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PubMed1992

Sequence of cDNA comprising the human pur gene and sequence-specific single-stranded-DNA-binding properties of the encoded protein.

The human Pur factor binds strongly to a sequence element repeated within zones of initiation of DNA replication in several eukaryotic cells. The protein binds preferentially to the purine-rich single strand of this element, PUR. We report here the cloning and sequencing of a cDNA encoding a protein with strong affinity for the PUR element. Analysis with a series of mutated oligonucleotides defines a minimal single-stranded DNA Pur-binding element. The expressed Pur open reading frame encodes a protein of 322 amino acids. This protein, Pur alpha, contains three repeats of a consensus motif of 23 amino acids and two repeats of a second consensus motif of 26 amino acids. Near its carboxy terminus, the protein possesses an amphipathic alpha-helix and a glutamine-rich domain. The repeat region of Pur cDNA is homologous to multiple mRNA species in each of several human cell lines and tissues. The HeLa cDNA library also includes a clone encoding a related gene, Pur beta, containing a version of the 23-amino-acid consensus motif similar, but not identical, to those in Pur alpha. Results indicate a novel type of modular protein with capacity to bind repeated elements in single-stranded DNA.

Amino Acid Sequence↗
PubMed1991

cDNA and genomic DNA sequence of the 21.3 kDa subunit of NADH:ubiquinone reductase (complex I) from Neurospora crassa.

The primary structure of a nuclear-encoded subunit of the respiratory chain NADH:ubiquinone reductase (complex I) from Neurospora crassa was determined by sequencing cDNA, genomic DNA and the N-terminus of the protein. The sequence correlates to a protein of 200 amino acids and a molecular mass of 21349 Da. The protein is synthesized without a cleavable presequence. It contains two alpha-helices predicted to traverse the bilayer and is a constituent of the membrane part of complex I.

Amino Acid Sequence↗
PubMed1996

cDNA cloning, sequencing and chromosomal assignment of the gene for mouse complement factor I (C3b/C4b inactivator): identification of a species specific divergent segment in factor I.

Factor I is an essential regulatory serine proteinase of the complement cascade. It cleaves and inactivates the C3b and C4b constituents of the C3 and C5 convertases and thereby regulates many complement-mediated activities. The human protein is a heterodimer composed of a 50 kDa non-catalytic subunit (which contains several domains, i.e. FIM, CD5, LDLr type A) disulfide linked to a 38 kDa catalytic subunit. Recent characterization of Xenopus factor I cDNA revealed a 29 residue negatively charged region in its heavy chain which is absent in the human protein (Kunnath-Muglia et al., Molec. Immun. 30, 1249-1256, 1993). We report the complete cDNA sequence of mouse factor I as well as a partial chicken factor I cDNA sequence. Alignment of these two sequences with the published sequences for human and Xenopus proteins (a) demonstrates an overall conservation of primary structure and domain organization of mouse factor I, and (b) defines a divergent segment (D segment) in each species. In Xenopus protein, the D segment includes the 29 residue negatively charged region. In each of the four species examined, the D segment differed in length, sequence, organization, and number of repeated subregions. These differences reflect a considerable evolution of D segment. The significance of the diversity of the D segment is at present unclear. We also report the chromosomal localization of the mouse factor I gene (Cfi) to distal chromosome 3 near Egf.

Amino Acid Sequence↗
PubMed2006

Isolation and partial sequencing of cDNA clones from rat incisor after Nd:YAG laser irradiation in root canal.

OBJECTIVE: The purpose of this study was to identify the genes expressed in odontoblast-like cells and related to osteodentin production of pulp cells in vivo during the repair process. METHODS: A pulsed Nd:YAG laser was irradiated in mandibular incisors of rats at 2 W and 20 pulses/s for 5 s. We constructed a unidirectional cDNA library from the non-calcified portion of irradiated incisors, sequenced cDNA clones and classified their sequences by homology search through the GenBank database. RESULTS: Of the sequences determined, 26% represented sequences of new genes that were not related to any previously reported gene. 65.5% of the clones strongly matched genes and proteins in the databases. Among them, sequences found more than once were amelogenin, ameloblastin, amelin, collagen alpha1 type 1, osteonectin, nestin, enamelin and osteocalcin. Sequences detected only once were transforming growth factor beta-1 (TGF-beta1), growth hormone receptor (Ghr), glypican, enamelysin (Mmp20), amelin 2, integral membrane protein 2B, amelogenin precursor (Ame1), splicing factor 3b, insuline-like growth factor binding protein 5. CONCLUSION: This library reflects the gene expression during the dentin repair process in vivo including the odontoblast-like cell specific gene expression and osteodentin-production. Using this library, we indicate the candidate genes which might be related to odontoblast-differentiation and formation of osteodentin.

