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PubMed1980

Characterization of cloned cDNA sequences derived from Xenopus laevis poly A(+) oocyte RNA.

Double-stranded cDNA sequences were prepared from Xenopus laevis ovary poly A(+) RNA with AMV reverse transcriptase and nuclease S1. They were inserted into the plasmid pBR 322 after ligation with a Hind III linker and were cloned in E. coli strain X1776. Plasmid pools containing a cDNA insert were identified by Hind II restriction and hybridization of the DNA fragments with radiolabelled pBR 322 DNA. Hybridization of the positive pools with ovary RNA labelled in vitro led to the identification of cloned cDNA sequences which represent RNA species of high to intermediate abundance in the ovary. Positive clones were further challenged with in vitro labelled mitochondrial DNA and RNA from different developmental stages. One clone of mitochondrial origin has been detected. The hybridization characteristics of the cDNA sequences with the RNA probes from later developmental stages is discussed.

Animals↗
PubMed1996

Analysis of pseudorabies virus genes by cDNA sequencing.

Cloning and sequencing of cDNA could provide a complementary approach to functional analysis of the pseudorabies virus (PrV) genome. Using colony hybridization, Southern hybridization, and DNA sequencing, four species of PrV-specific cDNA were identified. Among these four species of PrV-specific cDNA, three unidentified genes, UL26, UL29, and UL31, were mapped and a novel gI-11K bicistronic cDNA was confirmed. Thus, analysis of PrV-specific transcripts provided a way for identifying genes and a foundation to further study the roles of these transcripts in PrV infection.

Animals↗
PubMed1992

Clustering cDNA sequences.

A set of programs has been written to quantify the similarities between large numbers of cDNA sequences. This information is used to cluster similar sequences together. The main program can cluster thousands of cDNA sequences per day using a novel, computationally inexpensive algorithm. The clustering information is kept in a small index file so that disk storage requirements are negligible. Using this index file, subsidiary programs create various views and statistical summaries of the entire cDNA sequence collection.

Algorithms↗
PubMed1985

GTPase of bovine rod outer segments: the amino acid sequence of the alpha subunit as derived from the cDNA sequence.

The sequence of the 350 amino acids in the alpha subunit of GTPase of bovine rod outer segments has been determined. Enriched GTPase mRNA was used to prepare a cDNA library in the expression vector lambda gt11 and several overlapping cDNA clones corresponding to the alpha subunit of the GTPase were identified. The cDNA sequence determined contains 93 nucleotides upstream of the 5' end of the coding region, 1050 nucleotides that specify the amino acid sequence, and 45 nucleotides downstream from the 3' end. The previously described partial amino acid sequences and the sequences at the ADP-ribosylation sites for cholera and pertussis toxins are all confirmed and fitted into the present complete sequence. Homologies are found between the sequence of the alpha subunit and those of other guanine nucleotide-binding proteins, the ras proteins, peptide chain elongation factors EF-Tu and EF-G, and the initiation factor IF2.

Amino Acid Sequence↗
PubMed1988

cDNA sequence and homologies of the "57-kDa" nucleotide-binding subunit of the vacuolar ATPase from Arabidopsis.

Functional and structural similarities among a wide variety of endomembrane H+-ATPases suggest that they form a distinct class with a common origin. Immunological studies (Manolson, M. F., Percy, J. M., Apps, D. K., Xie, X. S., Stone, D. K., and Poole, R. J. (1987) in Proceedings of the Membrane Protein Symposium (Goheen, S. C., ed) pp. 427-434, Bio-Rad, Richmond, CA, and M. F. Manolson, J. M. Percy, D. K. Apps, X. S. Xie, D. K. Stone, M. Harrison, D. J. Clarke, R. J. Poole, unpublished data) support this idea and suggest an evolutionary relationship between the endomembrane and F0F1 ATPases. Further examination of relationships necessitates comparison of protein/nucleic acid sequence data. To this end, we have cloned and sequenced the cDNA encoding the 57-kDa polypeptide of the Arabidopsis vacuolar membrane H+-ATPase. To our knowledge, this is the first report of the sequence of a "57-kDa" subunit for plant or animal endomembrane H+-ATPase. This cDNA encodes a hydrophilic polypeptide containing a putative ATP binding site. Lack of a secretion signal sequence suggests it is not processed through the endoplasmic reticulum but translated on cytosolic ribosomes. Comparison of protein sequences shows the 57-kDa subunit from Arabidopsis to be nearly identical with the corresponding subunit in Neurospora vacuolar membrane H+-ATPase, very similar to the beta subunit of the archaebacterium Sulfolobus, and slightly, but nevertheless significantly, homologous to the alpha and beta subunits of the F0F1-ATPases. These results suggest that these different classes of ATPases have evolved from a common ancestor.

