Primary structure of the potato virus X genome: the region preceding the capsid protein cistron

DNA copies of the potato virus X (PVX) RNA corresponding to 2300 nucleotides at the 3'-end have been cloned. The cloned cDNA copies containing the nucleotides 445-1280 from the 3'-end have been sequenced. The 5'-terminal region of the PVX coat protein gene corresponds to residues 445-786 from the 3'-end. The amino acid sequences of two more open reading frames (ORF) have been deduced from the nucleotide sequence. The potential translation products of these ORF's would correspond to the nonstructural viral proteins. We have located the ORF1 within the region of residues 799-1009 preceding the coat protein cistron. The tentative protein is composed of 70 amino acids and has an aminoterminal segment which is markedly hydrophobic. ORF2 in the PVX sequence ends with UAG at nucleotides 942-944 and extends to the 5'-terminus for additional 340 nucleotides. The distant sequence homology exists between a carboxyterminal portion of PVX ORF2 and that of the nonstructural "30 K-proteins" of the plant tobamoviruses.     

Sequence of the cDNA and gene for angiogenin, a human angiogenesis factor

Human cDNAs coding for angiogenin, a human tumor derived angiogenesis factor, were isolated from a cDNA library prepared from human liver poly(A) mRNA employing a synthetic oligonucleotide as a hybridization probe. The largest cDNA insert (697 base pairs) contained a short 5'-noncoding sequence followed by a sequence coding for a signal peptide of 24 (or 22) amino acids, 369 nucleotides coding for the mature protein of 123 amino acids, a stop codon, a 3'-noncoding sequence of 175 nucleotides, and a poly(A) tail. The gene coding for human angiogenin was then isolated from a genomic lambda Charon 4A bacteriophage library employing the cDNA as a probe. The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (4688 base pairs) was then determined. The coding and 3'-noncoding regions of the gene for human angiogenin were found to be free of introns, and the DNA sequence for the gene agreed well with that of the cDNA. The gene contained a potential TATA box in the 5' end in addition to two Alu repetitive sequences immediately flanking the 5' and 3' ends of the gene. The third Alu sequence was also found about 500 nucleotides downstream from the Alu sequence at the 3' end of the gene. The amino acid sequence of human angiogenin as predicted from the gene sequence was in complete agreement with that determined by amino acid sequence analysis. It is about 35% homologous with human pancreatic ribonuclease, and the amino acid residues that are essential for the activity of ribonuclease are also conserved in angiogenin. This provocative finding is thought to have important physiological implications.     

The sequence of a cloned cDNA for the beta subunit of bovine thyrotropin predicts a protein containing both NH2-and COOH-terminal extensions

Cloned cDNAs containing sequences coding for the beta subunit of bovine thyrotropin have been identified. The complete nucleotide sequence of the largest of the beta subunit cDNA inserts has been determined. This cDNA contains 35 nucleotides from the 5' untranslated region of thyrotropin beta subunit mRNA and 60 nucleotides coding for an NH2-terminal precursor segment. This is followed by 339 nucleotides which code for the published amino acid sequence of the thyrotropin beta subunit. Following the 339 nucleotide beta subunit coding sequence, no termination codon is encountered for another 15 nucleotides. Thus, the cDNA codes for a thyrotropin beta subunit containing an additional 5 amino acids at the COOH terminus. The cDNA also contains 82 nucleotides of 3' untranslated sequence followed by a short poly(A) segment. Comparison of the bovine cDNA sequence to the recently described mouse thyrotropin beta subunit cDNA sequence reveals considerable homology throughout the coding sequence, including the COOH-terminal extension. These findings suggest the possibility that a thyrotropin beta subunit precursor is processed at both the NH2 and COOH termini.     

Amino acid sequence of human factor XI, a blood coagulation factor with four tandem repeats that are highly homologous with plasma prekallikrein

A lambda gtll cDNA library prepared from human liver poly(A) RNA has been screened with affinity-purified antibody to human factor XI, a blood coagulation factor composed of two identical polypeptide chains linked by a disulfide bond(s). A cDNA insert coding for factor XI was isolated and shown to contain 2097 nucleotides, including 54 nucleotides coding for a leader peptide of 18 amino acids and 1821 nucleotides coding for 607 amino acids that are present in each of the 2 chains of the mature protein. The cDNA for factor XI also contained a stop codon (TGA), a potential polyadenylation or processing sequence (AACAAA), and a poly(A) tail at the 3' end. Five potential N-glycosylation sites were found in each of the two chains of factor XI. The cleavage site for the activation of factor XI by factor XIIa was identified as an internal peptide bond between Arg-369 and Ile-370 in each polypeptide chain. This was based upon the amino acid sequence predicted by the cDNA and the amino acid sequence previously reported for the amino-terminal portion of the light chain of factor XI. Each heavy chain of factor XIa (369 amino acids) was found to contain 4 tandem repeats of 90 (or 91) amino acids plus a short connecting peptide. Each repeat probably forms a separate domain containing three internal disulfide bonds. The light chains of factor XIa (each 238 amino acids) contain the catalytic portion of the enzyme with sequences that are typical of the trypsin family of serine proteases. The amino acid sequence of factor XI shows 58% identity with human plasma prekallikrein.     

