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PubMed2004

[Isolation of differentially expressed cDNA sequences in human gastric carcinoma by cDNA microarray].

BACKGROUND & OBJECTIVE: Some gastric carcinoma-related genes haven't been identified yet. The study was designed to isolate and identify differentially expressed cDNA sequences in gastric carcinoma, and further clone gastric carcinoma-related genes. METHODS: The differences of gene expression profile between 30 gastric carcinoma tissues and their adjacent normal tissues were analyzed by cDNA microarray which representing about 4 892 cDNA sequences, the differentially expressed cDNA sequences were analyzed by bioinformatics, and selected cDNA sequences were confirmed by reverse transcripase-polymerase chain reaction (RT-PCR). RESULTS: A total of 33 differentially expressed cDNA sequences were identified in gastric carcinoma, among which 18 up-regulated in gastric carcinoma,while 15 down-regulated. MDSCBC11 clone,represented a novel gene, located in chromosome band 1p35-36, and significantly down-regulated in 13 of 30 (43%) gastric carcinomas. CONCLUSIONS: Some gastric carcinoma-related cDNA sequences have been identified,they might be involved in pathogenesis of gastric carcinoma. This study provides a useful clue for further clone gastric carcinoma-related genes.

Adult↗
PubMed1991

Primary structure of bovine chromogranin B deduced from cDNA sequence.

The cDNA sequence of bovine chromogranin B has been determined. Bovine chromogranin B consists of 626 amino acids and contains 16 pairs of basic amino acids which might be used for generation of biological active peptides. A comparison of the amino acid sequences of chromogranin B from various species identifies regions of high homology at the N- and C-termini as well as stretches in the middle of the chromogranin B sequence.

Amino Acid Sequence↗
PubMed1994

Isolation of unknown genes from human bone marrow by differential screening and single-pass cDNA sequence determination.

A cDNA sequencing project was initiated to characterize gene expression in human bone marrow and develop strategies to isolate novel genes. Forty-eight random DNAs from total human bone marrow were subjected to single-pass DNA sequence analysis to determine a limited complexity of mRNAs expressed in the bone marrow. Overall, 8 cDNAs (17%) showed no similarity to known sequences. Information from DNA sequence analysis was used to develop a differential prescreen to subtract unwanted cDNAs and to enrich for unknown cDNAs. Forty-eight cDNAs that were negative with a complex probe were subject to single-pass DNA sequence determination. Of these prescreened cDNAs, the number of unknown sequences increased to 23 (48%). Unknown cDNAs were also characterized by RNA expression analysis using 25 different human leukemic cell lines. Of 13 unknown cDNAs tested, 10 were expressed in all cell types tested and 3 revealed a hematopoietic lineage-restricted expression pattern. Interestingly, while a total of only 96 bone marrow cDNAs were sequenced, 31 of these cDNAs represent sequences from unknown genes and 12 showed significant similarities to sequences in the data bases. One cDNA revealed a significant similarity to a serine/threonine-protein kinase at the amino acid level (56% identity for 123 amino acids) and may represent a previously unknown kinase. Differential screening techniques coupled with single-pass cDNA sequence analysis may prove to be a powerful and simple technique to examine developmental gene expression.

Amino Acid Sequence↗
PubMed1991

The amino acid sequence of a glutamic acid-rich protein from bovine retina as deduced from the cDNA sequence.

cDNA clones encoding a glutamic acid-rich protein were isolated from a bovine retina cDNA expression library. The cDNA sequence contained an open reading frame of 1770 base pairs encoding a protein of 590 amino acids (64,509 Da) and untranslated regions of 60 and 490 base pairs at the 5' and 3' ends, respectively. The cDNA hybridized to a 2.4-kilobase retinal mRNA. The amino acid sequence derived from the cDNA sequence contains a glutamic acid-rich domain in which 68 of 109 amino acids are glutamic acid. In addition, this domain contains four repeats of a peptide of 11 amino acids and two repeats of a peptide of 26 amino acids. A polyclonal antibody raised against a decapeptide corresponding to the undecapeptide repeat sequence reacted with a protein in an extract of bovine rod outer segments, whose molecular mass, 65 kDa, corresponded to that of the above glutamic acid-rich protein. The retinal glutamic acid-rich protein showed homology with glutamic acid-rich proteins from bovine brain and the C-terminal region of mammalian neurofilaments.

