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At least 631 records · Page 35Linked to original sources
PubMed1985

Primary structure of the beta-subunit of bovine transducin deduced from the cDNA sequence.

DNA complementary to the bovine retinal mRNA coding for the beta-subunit of transducin has been cloned by screening a cDNA library with oligodeoxyribonucleotide probes. Nucleotide sequence analysis of the cloned cDNA has revealed that this polypeptide consists of 340 amino acid residues (including the initiating methionine). Furthermore, cDNA hybridizable with a transducin beta-subunit cDNA probe has been cloned from a library derived from bovine brain poly(A)+ RNA. Comparison of the cloned cDNAs, in conjunction with blot hybridization analysis and S1 nuclease mapping of poly(A)+ RNA from bovine retina, brain and liver, suggests that the mRNAs coding for the beta-subunits of transducin and other guanine nucleotide binding proteins have the same protein-coding sequence but partly different 5'-noncoding sequences.

Amino Acid Sequence↗
PubMed1992

Complete cDNA sequence of chicken vigilin, a novel protein with amplified and evolutionary conserved domains.

The complete cDNA (4375 bp), coding for a new protein called vigilin, was isolated from chicken chondrocytes. The cDNA shows an open reading frame of 1270 amino acids which are organized in 14 tandemly repeated homologous domains. Each domain consists of two subdomains, one with a conserved sequence motif of 35 amino acids (subdomain A) and another one with a presumptive alpha-helical structure of 21-33 amino acids (subdomain B). 149 amino acids at the N-terminus and 71 amino acids at the C-terminus of vigilin do not show the characteristic domain structure. No sequence characteristic of a signal peptide has been found, which argues for an intracellular localisation of vigilin. Vigilin is highly expressed in freshly isolated chicken chondrocytes but little in chondrocytes after prolonged time in culture. Vigilin mRNA exists in two size species, 4.4 kb and 6.5 kb in length due to the usage of different polyadenylation sites. Comparison of the vigilin sequence with data bases showed a remarkable similarity to protein HX from Saccharomyces cerevisiae [Delahodde, A., Becam, A. M., Perea, J. & Jacq, C. (1986) Nucleic Acids Res. 14, 9213-9214]. The yeast protein consists of eight homologous domains with 11 conserved amino acid residues within a set of 35 amino acids. The N-terminal and C-terminal regions of vigilin and protein HX do not reveal any sequence similarity. These results, together with the demonstration of the characteristic vigilin sequence motif in a human cDNA clone, suggest that the repeats represent evolutionary conserved autonomous domains within a family of proteins found in yeast, chicken and man.

Amino Acid Sequence↗
PubMed1991

cDNA sequence of a Drosophila melanogaster gene, Dfur1, encoding a protein structurally related to the subtilisin-like proprotein processing enzyme furin.

Screening a genomic library of Drosophila melanogaster DNA with a human fur cDNA probe resulted in the isolation of DNA clones that apparently belonged to two different DNA regions of the Drosophila genome. Subsequently, corresponding Drosophila cDNA clones were isolated. Nucleotide sequence analysis indicated that these cDNA clones originated from two different genes, which were called Dfur1 and Dfur2. From overlapping Dfur1 cDNA clones, a composite cDNA could be constructed and analysis of its nucleotide sequence revealed the coding sequence for a protein of 899 amino acid residues. This protein, designated Dfurin1, exhibited striking sequence homology to human furin and contained the same protein domains except for the cysteine-rich region. Furthermore, unlike human furin, Dfurin1 possessed an extended amino-terminal region in which a potential transmembrane anchor was present.

Amino Acid Sequence↗
PubMed1988

Primary structure of rat liver serine dehydratase deduced from the cDNA sequence.

The nucleotide sequence of serine dehydratase mRNA of rat liver has been determined from a recombinant cDNA clone, previously cloned in this laboratory, and from a recombinant cDNA clone screened from a primer-extended cDNA library. The sequence of 1322 nucleotides includes the entire protein coding region and noncoding regions on the 3'- and 5'-sides. The deduced polypeptide consists of 327 amino acid residues with a calculated molecular mass of 34,462 Da. Comparison of the amino acid sequences of the serine dehydratase polypeptide with those of biosynthetic threonine dehydratase of yeast and biodegradative threonine dehydratase of E. coli revealed various extents of homology. A heptapeptide sequence, Gly-Ser-Phe-Lys-Ile-Arg-Gly, which is the pyridoxal-binding site in the yeast and E. coli threonine dehydratases was found as a highly conserved sequence.