Cloning of a cDNA encoding a new ribosome-inactivating protein from Beta vulgaris vulgaris (mangold)

By means of a λZAP II cDNA library constructed from seedlings of Beta vulgaris vulgaris and immunoscreening, a cDNA clone containing a partial sequence of a new ribosome-inactivating protein (RIP) was obtained. As confirmed by Western blot analysis, this clone produced a RIP upon induction with IPTG. We called it betavulgin (Bvg). The recombinant protein (re-Bvg) was somewhat smaller than plant-derived RIP (28 versus 30 and 32 kDa), but showed the specific N-glycosidase activity on tobacco ribosomes, confirming its RIP character. The cDNA was sequenced and the missing 5'-end was established by RACE using bvg-specific primers. The entire cDNA was 1080 nucleotides in length and encoded a protein of 272 amino acids with a sequence identity of 26–40% with other RIP.     

cDNA clones completing the nucleotide and derived amino acid sequence of the alpha 1 chain of basement membrane (type IV) collagen from mouse

Six cDNA clones add 3549 nucleotides to the DNA sequence of the alpha 1 chain of basement membrane (type IV) collagen. Thus the complete nucleotide and derived amino acid sequence of the alpha 1 type IV collagen with 5007 nucleotides coding for 1669 amino acids with a calculated Mr of 160,827 is known. The six cDNA clones cover the putative N-terminal signal peptide, the 7 S region and two thirds of the helical region extending into the previously published murine nucleotide sequence [(1986) Gene 43, 301]. The protein sequence for 289 amino acids of the helical region adjacent to the 7 S region has not previously been published for any species.     

Rabbit C-reactive protein. Biosynthesis and characterization of cDNA clones

To study the biosynthesis of rabbit C-reactive protein (CRP), a cDNA library was constructed from CRP mRNA-enriched polysomal poly(A) RNA. Four recombinant plasmids, designated pCX9, pCX23, pCX28, and pCX39, from 39 positive clones were sequenced and found to represent overlapping clones. DNA sequencing of CRP cDNA and primer extension of the 5'-end of CRP mRNA have demonstrated that the complete length of rabbit CRP mRNA consists of 2331 nucleotides and a terminal poly(A) segment. Analysis of the resulting sequence indicated that rabbit CRP mRNA contained a 5'-noncoding region of 107 nucleotides, a leader sequence encoding 20 amino acids, a coding region covering 205 amino acids, and a 3'-noncoding region of 1549 nucleotides. The 3'-noncoding region contained a consensus AAUAAA sequence that is 105 nucleotides upstream from the 3'-terminal poly(A) segment. Using an in vitro translation system, we have confirmed that CRP is synthesized as a precursor polypeptide (Mr approximately equal to 26,000) which undergoes processing to form the mature polypeptide (Mr approximately equal to 23,500). The CRP precursor failed to display a calcium-dependent affinity for phosphorylcholine ligand as demonstrated by mature CRP, suggesting that the phosphorylcholine-binding site of CRP only formed after processing. Northern blot analysis suggested that following induction with turpentine, liver was the only site where CRP mRNA synthesis could be demonstrated and that the change in mRNA concentration correlated with the course of CRP production. Southern blot analysis of liver genomic DNA indicated a single gene copy for CRP.     

Cloning and sequencing of the rat preproepidermal growth factor cDNA: comparison with mouse and human sequences.

We have cloned and sequenced the cDNA corresponding to the rat preproepidermal growth factor (ppEGF) mRNA. The cDNA contained 4,801 nucleotides, similar to that reported for the mouse (4,749 nucleotides) and the human mRNAs (4,871 nucleotides). The predicted protein sequence would contain 1,133 amino acids, smaller than that reported for the mouse (1,217 amino acids) and the human sequences (1,207 amino acids). The results of the sequencing of several cDNA clones suggested the existence of more than one structural gene for ppEGF. In addition, there was an occurrence of alternative splicing events, resulting in deletions of entire exons from the mature mRNA. These alternative splicing events do not create frameshift mutations but cause a deletion of one or more of the "EGF-like" repeat units from the ppEGF. There is approximately the same homology between the rat and mouse amino acid sequences both in the EGF region and in the other regions of the ppEGF protein. We conclude that, because of this conservation of homology, there may be an important function performed by these other regions of the ppEGF besides their function as a precursor for the EGF protein.     