Amino Acid Sequence↗
PubMed2004

Whole genome sequence comparisons and "full-length" cDNA sequences: a combined approach to evaluate and improve Arabidopsis genome annotation.

To evaluate the existing annotation of the Arabidopsis genome further, we generated a collection of evolutionary conserved regions (ecores) between Arabidopsis and rice. The ecore analysis provides evidence that the gene catalog of Arabidopsis is not yet complete, and that a number of these annotations require re-examination. To improve the Arabidopsis genome annotation further, we used a novel "full-length" enriched cDNA collection prepared from several tissues. An additional 1931 genes were covered by new "full-length" cDNA sequences, raising the number of annotated genes with a corresponding "full-length" cDNA sequence to about 14,000. Detailed comparisons between these "full-length" cDNA sequences and annotated genes show that this resource is very helpful in determining the correct structure of genes, in particular, those not yet supported by "full-length" cDNAs. In addition, a total of 326 genomic regions not included previously in the Arabidopsis genome annotation were detected by this cDNA resource, providing clues for new gene discovery. Because, as expected, the two data sets only partially overlap, their combination produces very useful information for improving the Arabidopsis genome annotation.

Arabidopsis↗
PubMed2000

Detection of spurious interruptions of protein-coding regions in cloned cDNA sequences by GeneMark analysis.

cDNA is an artificial copy of mRNA and, therefore, no cDNA can be completely free from suspicion of cloning errors. Because overlooking these cloning errors results in serious misinterpretation of cDNA sequences, development of an alerting system targeting spurious sequences in cloned cDNAs is an urgent requirement for massive cDNA sequence analysis. We describe here the application of a modified GeneMark program, originally designed for prokaryotic gene finding, for detection of artifacts in cDNA clones. This program serves to provide a warning when any spurious split of protein-coding regions is detected through statistical analysis of cDNA sequences based on Markov models. In this study, 817 cDNA sequences deposited in public databases by us were subjected to analysis using this alerting system to assess its sensitivity and specificity. The results indicated that any spurious split of protein-coding regions in cloned cDNAs could be sensitively detected and systematically revised by means of this system after the experimental validation of the alerts. Furthermore, this study offered us, for the first time, statistical data regarding the rates and types of errors causing protein-coding splits in cloned cDNAs obtained by conventional cloning methods.

Alternative Splicing↗
PubMed1992

The primary structure of porcine aminoacylase 1 deduced from cDNA sequence.

A cDNA encoding the complete amino acid sequence of aminoacylase 1 (N-acylamino acid aminohydrolase, ACY-1) [EC 3.5.1.14], a dimeric metalloprotein having two Zn2+ in the molecule, which catalyzes the deacylation of N-acylated L-amino acids except L-aspartic acid, has been isolated from porcine kidney lambda gt10 cDNA library and sequenced. From sequence analysis of the cDNA and the N- and C-terminal amino acid analyses of the purified protein, it is deduced that porcine kidney ACY-1 consists of two identical subunits (M(r) 45,260), each of which consists of a single chain of 406 amino acids with acetylalanine at the N-terminus. A cDNA encoding porcine liver ACY-1 was also cloned. The amino acid sequence deduced from the nucleotide sequence of the cDNA from porcine liver was identical to that deduced for porcine kidney ACY-1. Northern blot analysis suggested that ACY-1 is more highly expressed in kidney than in liver. Comparison of the amino acid sequence of porcine ACY-1 with those of other Zn2+-binding metalloenzymes showed no significant homologies in either the overall sequence or the consensus sequences for the metal binding sites. This indicates that ACY-1 is a new type of metalloprotein.