Amino Acid Sequence↗
PubMed1995

Avian cathepsin B cDNA: sequence and demonstration that mRNAs of two sizes are produced in cell types producing large quantities of the enzyme.

Overlapping cDNA fragments encoding avian cathepsin B were cloned from an osteoclast cDNA library and sequenced. The primary structure of the prepro enzyme deduced from this sequence has 340 amino acids. The mature portion of the enzyme is 80% identical with murine cathepsin B; regions found in other papain superfamily enzymes are conserved. In osteoclasts and cultured macrophages, which produce large quantities of cathepsin B, mRNAs of 1.8 and 2.4 kb are produced in approximately equal quantities, while cells producing smaller quantities of the enzyme produce predominantly the 2.4 kb form. This variation in mRNAs suggests transcriptional differences related to production of large quantities of the enzyme.

Amino Acid Sequence↗
PubMed1992

Human glial fibrillary acidic protein (GFAP): molecular cloning of the complete cDNA sequence and chromosomal localization (chromosome 17) of the GFAP gene.

We isolated three glial fibrillary acidic protein (GFAP) cDNA clones from a glioma cell line, U-251 MG. One clone isolated from a U-251 MG cDNA library was long, but lacked both ends. Using poly(A)+ RNA and primers synthesized according to the sequence of this clone, we used the polymerase chain reaction-assisted rapid amplification of cDNA ends (PCR-RACE) method, which is a strategy to isolate cDNA ends, and obtained cDNA clones for the 5' and 3' ends. From the sequences of these overlapping clones, the complete nucleotide sequence of human GFAP cDNA was established. The start (ATG) and the stop (TGA) signals were seen at nucleotide positions 15 and 1311, respectively, and divided the entire sequence of 3027 bp into 14 bp of 5' non-coding, 1296 bp of coding and 1717 bp of 3' non-coding regions. Using cDNA probes made from both the coding and the 3' non-coding regions, Northern blot hybridization was performed with two different stringencies on RNAs from human and rodent brains and human GFAP-positive and -negative cells. It was shown that the 3' non-coding region probe was more specific for human GFAP than the coding region probe which was specific only under higher stringency conditions. This was also suggested by homology analysis of the sequence with those of various intermediate filament proteins. Based on these findings, we performed spot blot hybridization of sorted human chromosomes and Southern blot hybridization of PCR-amplified DNAs of a panel of hamster-human somatic cell hybrids and localized the human GFAP gene to chromosome 17.

Amino Acid Sequence↗
PubMed2002

Molecular cloning and complete cDNA sequence of UBH1 in mouse testis.

We have identified a full-length mouse UBH1 cDNA, encoding a putative deubiquitinating enzyme, from the testis by RT-PCR using primers prepared from sequences conserved amongst deubiquitinating enzymes. Sequence analysis predicts that the UBH1 cDNA encodes a 355 amino acid polypeptide with the molecular weight of approximately 39 kDa containing the highly conserved Cys, Asp, and His domains characteristic of the ubiquitin-specific processing proteases. Biochemical assay revealed that the mouse UBH1 has deubiquitinating enzyme activity and sequence analysis showed 98.3% amino acid identity with human UBH1.

Amino Acid Sequence↗
PubMed1988

Partial cDNA sequence encoding a nuclear pore protein modified by O-linked N-acetylglucosamine.

