleotides are identical in the two structures. The landmark cysteine residues are found in the same positions in both polypeptide chains, presumably forming the same disulfide bridges in AFP as those found in the albumin. The sequence homology reinforces the evidence that human AFP and albumin constitute a gene family, in analogy to the same family found in rodents. A comparison of the human and rodent sequence data suggests that the rate of molecular evolution has been faster for AFP than for albumin.     

Nucleotide sequence of cloned cDNA of human apolipoprotein A-I.

ApoA-I is the major human HDL apoprotein. By oligonucleotide hybridization, we have isolated 5 dscDNA clones to human hepatic apo A-I mRNA. One of these clones (pA1-3) was completely sequenced. It has 878 bp plus a poly A tail of 48 and includes all the coding and 3'-untranslated regions of the mRNA and part of the 5'-untranslated region. It predicts a peptide sequence of 267 amino acids (including the 24 amino acid prepropeptides) which is very similar to the sequence reported by Brewer et al., (1978) Biochem. Biophys. Res. Commun. 80:623-630. The predicted signal peptide sequence is highly homologous to the rat apoA-I signal peptide. There is no evidence for any internally repeated segments in apoA-I either at the amino acid or at the DNA level. Using pA1-3 as a probe, we have detected on Northern gels apo A-I mRNA sequences of approximately 1100 nucleotides in human hepatic and baboon hepatic and intestinal RNAs, but not in RNAs from baboon skeletal muscle, kidney or spleen. The demonstration of apo A-I mRNA sequences in specific organs is important to our concept of "reverse cholesterol transport".     

cDNA cloning of human apoA-I: Amino acid sequence of preproapoA-I

ds-cDNA to human liver apoA-I mRNA has been cloned. Nucleic acid sequence analysis revealed that apoA-I mRNA codes for a precursor apolipoprotein, preproapoA-I, which contains 24 amino acids on the NH₂-terminal end of the mature plasma apoA-I. Eighteen amino acids are contained within the hydrophobic prepeptide (Met-Lys-Ala-Ala-Val-Leu-Thr-Leu-Ala-Val-Leu-Phe-Leu-Thr-Gly-Ser-Gln-Ala) followed by a 6 amino acid propeptide (Arg-His-Phe-Top-Gln-Gln). Our results on human apoA-I are in agreement with the partial sequence of the precursor of rat apoA-I with respect to the length of the precursor sequence, location of the prepeptide, and the presence of an unusual propeptide sequence terminating in a neutral dipeptide Gln-Gln. A detailed analysis of the primary structure of normal human apoA-I mRNA will be essential to our ultimate understanding of the processing of human apoA-I and diseases characterized by molecular defects in apoA-I structure and function.     

Isolation of cDNA clones for human erythrocyte membrane sialoglycoproteins alpha and delta.

We have isolated almost full-length cDNA clones corresponding to human erythrocyte membrane sialoglycoproteins alpha (glycophorin A) and delta (glycophorin B). The predicted amino acid sequence of delta differs at two amino acid residues from the sequence determined by peptide sequencing. The sialoglycoprotein delta clone we have isolated contains an interrupting sequence within the region that gives rise to the cleaved N-terminal leader sequence for the protein and represents a product that is unlikely to be inserted into the erythrocyte membrane. Comparison of the cDNA sequences of alpha and delta shows very strong homology at the DNA level within the coding regions. The two mRNA sequences are closely related and differ by a number of clearly defined insertions and deletions.     

Cloning and nucleotide sequence of cDNA encoding human liver serine-pyruvate aminotransferase

Cloned cDNAs for human liver serine-pyruvate aminotransferase (Ser-PyrAT) were obtained by screening of a human liver cDNA library with a fragment of cDNA for rat mitochondrial Ser-PyrAT as a probe. Two clones were isolated from 50,000 transformants. Both clones contained approximately 1.5 kb cDNA inserts and were shown to almost completely overlap each other on restriction enzyme mapping and DNA sequencing. The nucleotide sequence of the mRNA coding for human liver Ser-PyrAT was determined from those of the cDNA clones. The mRNA comprises at least 1487 nucleotides, and encodes a polypeptide consisting of 392 amino acid residues with a molecular mass of 43,039 Da. The amino acid composition determined on acid hydrolysis of the purified enzyme showed good agreement with that deduced from the nucleotide sequence of the cDNA. In vitro translation of the mRNA derived from one of the isolated clones, pHspt12, as well as that of mRNA extracted from human liver, yielded a product of 43 kDa which reacted with rabbit anti-(rat mitochondrial Ser-PyrAT) serum. Comparison of the deduced amino acid sequences of human Ser-PyrAT and the mature form of rat mitochondrial Ser-PyrAT revealed 79.3% identity. Although human Ser-PyrAT appears to be synthesized as the mature size, the 5'-noncoding region of human Ser-PyrAT mRNA contains a nucleotide sequence which would encode, if translated, an amino acid sequence similar to that of the N-terminal extension peptide of the precursor for rat mitochondrial Ser-PyrAT.     