Amidohydrolases↗
PubMed1999

Subunit organization of the abalone Haliotis tuberculata hemocyanin type 2 (HtH2), and the cDNA sequence encoding its functional units d, e, f, g and h.

We have developed a HPLC procedure to isolate the two different hemocyanin types (HtH1 and HtH2) of the European abalone Haliotis tuberculata. On the basis of limited proteolytic cleavage, two-dimensional immunoelectrophoresis, PAGE, N-terminal protein sequencing and cDNA sequencing, we have identified eight different 40-60-kDa functional units (FUs) in HtH2, termed HtH2-a to HtH2-h, and determined their linear arrangement within the elongated 400-kDa subunit. From a Haliotis cDNA library, we have isolated and sequenced a cDNA clone which encodes the five C-terminal FUs d, e, f, g and h of HtH2. As shown by multiple sequence alignments, defg of HtH2 correspond structurally to defg from Octopus dofleini hemocyanin. HtH2-e is the first FU of a gastropod hemocyanin to be sequenced. The new Haliotis hemocyanin sequences are compared to their counterparts in Octopus, Helix pomatia and HtH1 (from the latter, the sequences of FU-f, FU-g and FU-h have recently been determined) and discussed in relation to the recent 2.3 A X-ray structure of FU-g from Octopus hemocyanin and the 15 A three-dimensional reconstruction of the Megathura crenulata hemocyanin didecamer from electron micrographs. This data allows, for the first time, an insight into the evolution of the two functionally different hemocyanin isoforms found in marine gastropods. It appears that they evolved several hundred million years ago within the Prosobranchia, after separation of the latter from the branch leading to the Pulmonata. Moreover, as a structural explanation for the inefficiency of the type 1 hemocyanin to form multidecamers in vivo, the additional N-glycosylation sites in HtH1 compared to HtH2 are discussed.

Amino Acid Sequence↗
PubMed1989

Primary structure of rat brain protein carboxyl methyltransferase deduced from cDNA sequence.

Two cDNA clones for protein carboxyl methyltransferase were isolated from a rat brain cDNA library in lambda gt 11 with synthetic oligonucleotides as probes. The two clones differ in size, but the nucleotide sequence including the whole coding region of the shorter cDNA is completely identical with the corresponding sequence of the longer cDNA. The open reading frame encodes a polypeptide of 227 amino acid residues, with a molecular weight of 24,626. This molecular weight is comparable to those reported for other protein carboxyl methyltransferases from several animals, which were determined by gel filtration chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Amino Acid Sequence↗
PubMed1984

Primary structure of beta subunit precursor of calf muscle acetylcholine receptor deduced from cDNA sequence.

Clones harbouring cDNA sequences for the beta subunit precursor of the acetylcholine receptor from calf skeletal muscle have been isolated. Nucleotide sequence analysis of the cloned cDNA has disclosed the primary structure of this polypeptide, which consists of 505 amino acids including a hydrophobic prepeptide of 24 amino acids. The beta subunit of the calf muscle acetylcholine receptor, like the alpha and gamma subunits of the same receptor and the alpha subunit of its human counterpart, exhibits structural features common to all four subunits of the Torpedo electroplax receptor, apparently being oriented across the membrane in the same manner as proposed for the fish receptor subunits. The degree of sequence homology between the calf and Torpedo beta subunits (59%) is comparable to that between the gamma subunits (56%), but is lower than that between the alpha subunits of the two species (81%). Some regions of the beta subunit molecule, including the region corresponding to the putative acetylcholine binding area on the alpha subunit and the region encompassing the clustered putative transmembrane segments M1, M2 and M3, are relatively well conserved between the two species.

Amino Acid Sequence↗
PubMed1985

Primary structure of delta subunit precursor of calf muscle acetylcholine receptor deduced from cDNA sequence.

