Sequence homology between human alpha 1-antichymotrypsin, alpha 1-antitrypsin, and antithrombin III

alpha 1-Antichymotrypsin mRNA was isolated by specific polysome immunoprecipitation from turpentine-treated baboon liver. The highly enriched mRNA was used for synthesis and cloning of the corresponding cDNA. Baboon alpha 1-antichymotrypsin cDNA clones were identified by hybrid-selected translation, and the insert DNA fragment from one of the putative clones was used as a probe to screen a human liver cDNA library comprised of 40 000 independent transformants. One of the human cDNA clones was unambiguously identified to contain alpha 1-antichymotrypsin DNA sequences by comparison of its 5'-terminal nucleotide sequence with the N-terminal amino acid sequence of the protein. This cDNA clone, designated phACT235, contains 1524 base pairs of human DNA, which was sequenced in its entirety. The inserted DNA codes for a 25 amino acid signal peptide sequence and the entire mature alpha 1-antichymotrypsin of 408 amino acid residues. Comparison of the amino acid sequence of alpha 1-antichymotrypsin with that of the human alpha 1-antitrypsin has revealed a homology level similar to that between chymotrypsin and trypsin.     

Primary structure of rat renal dipeptidase and expression of its mRNA in rat tissues and COS-1 cells.

Three cDNA clones of rat renal dipeptidase (rrDP) were isolated from rat renal and pulmonary cDNA libraries using a DNA fragment of human renal DP cDNA clone, MDP4, as a probe. The complete amino acid sequence deduced from the cDNA contains 410 amino acid residues, beginning with a signal peptide of 16 amino acid residues. RNA blot hybridization analysis showed that 1.6 and 2.2 kb mRNAs were expressed in lung and kidney, however, only 1.6 kb mRNA was detected in small intestine. COS-1 cells transfected with the cDNA expressed enzymatically active rrDP.     

Carp growth hormone: molecular cloning and sequencing of cDNA

cDNA clones of the fish Cyprinus carpio growth hormone (GH) mRNA have been isolated from a cDNA library prepared from carp pituitary gland poly(A)+RNA. The nucleotide sequence of one of the carp GH cDNA clones containing an insert of 1164 nucleotides (nt) was determined. The cDNA sequence was found to encode a polypeptide of 210 amino acids (aa) including a signal peptide of 22 aa and to contain 5' and 3' untranslated regions of the mRNA of 36 and 498 nt, respectively. The carp GH presents a 63% amino acid sequence homology with the salmon GH, has structural features common with other GH polypeptides of mammalian or avian origin and contains domains of conserved sequence near the N- and C-terminal regions. Southern blot hybridization of carp genomic DNA with GH cDNA probes shows the presence of at least two GH-coding sequences in the fish genome.     

Amino acid sequence of the a subunit of human factor XIII

Factor XIII is a plasma protein that plays an important role in the final stages of blood coagulation and fibrinolysis. The complete amino acid sequence of the a subunit of human factor XIII was determined by a combination of cDNA cloning and amino acid sequence analysis. A lambda gtll cDNA library prepared from human placenta mRNA was screened with an affinity-purified antibody against the a subunit of human factor XIII and then with a synthetic oligonucleotide probe that coded for a portion of the amino acid sequence present in the activation peptide of the a subunit. Six positive clones were identified and shown to code for the a subunit of factor XIII by DNA sequence analysis. A total of 3831 base pairs was determined by sequencing six overlapping cDNA clones. This DNA sequence contains a 5' noncoding region or a region coding for a portion of a pro-piece or leader sequence, the mature protein (731 amino acids), a stop codon (TGA), a 3' noncoding region (1535 nucleotides), and a poly(A) tail (10 nucleotides). When the a subunit of human factor XIII was digested with cyanogen bromide, 11 peptides were isolated by gel filtration and reverse-phase HPLC. Amino acid sequence analyses of these peptides were performed with an automated sequenator, and 363 amino acid residues were identified. These amino acid sequences were in complete agreement with those predicted from the cDNA. The a subunit of factor XIII contained the active site sequence of Tyr-Gly-Gln-Cys-Trp, which is identical with that of tissue transglutaminase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of a full-length cDNA for human alpha-N-acetylgalactosaminidase (alpha-galactosidase B)

In the process of molecular cloning of cDNA for proteins associated with a purified human placental sialidase fraction, we discovered one of the proteins with apparent molecular weight of 46 kDa is in reality alpha-N-acetylgalactosaminidase. The full length cDNA, pcD-HS1204, codes for 358 amino acids with the first 17 residues representing a putative signal peptide. The predicted amino acid sequence shows striking homology with human alpha-galactosidase A and yeast alpha-galactosidase. The substrate specificities as well as the behavior of the 46 kDa protein on hydroxylapatite chromatography confirmed that the 46 kDa protein is in reality alpha-N-acetylgalactosaminidase.     