Alignment of the amino terminal amino acid sequence of human cytochrome c oxidase subunits I and II with the sequence of their putative mRNAs.

Thirteen of the first fifteen amino acids from the NH2-terminus of the primary sequence of human cytochrome c oxidase subunit I and eleven of the first twelve amino acids of subunit II have been identified by microsequencing procedures. These sequences have been compared with the recently determined 5'-end proximal sequences of the HeLa cell mitochondrial mRNAs and unambiguously aligned with two of them. This alignment has allowed the identification of the putative mRNA for subunit I, and has shown that the initiator codon for this subunit is only three nucleotides away from the 5'-end of its mRNA; furthermore, the results have substantiated the idea that the translation of human cytochrome c oxidase subunit II starts directly at the 5'-end of its putative mRNA, as had been previously inferred on the basis of the sequence homology of human mitochondrial DNA with the primary sequences of the bovine subunit.     

The complete amino acid sequence of a human folate binding protein from KB cells determined from the cDNA

Two species of folate binding protein (FBP), an integral membrane-associated form and a soluble secreted form, have been previously purified from cultured human KB cells. The complete nucleotide sequence of the complementary DNA (cDNA) clone for the coding region of the mature membrane-associated FBP has now been determined, and the deduced amino acid sequence has been computer-analyzed for a prediction of the secondary structure of the protein. The clone has 857 nucleotides of which 678 comprise the coding region for 226 amino acids. The deduced amino sequence contains the identical sequence of the published 18 NH2-terminal amino acids of the purified FBP from KB cells and the published partial amino acid sequence of the human milk FBP except for 1 residue. There was also over 90% homology with the published amino acid sequence of the bovine milk FBP. A total of 16 cysteine residues has been conserved in the three proteins indicating that this amino acid may provide a tertiary structure which is required for its ligand binding function. Northern blot analysis using the cDNA probe identified a single band of 1.28-kilobase pair mRNA in KB cells which was 4.7-fold more intense in folate-depleted cells than in normal cells. These results indicate that the membrane FBP and the soluble FBP in the medium are translation products of the same gene. Computer analysis of the deduced amino acid sequence indicates that there is only one stretch of amino acids of sufficient hydrophobicity and length to span the lipid bilayer of the plasma membrane, but it lacked a predictable helical structure. Those regions of the sequence which did have a predictable helical structure lacked sufficient hydrophobicity required for a membrane anchor. Thus, it is likely that the fatty acids previously reported to be present in the membrane-associated FBP from these cells rather than a peptide sequence provide an important membrane anchoring function.     

Identification by molecular cloning of two forms of the alpha-subunit of the human liver stimulatory (GS) regulatory component of adenylyl cyclase

Two DNA molecules complementary to human liver mRNA coding for the alpha-subunit of the stimulatory regulatory component Gs of adenylyl cyclase were cloned. One of the two forms is a full-length cDNA of 1614 nucleotides plus a poly(A) tail of 59 nucleotides. The deduced sequence of 394 amino acids encoded by its open reading frame is essentially identical to that of the alpha-subunits of Gs identified by molecular cloning from bovine adrenals, bovine brain and rat brain. Two independent clones of the other type of cDNA were isolated. Both were incomplete, beginning within the open reading frame coding for the alpha s polypeptide. One codes for amino acids 5 through 394 and the other for amino acids 48 through 394 of the above described cDNA of 1614 nucleotides, and both have the identical 3'-untranslated sequence. They differ from the first cDNA, however, in that they lack a stretch of 42 nucleotides (numbers 214 through 255) and have nucleotides 213 (G) and 256 (G) replaced with C and A, respectively. This results in a predicted amino acid composition of another alpha-subunit of Gs that is shorter by 14 amino acids and contains two substitutions (Asp for Glu and Ser for Gly) at the interface between the deletion and the unchanged sequence. We call the smaller subunit alpha s1 and the larger alpha s2. This is the first demonstration of a structural heterogeneity in alpha s subunits that is due to a difference in amino acid sequence.     

绵羊FGF5基因的克隆、表达及RNA干扰

根据牛的成纤维细胞内生长因子5(Fibroblast growth factor 5,FGF5)基因cDNA序列设计引物,PCR扩增得到绵羊FGF5基因cDNA的开放阅读框序列,并比较和其他6种高等哺乳动物的序列同源性;同时研究该基因在绵羊多种组织的表达情况,以及研究以细胞模型RNA干扰下的表达情况。结果表明,绵羊FGF5基因ORF全长为813 bp,编码270个氨基酸,分子量约为29.58 kDa,理论等电点10.59。绵羊FGF5基因cDNA序列与牛、人、小鼠、大鼠、犬和猫的对应序列同源性高度保守,预测氨基酸序列同源性同样具有高度保守性。RT-PCR分析表明FGF5在绵羊皮肤、小肠、肾脏、心脏、肝脏、脾脏、胰脏和肺中均有表达,皮肤中表达量最高。构建该基因的原核表达载体和RNAi载体,IPTG诱导在大肠杆菌中融合表达获得55 kDa的蛋白条带,设计的RNA干扰片段能显著抑制FGF5基因的表达。文章为进一步阐明绵羊FGF5的功能尤其是在羊毛生长发育中的作用提供了理论和实验基础。     

Polypeptide chain elongation factor 1 alpha (EF-1 alpha) from yeast: nucleotide sequence of one of the two genes for EF-1 alpha from Saccharomyces cerevisiae.