Clones carrying cDNA sequences for the delta subunit precursor of the acetylcholine receptor from calf skeletal muscle have been isolated. Nucleotide sequence analysis of the cloned cDNA has indicated that this polypeptide consists of 516 amino acids including a hydrophobic prepeptide of 21 amino acids. The delta subunit of the calf muscle acetylcholine receptor, like the alpha, beta and gamma subunits of the same receptor as well as the alpha and gamma subunits of its human counterpart, exhibits structural features common to all four subunits of the Torpedo electroplax receptor, apparently being oriented across the membrane in the same manner as proposed for the fish receptor subunits. The degree of amino acid sequence homology between the calf and Torpedo delta subunits (60%) is comparable to that between the beta subunits (59%) and to that between the gamma subunits (56%), but is lower than that between the alpha subunits of the two species (81%). This suggests that the alpha subunit evolved more slowly than the three other subunits. A dendrogram representing the sequence relatedness among the four subunit precursors of the mammalian and fish acetylcholine receptors has been constructed. Some regions of the delta subunit molecule, including the region containing the putative disulphide bridge and that encompassing the clustered putative transmembrane segments M1, M2 and M3, are relatively well conserved between calf and Torpedo. The relative pattern of regional homology is similar for all four subunit precursors.

Amino Acid Sequence↗
PubMed1984

Primary structure of gamma subunit precursor of calf-muscle acetylcholine receptor deduced from the cDNA sequence.

Clones carrying cDNA sequences for the gamma subunit precursor of the acetylcholine receptor from calf skeletal muscle have been isolated. Nucleotide sequence analysis of the cloned cDNA has revealed the primary structure of this polypeptide, which consists of 519 amino acids including a hydrophobic prepeptide of 22 amino acids. The gamma subunit of the calf muscle acetylcholine receptor, like the alpha subunit of the calf as well as the human muscle receptor, shares features characteristic of all four subunits of the Torpedo electroplax receptor, such as the putative disulphide bridge corresponding to that in the alpha subunit proposed as being in close proximity to the acetylcholine binding site and the four putative, hydrophobic transmembrane segments M1-M4. Thus, the calf gamma subunit molecule apparently exhibits the same transmembrane topology as proposed for the fish receptor subunits. The degree of sequence homology between the calf and Torpedo gamma subunits (56%) is lower than that between the alpha subunits of the two species (81%). Some regions of the gamma subunit molecule, including the region encompassing the putative disulphide bridge and the region containing the putative transmembrane segments M1, M2 and M3, are relatively well conserved between the two species.

Amino Acid Sequence↗
PubMed1986

Primary structure of the alpha-subunit of human Na,K-ATPase deduced from cDNA sequence.

Clones carrying cDNA sequences for the alpha-subunit of the Na,K-ATPase from HeLa cells have been isolated. Nucleotide sequence analysis of the cloned cDNA has revealed the primary structure of this polypeptide, which consists of 1,023 amino acids. The alpha-subunit of the human Na,K-ATPase exhibited 87% homology with its Torpedo counterpart and 98% homology with its sheep counterpart. The six putative transmembrane segments M1-M6 showed higher conservation than the total segments. Total genomic Southern hybridization indicated the existence of at most two copies, possibly only one, of the gene encoding the Na,K-ATPase alpha-subunit in the human genome.

Amino Acid Sequence↗
PubMed1989

Primary structures of rat and bovine liver mitochondrial aldehyde dehydrogenases deduced from cDNA sequences.

The cDNA coding for rat liver mitochondrial aldehyde dehydrogenase was cloned and sequenced. It contained an open reading frame of 1557 bp. Of the deduced 519 amino acid residues, 19 were proposed to correspond to the signal peptide necessary to allow the protein to enter the mitochondria [Farrés, J., Guan, K.-L. and Weiner, H. (1987) Biochem. Biophys. Res. Commun. 150, 1083 - 1087]. The sequence of the 500 amino acid residues comprising the mature subunit was 96% identical to that of the corresponding human liver mitochondrial enzyme. The longest cDNA isolated coding for the bovine liver enzyme contained sequence information corresponding to residues 72 - 500 of the rat or human enzyme. The deduced protein sequence of these residues was 94% identical to those of the human enzyme. Over 60% of the bases in the coding region of the rat cDNA were G and C. These residues were clustered non-randomly and two potentially stable stem-loop structures could be calculated as existing in the mRNA. One would be found in the region coding for amino acid residues 64 - 79; a similar secondary structure could be responsible for the existing truncated cDNAs from the bovine-cDNA libraries. Comparisons with the amino acid sequences of other aldehyde dehydrogenases support the suggestion that the NAD-binding domain may be located in the middle portion of the mature enzyme.