Molecular cloning and nucleotide sequencing of human immunoglobulin epsilon chain cDNA.

DNA complementary to mRNA of human immunoglobulin E heavy chain (epsilon chain) isolated and purified from U266 cells has been synthesized and inserted into the PstI site of pBR322 by G-C tailing. This recombinant plasmid was used to transform E. coli chi 1776 to screen 1445 tetracycline resistant colonies. Nine clones (pGETI - 9) containing cDNA coding for the human epsilon chain were recognized by colony hybridization and Southern blotting analysis with a nick-translated human IgE genome fragment. The nucleotide sequence of the longest cDNA contained in pGET2 was determined. The results indicate that the sequence of 1657 nucleotides codes for 494 amino acids covering a part of the variable region and all of the constant region of the human epsilon chain. Most of the amino acid sequence deduced from the nucleotide sequence is in substantial agreement with that reported. Furthermore a termination codon after the -COOH terminal amino acid marks the beginning of a 3' untranslated region of 125 nucleotides with a poly A tail. Taking this into account, the structure of the human epsilon chain mRNA, except a part of the 5' end, is conserved fairly well in the cDNA insert in pGET2.     

Cloning and sequence analysis of cDNA clones for bovine aortic-endothelial-cell transglutaminase.

A cDNA clone encoding transglutaminase was isolated from a bovine-endothelial-cell cDNA library using oligonucleotide probes designed based on partial amino acid sequences of the purified protein. Sequencing of the cDNA insert revealed an open reading frame of 2061 bp coding for a protein of 687 amino acids. The sequence of bovine endothelial-cell transglutaminase was 88, 82, 80, 37, 37 and 37% identical with that of human endothelial, rat macrophage, guinea-pig liver, human and rat keratinocyte transglutaminases, and the human blood-coagulation factor XIIIa subunit, respectively. The cDNA clone was hybridized to a single mRNA species of 3.9 kb in the liver, lung, spleen and heart but not hybridized to RNA from the brain. Northern-blot analysis of mRNA from retinoid-treated cultured vascular endothelial cells revealed that retinoids were able to induce a large increase in the transglutaminase mRNA levels.     

Rat apolipoprotein E mRNA. Cloning and sequencing of double-stranded cDNA

A 900-base pair clone corresponding to rat liver apolipoprotein E (apo-E) mRNA, and containing a 3'-terminal poly(A) segment, was identified from a library of rat liver cDNA clones in the plasmid pBR322 by specific hybrid selection and translation of mRNA. A restriction endonuclease DNA fragment from this recombinant plasmid was used to clone the 5'-terminal region of the apo-E mRNA by primed synthesis of cDNA. A portion of the double-stranded cDNA corresponding to the 3'-terminal region of apo-E mRNA was subcloned into the bacteriophage M13mp7 and employed as a template for the synthesis of a radioactively labeled, cDNA hybridization probe. This cDNA probe was used in a RNA-blot hybridization assay that showed the length of the apo-E mRNA to be about 1200 nucleotides. The hybridization assay also demonstrated that apo-E mRNA is present in rat intestine, but at about a 100-fold lower level than that of the rat liver. The nucleotide sequence of rat liver apo-E mRNA was determined from the cloned, double-stranded cDNAs. The amino acid sequence of rat liver apo-E was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 311 amino acids. A comparison to the NH2-terminal amino acid sequence of rat plasma apo-E indicated that the first 18 amino acids of the primary translation product are not present in the mature protein and are probably removed during co-translational processing. The coding region was flanked by a 3'-untranslated region of 109 nucleotides, which contained a characteristic AAUAAA sequence that ended 13 nucleotides from a 3'-terminal poly(A) segment. At the 5'-terminal region of the mRNA, 23 nucleotides of an untranslated region were also determined. The inferred amino acid sequence of mature rat apo-E, which contains 293 amino acids, was compared to the amino acid sequence of human apo-E, which contains 299 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall, 69% of the amino acid positions are identical in both proteins. The amino acid identities are clustered in two broad domains separated by a short region of nonhomology, an NH2-terminal domain of 173 residues where 80% are identical, and a COOH-terminal domain of 84 residues where 70% are identical. These two domains may be associated with specific functional roles in the protein.     