Animals↗
PubMed1990

Molecular cloning, primary structure, and orientation of the vertebrate photoreceptor cell protein peripherin in the rod outer segment disk membrane.

Peripherin, a 39-kDa membrane protein, has been previously localized to the rim region of the vertebrate rod photoreceptor disk membrane by use of monoclonal antibodies and immunocytochemical labeling techniques. As an initial step in determining the structure and function of this protein, we have cloned and sequenced cDNA containing its complete coding sequence. A bovine retinal lambda gt11 expression library was screened with the antibodies, and a 583 base pair clone was initially isolated. The remaining part of the coding sequence was obtained from subsequent rescreenings of the same library and an independent lambda gt10 library. A C-terminal CNBr fragment of peripherin was purified by immunoaffinity chromatography and reverse-phase high-performance liquid chromatography. The amino acid sequence of the isolated C-terminal peptide and the N-terminal sequence analysis of immunoaffinity-purified peripherin are in agreement with the cDNA sequence. The cDNA sequence predicts that there are possibly four transmembrane domains. On the basis of immunocytochemical studies and sequence analysis, the hydrophilic C-terminal segment containing the antigenic sites for the antiperipherin monoclonal antibodies has been localized on the cytoplasmic side of the disk membrane. There are three consensus sequences for asparagine-linked glycosylation. Deglycosylation studies have indicated that at least one of these sites is utilized. The possible function of peripherin in relation to its primary structure is discussed.

Amino Acid Sequence↗
PubMed1984

Amino- and carboxy-terminal sequence of mouse J chain and analysis of tryptic peptides.

Mouse J chain was isolated from an IgM-producing hybridoma by gel filtration and ion-exchange chromatography. The sequence of the amino-terminal 25 residues was determined. At these positions, the results agree with the amino acid sequence deduced from the cDNA sequence determined previously by Koshland and co-workers and indicate that a leader sequence terminating in glycine is removed to form the mature J chain. Tryptic peptides of J chain were isolated by high pressure liquid chromatography and their amino acid compositions were compared with those expected from the cDNA sequence. The amino acid sequence of the carboxy-terminal peptide and a mixture of two other peptides was determined. The results were consistent with the cDNA sequence except that we found valine, not leucine, at position 67, and arginine, not glycine, at position 117. The presence of aspartic acid at the carboxy-terminus, as predicted from the cDNA, indicates that processing does not occur at this end of the polypeptide chain. Upon amino acid analysis, glucosamine was found in tryptic peptides 47-57 and 47-58. J chain was also cleaved at aspartylproline bonds with formic acid and the unfractionated digest was subjected to automated Edman degradation. The mixed sequence was consistent with the sequence deduced from the cDNA at positions 1 to 13, 28 to 40, 52 to 64, and 73 to 85. In conjunction with the results obtained previously by analysis of cDNA, these data show that mouse J chain is a polypeptide containing 137 amino acid residues, 93 of which are identical to residues in human J chain.

Amino Acid Sequence↗
PubMed1996

The entire genomic sequence and cDNA expression of mouse alpha-galactosidase A.