The nuclear pore complex contains a family of proteins ranging in molecular mass from 35 to 220 kDa that are glycosylated with O-linked N-acetylglucosamine (GlcNAc) residues. We sought to determine the primary sequence of a nuclear pore protein modified by O-linked GlcNAc. The major (62 kDa) nuclear pore glycoprotein (np62) was purified from rat liver nuclear envelopes by immunoaffinity chromatography and preparative gel electrophoresis. After CNBr fragmentation, a glycopeptide was isolated and microsequenced. An oligonucleotide probe based on this sequence information was used to screen a lambda gt11 cDNA library constructed from poly(A) mRNA of the rat thyroid cell line FRTL-5. A clone (B5) was isolated and shown to hybridize to a single 2.5-kilobase species in poly(A) mRNA from rat liver and FRTL-5. This insert was sequenced and found to contain a 691-base-pair cDNA encoding a 155-amino acid open reading frame. This open reading frame contained a CNBr fragment identical to the original glycopeptide sequence and a second CNBr fragment corresponding to a nonglycosylated peptide that was also isolated from the purified pore glycoprotein. The B5 cDNA produced a beta-galactosidase fusion protein of the size predicted by the open reading frame. Analysis of the residues making up a presumptive glycosylation site suggests that the sequence is unlike any known sites for enzymatic N- or O-linked glycosylation. The partial sequence of the 62-kDa nuclear pore glycoprotein shows little similarity to other characterized proteins and elucidates structural features of a member of the family of nuclear pore glycoproteins.

Acetylglucosamine↗
PubMed1990

Cloning and cDNA sequence of the beta-subunit component of human pyruvate dehydrogenase complex.

Two cDNA clones (lambda E1 beta 1, 1469 bp; lambda E1 beta 12, 1437 bp) encoding the beta-subunit of the pyruvate dehydrogenase (E1) component of the human pyruvate dehydrogenase complex were isolated from a human liver lambda gt11 cDNA library. The composite human liver E1 beta cDNA encoded the entire mature E1 beta [329 amino acids (aa)] as well as a portion (26 aa) of the E1 beta leader peptide. Significant discrepancies were identified between the nucleotide and deduced aa sequences of human liver E1 beta cDNAs and the corresponding sequences of a previously reported cultured human foreskin fibroblast E1 beta cDNA [Koike et al., Proc. Natl. Acad. Sci. USA 85 (1988) 41-45]. The composite human liver E1 beta cDNA generated in this study provides a reference sequence for investigating the structure-function relationship of human E1 beta and for characterizing genetic mutations in patients with E1 deficiency.

Amino Acid Sequence↗
PubMed1983

Identification of glucocorticoid-induced genes in rat hepatoma cells by isolation of cloned cDNA sequences.

The expression of specific cellular genes in M1.19 rat hepatoma cells involves glucocorticoid regulation by mechanisms that are not well understood. To approach this problem we cloned cDNA prepared from dexamethasone-induced poly(A)-RNA and used a comparative colony hybridization method to identify recombinant clones containing hormone-regulated sequences. Two such cDNA clones, p1394 and p255, hybridize to a homogeneous RNA species of 900 nucleotides that is present in high abundance in 24-hr-induced cells but is undetectable in uninduced cells. This RNA can be seen as early as 1 hr after dexamethasone stimulation. Inhibition of protein synthesis with cycloheximide significantly reduces the accumulation of the RNA but does not abolish the induction response. In normal adult rat liver the RNA is abundant, and this RNA is induced by dexamethasone in adrenalectomized rats. Plasmids p1394 and p255 contain sequences that are homologous to the mRNA coding for the acute-phase reactant protein alpha 1-acid glycoprotein. Two other cDNA clones, p655 and p333, hybridize to a more heterogeneous RNA species 200-400 nucleotides in size with a lower induction response to dexamethasone. Southern blot analysis of M1.19 genomic DNA indicates that p1394 and p255 are complementary to a single DNA fragment, whereas p655 and p333 are complementary to repetitive sequences in the M1.19 genome. It appears that the genetic domain of glucocorticoid control in M1.19 rat hepatoma cells involves low copy number genes such as alpha 1-acid glycoprotein as well as repetitive sequence elements.

Animals↗
PubMed1980

Brain tubulin and actin cDNA sequences: isolation of recombinant plasmids.

Rat brain mRNA enriched for tubulin and actin sequences was used to prepare double stranded cDNA. A library of recombinant clones was constructed by inserting the dsDNA into the Pst1 site of pBR322 plasmid and transformation of E. coli chi 1776 host. Clones bearing sequences coding for tubulin and actin were identified and characterized.