Human testis cDNA for the regulatory subunit RII alpha of cAMP-dependent protein kinase encodes an alternate amino-terminal region

Phosphorylations catalyzed by cAMP-dependent protein kinase are essential for sperm motility, and type II cAMP-dependent protein kinase in mature sperm has been shown to be firmly bound to the flagellum via the regulatory subunit, RII. The present study documents high-levelled expression of a human, testis-specific RII alpha mRNA (2.0 kb) analogous to the rat mRNA which is induced in haploid germ cells [(1988) FEBS Lett. 229, 391-394]. We report the molecular cloning of a full-length human cDNA corresponding to this unique testis mRNA, and the presence of an alternative amino-terminal region (amino acids 45-75) of the predicted RII alpha protein (404 amino acids) compared with the previously published mouse and rat sequences. However, this alternate region is also shown to be present in RII alpha mRNA (7.0 kb) of human somatic cells. Our data indicate the divergent amino-terminal sequence to be due to species differences, suggesting an active evolutionary pressure on this particular region, which could be involved in subcellular attachment of RII alpha and thereby localization of kinase activity to certain targets within the cell.     

Human tissue factor: cDNA sequence and chromosome localization of the gene

A human placenta cDNA library in lambda gt11 was screened for the expression of tissue factor antigens with rabbit polyclonal anti-human tissue factor immunoglobulin G. Among 4 million recombinant clones screened, one positive, lambda HTF8, expressed a protein that shared epitopes with authentic human brain tissue factor. The 1.1-kilobase cDNA insert of lambda HTF8 encoded a peptide that contained the amino-terminal protein sequence of human brain tissue factor. Northern blotting identified a major mRNA species of 2.2 kilobases and a minor species of approximately 3.2 kilobases in poly(A)+ RNA of placenta. Only 2.2-kilobase mRNA was detected in human brain and in the human monocytic U937 cell line. In U937 cells, the quantity of tissue factor mRNA was increased severalfold by exposure of the cells to phorbol 12-myristate 13-acetate. Additional cDNA clones were selected by hybridization with the cDNA insert of lambda HTF8. These overlapping isolates span 2177 base pairs of the tissue factor cDNA sequence that includes a 5'-noncoding region of 75 base pairs, an open reading frame of 885 base pairs, a stop codon, a 3'-noncoding region of 1141 base pairs, and a poly(A) tail. The open reading frame encodes a 33-kilodalton protein of 295 amino acids. The predicted sequence includes a signal peptide of 32 or 34 amino acids, a probable extracellular factor VII binding domain of 217 or 219 amino acids, a transmembrane segment of 23 amino acids, and a cytoplasmic tail of 21 amino acids. There are three potential glycosylation sites with the sequence Asn-X-Thr/Ser.(ABSTRACT TRUNCATED AT 250 WORDS)     

Guinea-pig casein A cDNA. Nucleotide sequence analysis and comparison of the deduced protein sequence with that of bovine alpha s2 casein

The nucleotide sequence (1036 bases) of guinea-pig casein A mRNA has been determined. Two cDNA recombinant plasmids contained a total of 993 base pairs, including part of the 5' noncoding region, and the complete coding and 3' noncoding region. The remaining 5' noncoding sequence was obtained by primer extension. The deduced 223-amino-acid-coding sequence of guinea-pig pre-casein A exhibited 30% homology with bovine alpha s2 casein, the most striking similarities being in the locations of potential phosphorylation sites.     

人SBK1 cDNA的克隆及其相互作用蛋白的筛选

首次克隆到人的SBK1（homo sapiens SH3-binding domain kinase 1,SBK1）的cDNA序列,并通过生物信息学的手段,电子克隆到人SBK1的基因组DNA序列.人的SBK1是鼠SBK1的直系同源物,两者基因组DNA结构相似,均含有4个外显子.人的sbk1基因ORF长1 275 bp,编码424个氨基酸,而鼠的ORF长1 254 bp,编码417个氨基酸.两者编码区的核苷酸序列同源性达87.7%,而氨基酸序列同源性达95.7%,在羧基端均有一个PV富集区,推测其能与含有SH3结构域的蛋白质结合.将RT－PCR所获得的长度为1 610 bp的sbk1cDNA序列搜索EST数据库,进行电子延伸,最终获得了约5 kb的人sbk1全长mRNA序列,它与鼠的sbk1全长mRNA大小一致;通过比较基因组学发现UniGene族Hs.97837实际上代表了sbk1基因UniGene族Hs.460471的3′UTR区域,而不是代表了一个新的UniGene族.采用酵母双杂交技术,以SBK1为“诱饵”,获得了与之相互结合的蛋白表皮生长因子受体EGFR和核孤儿受体蛋白NR4A1,它们之间的具体功能关系有待进一步研究.     