Structure of soybean Kunitz trypsin inhibitor mRNA determined from cDNA by using oligodeoxynucleotide primers

Oligodeoxynucleotides complementary to the deduced mRNA sequence of soybean Kunitz trypsin inhibitor (KTI) were used to prime the synthesis of cDNA from soybean cotyledon total poly(A) RNA. The primed cDNA was used to select clones from a Glycine max cotyledon cDNA library. Two out of twelve hybridizing clones were shown to contain KTI cDNA. The nucleotide sequence of one clone, pSTI 9-2, was determined and it was found to encompass the complete protein coding region of KTI excet for three C-terminal residues. Trypsin inhibitor is synthesized with a 25 amino acid hydrophobic N-terminal sequence presumed to be a signal peptide. The mature polypeptide encoded by pSTI 9-2 agrees with the published amino acid composition of KTI, but contains two discrepancies at the peptide sequence level.     

Cloning of a cDNA encoding porcine brain natriuretic peptide

Complimentary DNA (cDNA) clones encoding porcine brain natriuretic peptide (BNP) were isolated from a porcine atrial cDNA library. The longest of the cDNA clones (1507 nucleotides) apparently originated from an unprocessed messenger RNA, since the nucleotide sequence encoding BNP-26 was interrupted by an intron of 554 nucleotides. A partial cDNA clone representing processed BNP mRNA was prepared by polymerase chain reaction. A comparison of the sequence of these two cDNAs reveals the presence of an additional intron within the sequence encoding the BNP precursor. The identification of these introns suggests that the BNP gene structure differs from the atrial natriuretic peptide gene in the location of intron 2. BNP mRNA encodes a propeptide of 131 amino acids, including a signal peptide domain (25 amino acids) and a prohormone domain (106 amino acids). Like atrial natriuretic peptide, the bioactive BNP sequence is localized at the carboxyl terminus of the prohormone. Although the carboxyl-terminal peptide sequences of porcine atrial natriuretic peptide and BNP are well conserved, there is relatively little homology within their propeptide regions.     

Cloning and sequence analysis of cDNA for mouse prolactin

The present study was undertaken to find out whether or not sexual dimorphism in biological activities and amino acid compositions of mouse prolactin might be due to heterogeneity in mRNA for mouse prolactin Cloned cDNAs for mouse prolactin were first isolated from a mouse pituitary cDNA library by hybridization with a rat prolactin cDNA. Then, one clone of about 140 positive clones obtained from 2000 transformants was subjected to nucleotide sequence analysis and verified to contain a nearly full length of cDNA sequence coding for mouse prolactin precursor. The deduced complete amino acid sequence indicates that the precursor molecule consists of 31 amino acids as the signal peptide and 197 amino acids of prolactin, in which two amino acids were found to be different from the amino acid sequence previously published elsewhere. S1 nuclease mapping analysis using male and female pituitary RNAs indicates that mouse preprolactin is encoded by two mRNAs in both sexes. The two mRNAs differ from each other based upon the deletion of three nucleotides in the coding region for the signal peptide determined by the nucleotide sequence analysis in other cDNA clones. In the present study, no sexual difference was revealed in murine prolactin mRNA.     

Human serum amyloid A (SAA): biosynthesis and postsynthetic processing of preSAA and structural variants defined by complementary DNA

To study structural variants of human serum amyloid A (SAA), an apoprotein of high-density lipoprotein, complementary DNA clones were isolated from a human liver library with the use of two synthetic oligonucleotide mixtures containing sequences that could code for residues 33-38 and 90-95 of the protein sequence. The SAA-specific cDNA clone (pA1) contains the nucleotide sequence coding for the mature SAA and 10 amino acids of the 18-residue signal peptide. It also includes a 70 nucleotide long 3'-untranslated region and approximately 120 bases of the poly(A) tail. The derived amino acid sequence of pA1 is identical with the alpha form of apoSAA1. A fragment of pA1 containing the conserved (residues 33-38) region of SAA also hybridized with RNA from human acute phase liver and acute phase stimulated, but not unstimulated, mouse and rabbit liver. In contrast, a fragment corresponding to the variable region hybridized to a much greater extent with human than with rabbit or murine RNA. Human acute phase liver SAA mRNA (approximately 600 nucleotides in length) directs synthesis of preSAA (Mr 14 000) in a cell-free translating system. In a Xenopus oocyte translation system preSAA is synthesized and processed to the mature Mr 12 000 product. The complete 18 amino acid signal peptide sequence of preSAA was derived from sequencing cDNA synthesized by "primer extension" from the region of SAA mRNA corresponding to the amino terminus of the mature product. Two other SAA-specific cDNA clones (pA6 and pA10) differed from pA1 in that they lack the internal PstI restriction enzyme site spanning residues 54-56 of pA1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and characterization of the growth hormone-dependent insulin-like growth factor binding protein (IGFBP-3) in the rat.