Actins↗
PubMed2002

Diminished expression of the type II receptor for TGFbeta (TGFbetaRII) in T lymphocytes from patients with Sezary syndrome is not due to mutations in the receptor's poly-A tract: limitations of the standard RT-PCR in cDNA sequence analysis of homopolymeric base stretches.

Peripheral blood lymphocytes from patients with Sezary syndrome (SzS) frequently demonstrate decreased surface expression of transforming growth factor beta receptor II (TGFbetaRII). The mechanism of this low TGFbetaRII expression remains unknown. Because mutations within the poly-A tract of the TGFbetaRII sequence (nucleotides 709-718) were shown to result in diminished TGFbetaRII expression in other types of malignant tumors, we examined the sequence of the TGFbetaRII poly-A tract in two SzS-derived cell lines and in peripheral blood SzS cells from 17 SzS patients and 4 control, healthy individuals using DNA sequencing and single-stranded conformation polymorphism (SSCP) analysis. A standard bidirectional, automated sequence analysis of the RT-PCR-generated cDNA TGFbetaRII fragment showed a heterogenous population of the normal length, 10-, with admixed, shortened, 9-base poly-A stretches. Surprisingly, this mixture was present not only in the cells from 5 SzS patients and 2 SzS cell lines, but also in cells from 2 healthy control individuals. Importantly, the proportion of the shortened, 9-base fragments was markedly reduced or practically eliminated when the procedure was modified by usage of high-fidelity DNA polymerase, labeled primers and/or cloned RT-PCR products, which indicates that the presence of the shortened, 9-base fragments represented a procedural phenomenon rather than a true deletional mutation within an allele of the TGFbetaRII gene. Accordingly, SSCP analysis of genomic DNA did not reveal any mutations within the poly-A tract-containing region. These results indicate that a mechanism different from mutations in the polyadenine tract underlies the diminished TGFbetaRII expression in SzS cells and that the results of an unmodified, direct sequence analysis of homopolymeric base streaches in RT-PCR-derived cDNA should be interpreted with caution.

Base Sequence↗
PubMed1997

Mouse p150Glued (dynactin 1) cDNA sequence and evaluation as a candidate for the neuromuscular disease mutation mnd2.

p150Glued (Dynactin 1) is a component of the dynactin complex that is essential for retrograde axonal transport in neurons. The mouse p150Glued cDNA was isolated from mouse brain by RT-PCR. The complete sequence of the full length mouse cDNA was determined, including 19 bp of 5' UTR, 3843 bp of coding sequence, and 162 bp of 3' UTR. The predicted protein has a molecular mass of 142 kDa and exhibits 95% amino acid sequence identity to the predicted amino acid sequence of human p150Glued (DCTN1). The mouse Dctn1 gene was previously mapped in the central region of mouse chromosome 6, close to the neuromuscular disease gene mnd2. Northern blot analysis, complete sequencing of the cDNA, Western blot, and functional tests of the protein did not detect any abnormalities of p150Glued in mnd2 mice.

Amino Acid Sequence↗
PubMed2005

Isolation and analysis of partial cDNA sequence coding for superoxide dismutase in Wuchereria bancrofti.

Molecular characterization of Wuchereria bancrofti is essential to develop suitable anti-filarial drugs and vaccines. We describe here isolation, sequence analysis and cloning of a partial cDNA of an enzyme superoxide dismutase from this parasite. The immunoscreening of a lambda zap W. bancrofti microfilarial (Mf) cDNA library with microfilaremic sera had resulted in the isolation of several seroreactive clones including, WbSOD. This clone contained a 309 bp insert and showed significant nucleotide and deduced amino acid sequence homologies to the superoxide dismutases of other nematode parasites. The antioxidant property of this enzyme may have important contribution in the defense mechanism of the parasite against host immune response.

Amino Acid Sequence↗
PubMed1989

Cloning and cDNA sequence of the rat X-chromosome linked phosphoglycerate kinase.