Adenosine Triphosphate↗
PubMed1999

cDNA representational difference analysis of differentially expressed cDNA sequences in human nasopharyngeal carcinoma.

OBJECTIVE: To search differentially expressed sequences correlated with pathogenesis of human nasopharyngeal carcinoma (NPC), including the candidates of tumor suppressor genes. METHODS: Representational difference analysis (RDA) was performed to isolate differentially expressed sequences between cDNA from normal human primary cultures of nasopharyngeal epithelial cells and cDNA from NPC cell line HNE1. The source of differentially expressed products were proved by Southern blot, Northern blot and in situ hybridization. The fragments were cloned with pGEM-T easy kit and sequenced by the chain termination reaction. RESULTS: Four differentially expressed cDNA fragments were isolated in the fourth subtractive hybridization using cDNA from normal human nasopharyngeal epithelial cells as tester amplicon and cDNA from NPC cell line HNE1 as driver amplicon by cDNA RDA. These differential cDNA fragments revealed that they really came from the tester amplicon and were not expressed or down-regulated in the NPC HNE1 cells. Some of the genes were expressed only in human nasopharyngeal epithelial cells but deleted or down-regulated in the biopsies of NPC. Of these obtained clones, some were the sequences of the human known genes including house-keeping genes, the others represented novel gene sequences. CONCLUSION: The differentially expressed products including the candidates of tumor-suppressor genes may be associated with the initiation of the NPC.

DNA, Complementary↗
PubMed2000

Antibacterial properties and partial cDNA sequences of cecropin-like antibacterial peptides from the common cutworm, Spodoptera litura.

The antibacterial properties and cDNA sequences of two types of antibacterial peptides from the haemolymph of immunized common cutworm, Spodoptera litura larvae, were determined. Since the primary structures of peptides deduced from cDNA sequences showed significant homologies to cecropins A and B, they were named as Spodoptera cecropins A and B. Spodoptera cecropins were broadly effective against Gram-positive and negative bacteria. They also retained antibacterial activities in all conditions tested (at pH 5.6-8.0 and in the presence of 50-150 mM NaCl) that was adapted to confirm the antibacterial properties of Spodoptera cecropins. These results indicate that the change of pH and the increase of salt concentration in the media do not influence the activities of Spodoptera cecropins. For the reverse transcription (RT)-polymerase chain reaction (PCR) to obtain the complete primary sequence, the primer designed according to the conserved region of the cecropin leader sequences was used, which was determined by the comparison of the cDNA sequences of known cecropins. The results from RT-PCR presented that the partial cDNAs of Spodoptera cecropins A and B encode 57 and 58 amino acids, including the sequences of mature peptides, respectively. In addition, Northern blot analysis with (32)P-labeled PCR product coding for Spodoptera cecropin A revealed that Spodoptera cecropin genes are expressed in immunized fat body, but not in normal fat body.

Amino Acid Sequence↗
PubMed1990

Mouse preproacrosin: cDNA sequence, primary structure and postmeiotic expression in spermatogenesis.

The primary structure of mouse preproacrosin was deduced by nucleotide sequencing of cDNA clones isolated from a mouse testis cDNA library. The largest cDNA, with 1373 bp, consists of a 11-bp 5'untranslated sequence, a 1254-bp open reading frame terminated by a TGA triplet and a 105-bp 3' untranslated end, including one potential polyadenylation signal. The NH2-terminus of the polypeptide contains a hydrophobic 15-amino acid signal peptide. This cleavable signal sequence is followed by 403 amino acids, representing the acrosin light and the heavy chain of 23 and 380 amino acid residues, respectively. The proteolytic active site segments His, Asp and Ser are part of the heavy chain, as well as a proline-rich COOH-terminus, which is not present in any other serine proteinase studied so far. Furthermore the postmeiotic expression of the preproacrosin gene during mouse spermatogenesis was studied.

Acrosin↗
PubMed1992

Sequence of horse pancreatic lipase as determined by protein and cDNA sequencing. Implications for p-nitrophenyl acetate hydrolysis by pancreatic lipases.