We report for the first time the complete amino acid sequence for the growth hormone dependent insulin-like growth factor binding protein (IGFBP-3) in the rat. A human IGFBP-3 clone was generated using the polymerase chain reaction (PCR) and used to screen a rat liver cDNA library. cDNA clones of the rat IGFBP-3 were isolated and the full amino acid sequence deduced. The sequence begins with a putative, 26 amino acid signal peptide followed by a 265 amino acid binding protein. The amino acid sequence is over 80% homologous with the equivalent human IGFBP-3 form and shows complete conservation of 18 cysteine residues that are clustered at the amino and carboxy ends of the protein. IGFBP-3 is the binding subunit of the major circulating IGFBP in the rat, and hence the availability of precise structural data and cDNA probes provides an important opportunity for a detailed study of the control of IGFBP-3 synthesis at the level of gene expression.     

Structure and expression of a cloned cDNA for mouse interferon-beta

A unique sequence in the mouse genome which cross-hybridized to a cloned human interferon-beta 1 gene was detected by DNA blot analysis. Taking advantage of this, a cDNA library prepared from partially purified mRNA for mouse interferon-beta was screened using human interferon-beta 1 DNA as a probe. One of the positive clones, pM beta-3, contained a 680-base pair cDNA insert, whose base sequence contained a single large open reading frame for 182 amino acids. The coding sequences of the cDNA showed homologies of 63% at the nucleotide and 48% at the amino acid level with respect to human interferon-beta 1 cDNA (Taniguchi, T., Ohno, S., Fujii-Kuriyama, Y., and Muramatsu, M. (1980) Gene 10, 11-15). The first 21 amino acids, considered to be the signal peptide, were followed by 24 amino acids, whose sequence was identical with the NH2-terminal sequence that had been reported for mouse interferon-beta from Ehrlich ascites tumor cells (Taira, H., Broeze, R. J., Jayaram, B. M., Lengyel, P., Hunkapiller, M. W., and Hood, L. E. (1980) Science (Wash. D.C.) 207, 528-530). The complete primary sequence of mature interferon-beta polypeptide consisting of 161 amino acids (Mr = 19,700) was deduced. There are three N-glycosylation sites, and this offers an explanation for the larger molecular size (Mr = 26,000-40,000) of natural mouse interferon-beta in comparison to the deduced interferon polypeptide. The cDNA, when fused to a SV40 promoter sequence and then introduced into COS-7 cells, directed the synthesis and secretion of a protein product indistinguishable from the authentic mouse interferon-beta.     

Cloning of the grass carp growth hormone cDNA

We have constructed a cDNA library in lamda gt11 using mRNA isolated from the pituitary glands of the grass carp (Ctenopharyngodon idellus). Based on the published sequence of the rainbow trout growth hormone cDNA, we synthesized two oligonucleotide probes. One of these hybridized strongly with a specific mRNA fragment from the grass carp pituitary. Using this probe, we have isolated six positive clones carrying an insert of approximately 1.2 Kb. By restriction enzyme digestion, all the clones were determined to be identical. Sequence determination on one of them indicated that it has an open reading frame coding for 210 amino acids. Both the nucleotide and translated amino acid sequence are highly homologous to those of the salmon growth hormone and the common carp. A putative signal peptide consisting of hydrophobic amino acids can be identified at the 5' end of the sequence. A polyadenylation signal, ATTAAA, was also present 12 base upstream from the poly A tail.     

Molecular cloning of human immune interferon cDNA and its expression in eukaryotic cells.

Starting with mRNA derived from Staphylococcal enterotoxin A induced human splenocytes, dsDNA was synthesized and inserted into unique BamHI site of the eukaryotic expression vector pSV529 (1). A recombinant plasmid containing human immune interferon (IFN-gamma) cDNA was identified by hybridization of plasmid inserted DNA bound onto nitrocellulose filters with mRNA derived from SEA-induced splenocytes, translation of the eluted RNA in Xenopus laevis oocytes and assaying for IFN activity. Plasmids containing the entire human IFN-gamma cDNA sequence were identified by colony hybridization and were sequenced. A unique coding region was identified which predicted a protein of 166 amino acids, the 20 N-terminal amino acids of which presumably represent a signal peptide. After transfection of monkey cells with plasmid DNA isolated from one of the recombinant clones (pHIIF-SV-gamma 1), IFN was excreted into the culture medium. This IFN was not distinguishable from human IFN-gamma by serological criteria or by cell target species specificity.     