The full-length cDNA and genomic sequences encoding mouse alpha-galactosidase A (alpha-Gal A; EC 3.2.1.22), a lysosomal galactohydrolase, were isolated and characterized. The cDNA's open reading frame encoded 419 amino acids and had 82% nucleotide (nt) and 78% amino acid identity with the human sequence, although the carboxy terminus of the mouse alpha-Gal A polypeptide was 10 amino acids shorter. The functional integrity of the mouse cDNA was demonstrated by transient expression in COS-1 cells. Northern analysis revealed two mRNA species of about 1.6 and 3.4 kb due to alternative polyadenylation signals. The entire 14.4-kb mouse genomic sequence was determined; each of its seven exons was interrupted by intronic sequence at the identical positions as the exons in the human gene. The mouse 5' flanking region (250 nt) had one Sp1, site, five CAAT boxes, and no TATA box and had 67% identity with the human promoter region. The gene contained 18 complete or partial Alu-repetitive elements (13 type 1 and 5 type 2 repeats), and three putative functional AATAAA consensus polyadenylation signals were identified 72, 1668, and 1682 nt after the TAA termination codon. Use of the 72-nt site and the 1866 and/or 1682 sites were consistent with the shorter and longer transcripts. The availability of the full-length cDNA and genomic sequence encoding mouse alpha-Gal A should facilitate structure/function studies of this lysosomal glycosidase and the construction of alpha-Gal A-deficient mice by targeted gene disruption.

Amino Acid Sequence↗
PubMed1989

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↗
PubMed1993

Sequence studies on pyridoxal phosphate-dependent enzymes.

The amino acid sequences of two pyridoxal phosphate-dependent enzymes, namely glutamate decarboxylase and 4-aminobutyrate aminotransferase, were solved by a combination of direct protein sequencing and cDNA sequencing. In the case of glutamate decarboxylase from Escherichia coli, correct ordering of three internal peptides was achieved by sequencing a fragment of DNA generated by PCR. In the case of 4-aminobutyrate aminotransferase from pig liver, chemical studies on the reaction of the enzyme with a suicide inhibitor allowed a detailed description of the mechanism of inactivation and collection of partial sequence information on the protein. The sequence of the protein was completely reconstructed after analysis of further peptide fragments derived by proteolytic and chemical cleavages, with the help of the information from the cDNA-deduced sequence of pig brain 4-aminobutyrate aminotransferase performed by another group (Kwon, Park and Churchich [1992] J. Biol. Chem. 267, 7215-7216).

4-Aminobutyrate Transaminase↗
PubMed1996

Identification, characterization, cloning, and expression of apolipoprotein C-IV, a novel sialoglycoprotein of rabbit plasma lipoproteins.

We have identified and characterized a novel proline- and arginine-rich protein component of lipoproteins, present in up to five sialylated isoforms, in rabbit blood plasma. The pI of the desialylated protein is 5.7. Based upon its N-terminal sequence, a complete cDNA sequence of 555 nucleotides was cloned from rabbit liver. The synthesized protein is predicted to contain 124 amino acids, including a typical signal peptide of 27 residues. The mature protein of 97 amino acids, designated apolipoprotein C-IV, is associated with the lipoproteins of blood plasma, primarily very low density and high density lipoproteins. It contains two potential amphipathic helices characteristic of plasma apolipoproteins and forms discoidal micelles with phosphatidylcholine. Northern analysis shows a single 0.6-kilobase apolipoprotein C-IV mRNA, detected only in the liver, and Southern analysis suggests a single copy gene. Sialylated apolipoprotein C-IV is secreted from transfected mammalian cells. Nucleotide sequence comparisons demonstrate a strong homology to portions of the upstream regions of the mouse and human apolipoprotein C2 genes, within each of which a distinct gene has recently been identified. The nucleotide sequences and the predicted amino acid sequences, as well as corresponding cDNA sequences in the rat and monkey, indicate that the apolipoprotein C4 gene has been highly conserved during mammalian evolution.

Amino Acid Sequence↗
PubMed1995

Tissue-type transglutaminase from red sea bream (Pagrus major). Sequence analysis of the cDNA and functional expression in Escherichia coli.

A cDNA clone encoding a tissue-type transglutaminase (TGase) was isolated from a cDNA library prepared from the liver of red sea bream (Pagrus major). The cDNA sequence had an open reading frame coding for a protein of 695 amino acids and showed 43% identity to the sequence of guinea pig liver TGase, revealing a relatively low overall similarity. However, the 25-amino-acid sequence containing the putative active site (Cys272) of the enzyme was completely conserved between the two species, and was also identical to the corresponding regions of human and bovine endothelial cell TGases. In addition, the critical residues (His332 and Asp355) thought to form the catalytic-center triad together with Cys272, were found in the highly conserved region. The red sea bream TGase had an extension of 11 amino acids in the C-terminal region and some differences in the N-terminal region when compared with guinea pig TGase. From the cloned cDNA, a semi-synthetic TGase gene suitable for overexpression in Escherichia coli was constructed (pTTG2-22). At a reduced temperature (28 degrees C), E. coli cells transformed with pTTG2-22 could produce soluble TGase which exhibited catalytic activity in the presence of calcium. E. coli extracts containing the recombinant red sea bream TGase induced gelation of actomyosin solutions, accompanied by a significant increase of epsilon-(gamma-glutamyl)lysine bonds, which are predominantly derived from the cross-linking of myosin heavy chains. These results indicate that this fish TGase should be useful for further analysis of TGase structure/function relationships and that it could also be employed to enhance the viscoelastic properties of proteinaceous materials.