Aldehyde Dehydrogenase↗
PubMed1998

Research notes: Identification and isolation of chicken sucrase-isomaltase cDNA sequence.

The intestinal sucrase-isomaltase (SI) complex is a glycoprotein of the small intestine brush border membrane that plays an important role in the final degradation of carbohydrate. To clone the chicken SI, we employed reverse transcriptase polymerase chain reaction (RT-PCR). Agarose gel electrophoresis of the PCR products exhibited one amplified band of approximately 800 bp. The fragment was extracted from the gel and sequenced. The cDNA sequence of the chicken SI is 786 bp in length and exhibits 99% identity at the nucleotide level to the Homo sapiens SI mRNA. Using our cDNA as a probe, Northern analysis revealed a transcript of approximately 6.0 kb in chicken jejunum and ileum tissues.

Amino Acid Sequence↗
PubMed2003

Human disease genes and their cloned mouse orthologs: exploration of the FANTOM2 cDNA sequence data set.

The FANTOM2 cDNA sequence data set is an excellent model to demonstrate the power of large-scale cDNA sequencing, with the goal of providing a full-length transcript sequence for each mouse gene. This data set enhances the use of the mouse as a model for human disease. Here we identify mouse cDNA sequences in the FANTOM2 data set for a set of 67 human disease genes that as of May 2002 had no corresponding mouse cDNA annotated in the Mouse Genome Informatics (MGI) database. These 67 human disease genes include genes related to neurological and eye disorders and cancer. We also present a list of the human disease genes and their cloned mouse orthologs found in two public databases, LocusLink and MGI. Allelic variant and gene functional information available in MGI provides additional information relative to these mouse models, whereas computed sequence-based connections at NCBI support facile navigation through multiple genomes.

Alleles↗
PubMed1988

Primary structure of nonspecific crossreacting antigen (NCA), a member of carcinoembryonic antigen (CEA) gene family, deduced from cDNA sequence.

A cDNA containing the entire coding region for a member of carcinoembryonic antigen (CEA) gene family has been cloned from cDNA library of HLC-1 cells by immunochemical screening with the antibody specific to nonspecific crossreacting antigen (NCA). The cDNA encodes a precursor form of a polypeptide consisting of a 34-residue signal sequence, a 108-residue N-terminal (N-) domain, a 178-residue domain (NCA-I domain) and a 24-residue domain rich in hydrophobic amino acids (M-domain). Each domain has a distinct but homologous amino acid sequence to that of the corresponding domain of CEA. Unlike the coding sequences, the 3'-untranslated sequences differ markedly in the NCA and CEA cDNAs facilitating the preparation of probes that will discriminate between nucleotide sequences for CEA and NCA.

Amino Acid Sequence↗
PubMed2005

Evaluating and improving cDNA sequence quality with cQC.

SUMMARY: Errors are prevalent in cDNA sequences but the extent to which sequence collections differ in frequencies and types of errors has not been investigated systematically. cDNA quality control, or cQC, was developed to evaluate the quality of cDNA sequence collections and to revise those sequences that differ from a higher quality genomic sequence. After removing rRNA, vector, bacterial insertion sequence and chimeric cDNA contaminants, small-scale nucleotide discrepancies were found in 51% of cDNA sequences from one Arabidopsis cDNA collection, 89% from a second Arabidopsis collection and 75% from a rice collection. These errors created premature termination codons in 4 and 42% of cDNA sequences in the respective Arabidopsis collections and in 7% of the rice cDNA sequences.

5' Untranslated Regions↗