Characterization of a cDNA for human protein C inhibitor. A new member of the plasma serine protease inhibitor superfamily

A cDNA library in lambda-phage lambda gt11 containing DNA inserts prepared from human liver mRNA was screened with monoclonal antibodies to human protein C inhibitor. Six positive clones were isolated from 6 X 10(6) phages and plaque purified. The cDNA in the phage containing the largest insert, which hybridized to a DNA probe prepared on the basis of the amino-terminal amino acid sequence of the mature inhibitor, was sequenced. This cDNA insert contained 2106 base pairs coding for a 5'-noncoding region, a 19-amino acid signal peptide, a 387-amino acid mature protein, a stop codon, and a long 3'-noncoding region of 839 base pairs. Based on the amino acid sequence of the carboxyl-terminal peptide released by cleavage of protein C inhibitor by activated protein C as well as by thrombin, the reactive site peptide bond of protein C inhibitor is Arg354-Ser355. Five potential carbohydrate-binding sites were found in the mature protein. The high homology of the amino acid sequence of protein C inhibitor to the other known inhibitors clearly demonstrates that protein C inhibitor is a member of the superfamily of serine protease inhibitors including alpha 1-antichymotrypsin, alpha 1-antitrypsin, antithrombin III, ovalbumin, and angiotensinogen. Based on the difference matrices for these proteins, we present possible phylogenetic trees for these proteins.     

Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning.

A lambda gt11 library constructed from poly(A+) RNA isolated from aortic tissue of neonatal rats was screened for rat tropoelastin cDNAs. The first screen, utilizing a human tropoelastin cDNA clone, provided rat tropoelastin cDNAs spanning 2.3 kb of carboxy-terminal coding sequence and extended into the 3'-untranslated region. A subsequent screen using a 5' rat tropoelastin cDNA clone yielded clones extending into the amino-terminal signal sequence coding region. Sequence analysis of these clones has provided the complete derived amino acid sequence of rat tropoelastin and allowed alignment and comparison with published bovine cDNA sequence. While the overall structure of rat tropoelastin is similar to bovine sequence, numerous substitutions, deletions, and insertions demonstrated considerable heterogeneity between species. In particular, the pentapeptide repeat VPGVG, characteristic of all tropoelastins analyzed to date, is replaced in rat tropoelastin by a repeating pentapeptide, IPGVG. The hexapeptide repeat VGVAPG, the bovine elastin receptor binding peptide, is not encoded by rat tropoelastin cDNAs. Variations in coding sequence between rat tropoelastin cDNA clones were also found which may represent mRNA heterogeneity produced by alternative splicing of the rat tropoelastin pre-mRNA.     

鲑鱼生长激素cDNA的分子克隆和序列分析

从太平洋切奴克鲑鱼(Pacific Chinook Salmon,Oncorthychus tschawytscha)垂体poly(A)~+ RNA构建cDNA文库。按照鲑鱼生长激素(sGH)部分氨基酸序列合成两个寡聚脱氧核苷酸探针,它们分别与编码第1—7和第166—172氨基酸序列互补。用探针筛查cDNA文库,得到了完整的sGH cDNA克隆。cDNA序列已测定,包括编码210个氨基酸的编码序列。其中含有22个氨基酸的信号肽序列和188个氨基酸的成熟GH序列。该克隆还包括了5'端和3'端非翻译区,分别为72个和438个碱基对长。与Chum鲑鱼比较表明,核酸序列和氨基酸序列的同源性分别为97%和99%。     

Isolation and characterization of the complete complementary and genomic DNA sequences of human serum amyloid P component

Complementary and genomic DNA clones corresponding to the human serum amyloid P component (SAP) mRNA have been isolated and analyzed. The nucleotide sequences of the cDNA and the corresponding regions of the genomic SAP DNA reported here were identical, and revealed that after coding for a signal peptide of 19 amino acids and the first two amino acids of the mature SAP protein, there is one small intron of 115-base pairs (bp), followed by a nucleotide sequence coding for the remaining 202 amino acid residues. The SAP gene has an ATATAAA sequence 29-bp upstream from the cap site, but there is no CAAT box-like sequence. A possible polyadenylation signal sequence, ATTAAA, was found to be located 28-bp upstream from the polyadenylation site. A comparison of the genomic SAP DNA sequence with that of human C-reactive protein (CRP) revealed a striking overall homology which was not uniform: several highly conserved regions were bounded by non-homologous regions. This comparison provides further support for the hypothesis that SAP and CRP are products of a gene duplication event.