Actomyosin↗
PubMed2002

[Construction and application of an in silico elongation system of nucleic acid sequence].

Normally it is difficult to obtain full-length cDNA sequence of novel genes. More and more expressed sequence tags(ESTs) have been obtained since the start-up of human genome project. Powerful system is badly needed for data mining on these EST sequences. Based on a personal computer coupled with Linux operating system and EST database, the Blast software and Phrap software were used to construct a platform for in silico elongation of ESTs in our lab. The performance was tested using 11386 EST sequences and 511 partial-length cDNA sequences. Results demonstrated that 8373 EST and 389 cDNA sequence were elongated using this system. Thus the platform seems to be a fast way for full-length cDNA sequence cloning of new genes.

English Abstract↗
PubMed1998

Cloning and sequence analysis of the cDNA encoding the elongation factor-1 beta of Dictyostelium discoideum.

A cDNA encoding elongation factor-1 beta (DdEF-1 beta) for protein synthesis was isolated from Dictyostlium discoideum cells by the differential display (DD) method. The DdEF-1 beta cDNA sequence with a length of 733 bp encodes a protein (M(r) 24.1 kDa) consisting of 214 amino acids. Comparison of the predicted amino acid sequence revealed relatively high homology to that of DdEF-1 beta from silkworm (59%), human (57%) and Xenopus (55%). Northern analysis showed that the DdEF-1 beta mRNA is maximally expressed during the vegetative growth phase, followed by marked decrease in response to cell differentiation induced by starvation.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and sequence analysis of the cDNA for ancrod, a thrombin-like enzyme from the venom of Calloselasma rhodostoma.

The 1.54 kb cDNA for ancrod, a thrombin-like enzyme, was cloned from a lambda ZAP cDNA library derived from the venom glands of Calloselasma (Agkistrodon) rhodostoma. The cDNA sequence reveals that ancrod is synthesized as a pre-zymogen of 258 amino acids, including a putative secretory peptide of 18 amino acids and a proposed zymogen peptide of 6 amino-acid residues. The amino-acid sequence of the predicted active form of the enzyme exhibits a high degree of sequence similarity to those of mammalian serine proteases (trypsin and pancreatic kallikrein) and other thrombin-like enzymes (batroxobin and flavoxobin). Key amino-acid residues (His43, Asp88, Ser182 and Asp176) that are thought to be involved in the substrate cleavage and in the substrate-binding reaction are conserved. Ancrod contains 13 cysteine residues. Based on alignment with the amino-acid sequences of trypsin and batroxobin, six disulphide bridges can be predicted to be present in the ancrod protein. The existence of a free cysteine, which changes the common sequence surrounding the Ser182 active site from Gly-Asp-Ser-Gly-Gly-Pro to Cys-Asp-Ser-Gly-Gly-Pro, is unusual for a serine protease.

Amino Acid Sequence↗
PubMed1996

Cloning and sequence analysis of a cDNA encoding salmon (Onchorhynchus keta) liver transglutaminase.

We isolated cDNA clones encoding a transglutaminase (TGase: EC 2.3.2.13) from a salmon (Onchorhynchus keta) cDNA library prepared from the liver. In the cDNA sequence combined, an open reading frame coding for a protein of 680 aa was found. The deduced sequence showed a considerable similarity (62.4%) to that of red sea bream TGase. By comparison of sequence similarity to other TGases, the structure of salmon TGase was like tissue type TGases, rather than membrane-associated type or plasma type TGases. As a structural feature of salmon TGase, 3 aa residues were substituted in the 25 aa sequence around the active site Cys residue, which is conserved among several tissue type TGases. The critical residues thought to form the catalytic-center triad (Cys272, His331, and Asp301) were found in the highly conserved region, but the region surrounding Tyr511, which corresponds to the residue participates in hydrogen-bond interactions of active center domain, was less similar to other TGases, except for red sea bream TGase. These finding suggests that the overall structure of fish TGase resembles tissue-type TGases, but has some unique structure.