This paper reports the isolation and the sequence determination of rat phosphoglycerate kinase (PGK) cDNA clones. This cDNA, derived from an X-linked PGK gene transcript, contains a reading frame of 1254 nt and 5' and 3' non coding regions of 40 and 380 nt respectively. Analysis of the nucleotide sequence at the three codon position shows a biased codon usage with a prevalence of the triplet G non G N. Comparison of the inferred rat amino acid sequence with that of other organisms makes possible the calculation of the unit evolutionary period (UEP) for this enzyme, placing it at around 40 million years (My). Thus PGK is one of the oldest housekeeping enzymes.

Amino Acid Sequence↗
PubMed1990

Demonstration of an unusual allelic variation of mouse factor H by the complete cDNA sequence of the H.2 allotype.

Three allotypes of murine factor H have been identified serologically in the previous study (denoted H.1, H.2, and H.3). A cDNA clone coding for the entire length of murine factor H was isolated from a library constructed from the livers of STR/N mice which have H.2 allotype and was fully sequenced. The insert of this clone (STR309) contained 4184 nucleotides and consisted of a 47-bp 5' noncoding region, a 54-bp coding for leader peptide, a 3648 bp for the mature factor H protein, and a 435-bp 3' noncoding region. Compared with the previously reported sequence of the cDNA clone (MH8) isolated from B10.WR mice that have H.1 allotype, the size of the protein coding region was exactly the same, but 21 nucleotide substitutions resulting in 15 amino acid replacements were observed. The amino acid replacement/nucleotide substitution ratio (0.71) is far higher than those observed in the allotypic variations of other proteins. Four 15-base oligonucleotide probes specific for either STR309 or MH8 were synthesized and used in Northern blot analysis. The probes specific for STR309 hybridized with mRNA isolated from the livers of STR/N mice but not with mRNA from the livers of BALB/c mice that have H.1 allotype, whereas the reverse pattern was observed with the oligonucleotide probes specific for MH8. These results strongly suggest that the nucleotide sequence of STR309 represents H.2 allotype of factor H protein, providing an example of an unusual allotype with high ratio of amino acid replacements to nucleotide substitutions.

Alleles↗
PubMed1989

cDNA sequence, interspecies comparison, and gene mapping analysis of argininosuccinate lyase.

A cDNA clone of the argininosuccinate lyase gene (ASL) was isolated from an adult human liver library by probing with synthetic oligonucleotide probes. This clone and a yeast genomic DNA fragment containing the ASL gene were sequenced using the M13-dideoxynucleotide method. Comparison of the yeast and human clones at the nucleotide and putative amino acid sequence levels indicated identities of 50 and 54%, respectively. The most conserved region of the yeast gene was used to detect human clones in the liver cDNA library to test phylogenetic screening capabilities of conserved genes. ASL was mapped to human chromosome 7pter----q22 using human-mouse somatic cell hybrid DNA and further mapped by in situ hybridization to chromosome 7cen----q11.2 on human metaphase chromosomes. The probe also detected a sequence on chromosome 22. Somatic cell hybrid DNA digested with PvuII revealed a mouse polymorphism between Balb/c and C3H mice in the ASL gene.

Amino Acid Sequence↗
PubMed1991

cDNA sequence and deduced amino acid sequence of human preprocolipase.

Complementary DNA clones for human pancreatic colipase were identified in human pancreatic cDNA libraries by hybridization with a pool of synthetic oligonucleotides containing all possible coding sequences for amino acids 75 to 80 of the partial human colipase protein sequence (Sternby, et al. Biochim Biophys Acta 1984;784:75). Alignment of overlapping cDNA clones yielded an mRNA sequence of 504 nucleotides [not including the poly(A) tail] encoding a polypeptide of 112 amino acids. The prepeptide comprised 17 amino acids, with an amino-terminal cluster of charged residues followed by a hydrophobic core of 12 residues typical of leader sequences. The deduced human procolipase sequence comprised 95 residues, including a propeptide of 5 residues. It was in complete agreement with the partial sequence previously obtained by protein sequencing. Northern blot analysis revealed that the polyadenylated preprocolipase transcript had a length of approximately 680 nucleotides.

Amino Acid Sequence↗