Cloning, expression and sequence homologies of cDNA for human gamma enolase

The nucleotide sequence of the human gamma-enolase mRNA was determined from recombinant cDNA clones. The sequence spans 2273 bp and includes the complete coding region of 1299 bp, a 5'-noncoding region of 74 bp and a 897-bp-long 3'-noncoding region containing a variant polyadenylation signal (ATTAAA). The deduced amino acid (aa) sequence is 433 aa long and shows a 97% similarity with rat gamma-enolase. Both the 5'- and 3'-untranslated regions are similar (82% and 68%, respectively) to the analogous regions of the rat gamma-enolase gene, suggesting that a strong selective pressure operates on noncoding segments of gamma-enolase mRNAs. The size of the gamma-enolase mRNA expressed in human brain is 2.4 kb. A crosshybridizing 1.5-kb message is detected in human skeletal muscle which may be derived from the beta-enolase-coding gene.     

Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene

A human umbilical vein endothelial cell cDNA library in lambda gt11 was screened for expression of thrombomodulin antigens with affinity-purified rabbit polyclonal anti-thrombomodulin immunoglobulin G (IgG) and mouse monoclonal anti-human thrombomodulin IgG. Among 7 million recombinant clones screened, 12 were recognized by both antibodies. Two of these, lambda HTm10 and lambda HTm12, were shown to encode thrombomodulin by comparison of the amino acid sequence deduced from the nucleotide sequence to the amino acid sequence determined directly from tryptic peptides of thrombomodulin. Thrombomodulin mRNA was estimated to be 3.7 kilobases in length by Northern blot analysis of endothelial cell and placental poly(A)+ RNA. Thrombomodulin mRNA was not detected in human brain, HepG2 hepatoma cells, or the monocytic U937 cell line. Additional cDNA clones were selected by hybridization with the 1.2-kilobase insert of lambda HTm10. One isolate, lambda HTm15, contained a 3693 base pair cDNA insert with an apparent 5'-noncoding region of 146 base pairs, an open reading frame of 1725 base pairs, a stop codon, a 3'-noncoding region of 1779 base pairs, and a poly(A) tail of 40 base pairs. The cDNA sequence encodes a 60.3-kDa protein of 575 amino acids. The predicted protein sequence includes a signal peptide of approximately 21 amino acids, an amino-terminal ligand-binding domain of approximately 223 amino acids, an epidermal growth factor (EGF) homology region of 236 amino acids, a serine/threonine-rich segment of 34 amino acids, a membrane-spanning domain of 23 amino acids, and a cytoplasmic tail of 38 amino acids. The EGF-homology region consists of six tandemly repeated EGF-like domains.(ABSTRACT TRUNCATED AT 250 WORDS)     

cDNA cloning of porcine transforming growth factor-beta 1 mRNAs. Evidence for alternate splicing and polyadenylation

Most eukaryotic cells encode principally a 2.5-kilobase (kb) transforming growth factor (TGF)-beta 1 mRNA. However, we have found two major TGF-beta 1 RNA species, 3.5 and 2.5 kb long, in porcine tissues. The 3.5-kb species has a longer 3'-untranslated sequence generated by the selection of an alternate polyadenylation site. There is a 117-nucleotide sequence within this unique 3' region, which is similar to the PRE-1 repetitive sequence of unknown function, reported earlier in the porcine genome. We have also cloned and characterized an alternately spliced mRNA species specific for the TGF-beta 1 gene, in which exons IV and V of the corresponding human TGF-beta 1 gene are deleted. The nucleotide sequence of this cDNA clone predicts a putative precursor protein of 256 amino acids; the N-terminal 211 amino acids of this putative protein are identical to the TGF-beta 1 precursor protein (exons I, II, and III of the human TGF-beta 1 gene), but the C-terminal 45 amino acids are distinct, due to a frameshift in the translation of exons VI and VII. In addition we provide data for the existence of other mRNA species generated in a tissue-specific manner either by alternate splicing or by heterogeneous 5' leader sequences.