The complete sequence of the horse pancreatic lipase was elucidated by combining polypeptide chain and cDNA sequencing. Among the structural features of horse lipase, it is worth mentioning that Lys373 is not conserved. This residue, which is present in human, porcine and canine lipases, has been assumed to be involved in p-nitrophenyl acetate hydrolysis by pancreatic lipases. Kinetic investigation of the p-nitrophenyl acetate hydrolysis by the various pancreatic lipases and by the C-terminal domain (336-449) of human lipase reveals that this hydrolysis is the result of the superimposition of independent events; a specific linear hydrolysis occurring at the active site of lipase, a fast acylation depending on the presence of Lys373 and a non-specific hydrolysis most likely occurring in the C-terminal domain of the enzyme. This finding definitely proves that pancreatic lipase bears only one active site and raises the question of a covalent catalysis by pancreatic lipases. Moreover, based on sequence comparison with the above-mentioned pancreatic lipases, three residues located in the C-terminal domain, Lys349, Lys398 and Lys419, are proposed as possible candidates for lipase/colipase binding.

Amino Acid Sequence↗
PubMed2000

cDNA sequence and genomic structure of the rat RET proto-oncogene.

The RET proto-oncogene, a member of the Receptor Tyrosine Kinase family, plays a crucial role during the development of the excretory system and the enteric nervous system, as demonstrated by in vivo animal studies and by its involvement in the pathogenesis of several human neurocristopathies like Hirschsprung disease and Multiple Endocrine Neoplasia type 2. Using a multistep RT-PCR approach we have isolated and sequenced the cDNA of the whole rat RET proto-oncogene, reporting the deduced amino acid sequence in comparison with the human and mouse counterparts. Moreover, two different isoforms (RET9 and RET51) have been confirmed in the rat, while a third RET isoform demonstrated in human (RET43) has not resulted to be conserved in this species. Finally, we have determined the genomic structure of the rat RET proto-oncogene comparing the exon-intron boundaries and intron sizes with the known structure of the human homologous gene. Our findings will facilitate the molecular study of appropriate rat models of RET related human diseases.

Alternative Splicing↗
PubMed1994

Primary structure of bovine interstitial collagenase deduced from cDNA sequence.

Interstitial collagenase (EC 3.4.24.7, MMP-1) is a member of a family of metalloproteinases and is though to play a role in extracellular matrix remodeling. We have isolated and sequenced a cDNA for bovine interstitial collagenase from a periodontium fibroblast cDNA library. An insert of the cDNA we isolated was 2,025 bp containing an open reading frame which encodes a sequence of 469 amino acids. The identity at the amino acid level between bovine and human interstitial collagenase was 88%, between bovine and rabbit 85% and between bovine and porcine 87%. However, sequence similarity of mouse and rat interstitial collagenases to that of bovine was revealed 55%.

Amino Acid Sequence↗
PubMed1996

Identification of two prolactin cDNA sequences from a goldfish pituitary cDNA library.

We have identified two different cDNA clones encoding for goldfish prolactin (gfPRL) from a pituitary cDNA library. The coding regions of these clones are predicted to encode for an identical amino acid (aa) sequence with four silent mutations. The 3'-untranslated regions (UTRs) of these clones show only 72% nucleotide (nt) sequence identity. The two genes each encoding gfPRL might have derived from recent gene duplication before the divergence of goldfish from other Cypriniforms. Genomic Southern blot analysis of goldfish DNA also demonstrated that there is a small family of two genes for prolactin in the genome of goldfish.

Amino Acid Sequence↗
PubMed1992

Molecular evolution of phosphoenolpyruvate carboxylase for C4 photosynthesis in maize: comparison of its cDNA sequence with a newly isolated cDNA encoding an isozyme involved in the anaplerotic function.

Molecular events associated with the evolution of an enzyme for C4 photosynthesis were investigated. In maize, at least three isozymes of phosphoenolpyruvate carboxylase [EC 4.1.1.31] are known: the C4-form, the C2-form and the root-form, being named according to their physiological roles and pattern of tissue distribution [Ting, I.P. & Osmond, C.B. (1973) Plant Physiol. 51, 448-453]. A cDNA clone which presumably encodes the C3-form isozyme was newly isolated and analyzed. Comparison of the sequences of the C3-form and C4-form isozymes revealed that (i) the homologies in the nucleotide and deduced amino acid sequences were 71 and 77%, respectively, and (ii) the gene for the C4-form isozyme evolved under strong G/C pressure. The genes for these isozymes were found to be located apart on different chromosomes. A phylogenetic tree was constructed using 8 amino acid sequences of phosphoenolpyruvate carboxylases from various sources. The topology of the tree indicated that, at least in monocots such as maize and sorghum, the genes for the C4-form and C3-form isozymes diverged from their common ancestral gene earlier than the monocot-dicot divergence (about 2 x 10(8) yr ago), though the divergence of maize (C4 plant) from wheat (C3 plant) is supposed to have occurred much later (6 x 10(7) yr ago).

Amino Acid Sequence↗
PubMed1985

Murine sex-limited protein: complete cDNA sequence and comparison with murine fourth complement component.