Messenger RNA for yeast cytosolic polypeptide chain elongation factor 1 alpha (EF-1 alpha) was partially purified from Saccharomyces cerevisiae. Double-stranded complementary DNA (cDNA) was synthesized and cloned in Escherichia coli with pBR327 as a vector. Recombinant plasmid carrying yEF-1 alpha cDNA was identified by cross-hybridization with the E. coli tufB gene and the yeast mitochondrial EF-Tu gene (tufM) under non-stringent conditions. A yeast gene library was then screened with the EF-1 alpha cDNA and several clones containing the chromosomal gene for EF-1 alpha were isolated. Restriction analysis of DNA fragments of these clones as well as the Southern hybridization of yeast genomic DNA with labelled EF-1 alpha cDNA indicated that there are two EF-1 alpha genes in S. cerevisiae. The nucleotide sequence of one of the two EF-1 alpha genes (designated as EF1 alpha A) was established together with its 5'- and 3'-flanking sequences. The sequence contained 1374 nucleotides coding for a protein of 458 amino acids with a calculated mol. wt. of 50 300. The derived amino acid sequence showed homologies of 31% and 32% with yeast mitochondrial EF-Tu and E. coli EF-Tu, respectively.     

Glycinin A3B4 mRNA. Cloning and sequencing of double-stranded cDNA complementary to a soybean storage protein

The cDNA clones encoding the precursor form of glycinin A3B4 subunit have been identified from a library of soybean cotyledonary cDNA clones in the plasmid pBR322 by a combination of differential colony hybridizations, and then by immunoprecipitation of hybrid-selected translation product with A3-mono-specific antiserum. A recombinant plasmid, designated pGA3B41425, from one of six clones covering codons for the NH2-terminal region of the subunit was sequenced, and the amino acid sequence was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 516 amino acids. Analysis of this cDNA also showed that it contained 1786 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 46 nucleotides, a signal peptide region corresponding to 24 amino acids, an A3 acidic subunit region corresponding to 320 amino acids followed by a B4 basic subunit region corresponding to 172 amino acids, and a 3'-terminal nontranslated region of 192 nucleotides, which contained two characteristic AAUAAA sequences that ended 110 nucleotides and 26 nucleotides from a 3'-terminal poly(A) segment, respectively. Our results confirm that glycinin is synthesized as precursor polypeptides which undergo post-translational processing to form the nonrandom polypeptide pairs via disulfide bonds. The inferred amino acid sequence of the mature basic subunit, B4, was compared to that of the basic subunit of pea legumin, Leg Beta, which contained 185 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall 42% of the amino acid positions are identical in both proteins. These results led us to conclude that both storage proteins have a common ancestor.     

Cloning of cDNAs coding for the heavy chain region and connecting region of human factor V, a blood coagulation factor with four types of internal repeats

Human factor V is a high molecular weight plasma glycoprotein that participates as a cofactor in the conversion of prothrombin to thrombin by factor Xa. Prior to its participation in the coagulation cascade, factor V is converted to factor Va by thrombin generating a heavy chain and a light chain, and these two chains are held together by calcium ions. A connecting region originally located between the heavy and light chains is liberated during the activation reaction. In a previous study, a cDNA of 2970 nucleotides that codes for the carboxyl-terminal 938 amino acids of factor V was isolated and characterized from a Hep G2 cDNA library [Kane, W. H., & Davie, E. W. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6800-6804]. This cDNA has been used to obtain additional clones from Hep G2 and human liver cDNA libraries. Furthermore, a Hep G2 cDNA library prepared with an oligonucleotide from the 5' end of these cDNAs was screened to obtain overlapping cDNA clones that code for the amino-terminal region of the molecule. The composite sequence of these clones spans 6911 nucleotides and is consistent with the size of the factor V message present in Hep G2 cells (approximately 7 kilobases). The cDNA codes for a leader sequence of 28 amino acids and a mature protein of 2196 amino acids. The amino acid sequence predicted from the cDNA was in complete agreement with 139 amino acid residues that were identified by Edman degradation of cyanogen bromide peptides isolated from the heavy chain region and connecting region of plasma factor V.(ABSTRACT TRUNCATED AT 250 WORDS)