Amino Acid Sequence↗
PubMed1996

Application of an optimised reverse transcriptase-polymerase chain reaction method to determine the cDNA nucleotide sequence of porcine basic fibroblast growth factor.

In the present investigation, oligonucleotide primers of high hybridisation stringency have been used in combination with optimised reverse transcriptase-polymerase chain reaction (RT-PCR) methods for the determination of the cDNA sequence corresponding to porcine FGF-2 mRNA present in brain and uterine tissue. Application of these optimised methods have overcome previous limitations associated with the low abundance of the porcine FGF-2 mRNA, and allowed as little as 100 micrograms of tissue to be employed to generate the complete cDNA nucleotide sequences as well as to provide specific template fragments selected for their suitability in subsequent ligation and mutagenesis studies with conventional expression vectors. Comparisons of the cDNA nucleotide and the deduced amino-acid sequence of porcine FGF-2 and the known FGF-2s from other species have indicated nucleotide sequence homologies of 95.5% with the bovine, 94.7% with the human and 88.7% with the rat FGF-2 cDNA whilst amino-acid sequence homologies of 100% with the bovine, 98.7% with the human and 96.8% with the rat FGF-2, respectively, were found. Based on these investigations, application of analogous strategies and methods with low abundance mRNAs related to other members of this family of growth factors, as well as very low abundance mRNAs of other protein growth factor, in the pig should now be readily realised.

Amino Acid Sequence↗
PubMed1986

cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts.

The entire genome of tobacco mosaic virus (TMV) was copied into a series of subgenomic cDNA clones. cDNA sequences of the 5' and 3' ends of TMV were cloned separately. A synthetic oligonucleotide primer was used to generate a Pst I site at the 5' terminus, whereas a different primer was used to generate an Nde I site at the 3' terminus. This strategy permitted removal of non-TMV sequences from cloned cDNA inserts by treatment with exonuclease VII following restriction endonuclease cleavage. Pst I linkers were added to TMV 3' terminal cDNAs. Subgenomic cDNA fragments were ligated together into several independent full-genomic constructions from which TMV cDNA sequences could be cleanly excised as a single fragment by Pst I digestion. Full-genomic TMV cDNA was ligated immediately downstream from the lambda phage promoter from pPM1 and transcribed in vitro with Escherichia coli RNA polymerase. RNA transcripts from three of four full-genomic cDNA constructions were infectious, even though they contained 6 non-TMV nucleotides at the 3' end. Transcripts from a construction with 6 extra nucleotides at the 5' end also were infectious. Progeny virus from plants infected with cDNA transcripts appeared identical to the parental virus. Restriction maps of independent cDNA clones of the same regions of the genome were identical to each other and as predicted from the reported nucleotide sequence of TMV. Also, sequences of the 200 nucleotides proximal to the 5' termini of four independent cDNA clones were identical to each other and to published sequences, suggesting that independent isolates of TMV may have remarkably similar sequences.

Journal Article↗
PubMed1983

The vicilin gene family of pea (Pisum sativum L.): a complete cDNA coding sequence for preprovicilin.

A cDNA plasmid bank has been constructed using mRNA from developing pea seeds and three cDNAs coding for vicilin polypeptides have been selected. These cDNAs have been sequenced and between them cover the whole of the coding sequence plus part of the 5' and 3' untranslated regions. Comparison with amino acid sequence data from the protein indicates that vicilin is synthesised as preprovicilin with subsequent removal of a signal peptide and a C-terminal peptide as well as post translational endo-proteolytic cleavage. The cDNAs represent two different classes of vicilin genes whilst amino acid data show that there are at least three major classes of vicilin polypeptide. The vicilin sequences show extensive homology with conglycinin and phaseolin except in the regions of the internal proteolytic cleavages. The evolutionary significance of this relationship is discussed.

Amino Acid Sequence↗