Murine sex-limited protein (Slp) is a structural homologue of the murine fourth complement component (C4) that lacks C4 activity and has no known function. The genes for C4 and Slp lie closely linked in the S region of the murine major histocompatibility complex. We have sequenced a cDNA clone that spans the entire protein-coding region of Slp from the mouse strain B10.WR. The sequence contains a 1735 amino acid-long open reading frame encoding a putative prepro-Slp flanked by 51 and 103 untranslated nucleotides at the 5' and 3' ends respectively; it shows 96% nucleotide and 94% amino acid identity with our previously reported complete sequence of murine C4 from the same mouse strain. The present complete Slp sequence differs slightly from our previously reported partial sequence from the same mouse strain; this suggests that at least two distinct Slp genes are transcribed in B10.WR mice. We suggest, by analogy with procaryotic DNA-binding proteins, that a three amino acid deletion in Slp, close to the Cls cleavage site, makes that site resistant to proteolysis; this renders Slp inactive. We also speculate on the possibility that Slp might be a gene in evolutionary transition; one that is midway in the evolution of a completely silent pseudogene or a new gene with a novel function.

Amino Acid Sequence↗
PubMed1996

Interpreting cDNA sequences: some insights from studies on translation.

This review discusses some rules for assessing the completeness of a cDNA sequence and identifying the start site for translation. Features commonly invoked-such as an ATG codon in a favorable context for initiation, or the presence of an upstream in-frame terminator codon, or the prediction of a signal peptide-like sequence at the amino terminus-have some validity; but examples drawn from the literature illustrate limitations to each of these criteria. The best advice is to inspect a cDNA sequence not only for these positive features but also for the absence of certain negative indicators. Three specific warning signs are discussed and documented: (i) The presence of numerous ATG codons upstream from the presumptive start site for translation often indicates an aberration (sometimes a retained intron) at the 5' end of the cDNA. (ii) Even one strong, upstream, out-of-frame ATG codon poses a problem if the reading frame set by the upstream ATG overlaps the presumptive start of the major open reading frame. Many cDNAs that display this arrangement turn out to be incomplete; that is, the out-of-frame ATG codon is within, rather than upstream from, the protein coding domain. (iii) A very weak context at the putative start site for translation often means that the cDNA lacks the authentic initiator codon. In addition to presenting some criteria that may aid in recognizing incomplete cDNA sequences, the review includes some advice for using in vitro translation systems for the expression of cDNAs. Some unresolved questions about translational regulation are discussed by way of illustrating the importance of verifying mRNA structures before making deductions about translation.

Animals↗
PubMed2004

Transcriptome analysis for the chicken based on 19,626 finished cDNA sequences and 485,337 expressed sequence tags.

We present an analysis of the chicken (Gallus gallus) transcriptome based on the full insert sequences for 19,626 cDNAs, combined with 485,337 EST sequences. The cDNA data set has been functionally annotated and describes a minimum of 11,929 chicken coding genes, including the sequence for 2260 full-length cDNAs together with a collection of noncoding (nc) cDNAs that have been stringently filtered to remove untranslated regions of coding mRNAs. The combined collection of cDNAs and ESTs describe 62,546 clustered transcripts and provide transcriptional evidence for a total of 18,989 chicken genes, including 88% of the annotated Ensembl gene set. Analysis of the ncRNAs reveals a set that is highly conserved in chickens and mammals, including sequences for 14 pri-miRNAs encoding 23 different miRNAs. The data sets described here provide a transcriptome toolkit linked to physical clones for bioinformaticians and experimental biologists who wish to use chicken systems as a low-cost, accessible alternative to mammals for the analysis of vertebrate development, immunology, and cell biology.

Animals↗
PubMed1987

A complete cDNA sequence for the major epidermal growth factor binding protein in the male mouse submandibular gland.

The complete cDNA sequence of the major epidermal growth factor binding protein (EGF-BP), isolated from the mouse submandibular gland, has been determined. Oligonucleotide probes complementary to unique, nonconserved, regions of homogeneous preparations of EGF-BP were used to identify the correct cDNA clone from a male mouse submandibular gland cDNA library. The nucleotide sequence codes for a glandular kallikrein that is the main arginine esterase complexed with epidermal growth factor. The mRNA coding for this EGF-BP is estimated at 0.24% of the total mRNA of the adult male mouse submandibular gland, thus representing an abundant member of the kallikrein family in this tissue. In addition, the cDNA sequence defines a putative transcription start site. The reported cDNA sequence is clearly different from, and not an allelic form of, a previously reported cDNA sequence for EGF-BP. The present work reconciles conflicting information in the literature regarding the identity of EGF-BP.

Amino Acid Sequence↗