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At least 37 records · Page 2Linked to original sources
PubMed1994

Molecular cloning of a novel luteinizing-hormone/human-chorionic-gonadotropin-receptor cDNA. Identification of a long 3' untranslated region and cDNA sequence of the major transcript in rat ovary.

The biological action of luteinizing hormone/human chorionic gonadotropin (lutropin/choriogonadotropin) in the ovary is mediated by interaction with its specific receptor. Lutropin/choriogonadotropin-receptor hnRNA is processed into multiple mRNAs. However, nucleotide sequences for many of the transcripts, including the major form (6.7 kb), have yet to be determined. In an attempt to identify a cDNA encoding the major transcript, we have isolated a 3.5-kb cDNA clone from a rat ovary cDNA library. The 3.5-kb cDNA recognized only two (6.7 kb, 4.4 kb) of the three (6.7, 4.4, 2.6 kb) ovarian lutropin/choriogonadotropin-receptor transcripts when used as a probe. The first 732 nucleotides of the newly identified 3.5-kb cDNA showed 98% identity to the 3' untranslated region (3' UTR) of the previously cloned cDNA corresponding to the 4.4-kb transcript. Southern blot analysis indicated that the 3.5-kb cDNA and the C-terminal domain of the lutropin/choriogonadotropin-receptor originate from the same gene. Oligonucleotide-directed cleavage of the 6.7-kb lutropin/choriogonadotropin-receptor mRNA by RNase H revealed that the newly identified 3.5-kb cDNA is a 3' extension of the 4.4-kb transcript. We propose that the nucleotide sequence of the 6.7-kb lutropin/choriogonadotropin-receptor transcript, the major form found in rat ovary, contains a long 3' UTR, which has not been previously identified.

Animals↗
PubMed2004

Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection.

Phage display technologies are powerful tools for selecting binding ligands against purified molecular targets, live cells, and organ vasculature. However, the selection of natural ligands using phage display has been limited because of significant problems associated with the display of complex cDNA repertoires. Here we describe the use of cDNA fragmentation and open reading frame (ORF) selection to display a human placental cDNA library on the pIII coat protein of filamentous phage. The library was enriched for ORFs by selecting cDNA-beta-lactamase fusion proteins on ampicillin, resulting in a cDNA population having 97% ORFs. The ORF-selected cDNAs were fused to pIII in the phagemid vector, pUCMG4CT-198, and the library was rescued with a pIII-deleted helper phage for multivalent display. The resulting phagemid particle library consisted of 87% ORFs, compared to only 6% ORFs when prepared without ORF selection. Western blot analysis indicated cDNA-pIII fusion protein expression in eight out of nine ORF clones tested, and seven of the ORF encoded peptides were displayed multivalently. The high level of cDNA expression obtained by ORF selection suggests that ORF-enriched phage cDNA libraries prepared by these methods will be useful as functional genomics tools for identifying natural ligands from various source tissues.

DNA, Complementary↗
PubMed2001

[The construction of rapid amplification of cDNA ends cDNA libraries from human fetal bone and joint].

OBJECTIVE: To construct rapid amplification cDNA ends(RACE) cDNA libraries from human fetal bone and joint and provide resources for isolation of bone- and joint-specific development-related genes. METHODS: Total RNA of bone and joint were extracted with the modified single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. The double-stranded end-blunted cDNA were synthesized using TaKaRa's cDNA synthesis kit and ligated to cassette adaptors. All of the cDNA molecules were amplified by a pair of common primers. RESULTS: A protocol for RACE cDNA library construction from bone and joint was established and two RACE cDNA libraries from human fetal bone and joint were successfully constructed. CONCLUSION: The protocol of RACE cDNA library construction from limited materials proved to be simple and efficient and the library was suitable for RACE to isolate tissue-specific genes.

Bone and Bones↗
PubMed1998

[Primary study of differentially expressed cDNA sequences in cell line HNE1 of human nasopharyngeal carcinoma by cDNA representational difference analysis].

OBJECTIVE: To search differentially expressed sequences correlated with pathogenesis of human nasopharyngeal carcinoma (NPC), including the candidates of tumor suppressor genes. METHODS: cDNA 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 sources of differentially expressed products were proved by Southern blot and Northern blot. 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 primary cultures of 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. Of these obtained clones, some are the fragments of the human known genes including house-keeping genes, the others are novel genes. CONCLUSION: NPC involves alteration of multiple genes. Some of known genes matched with the differentially expressed sequences have an effective suppressive ability on the carcinoma.

Base Sequence↗
PubMed1996

The 5' untranslated region of Arabidopsis thaliana calmodulin cDNA is an independent cDNA containing an open reading frame.

A cDNA clone, pAUK1, with an open reading frame (ORF) coding for a hypothetical 164-amino-acid protein was isolated from an Arabidopsis thaliana (L.) Heynh cDNA library. The clone was attached, tail to tail, to the 3' end of A. thaliana hexokinase cDNA. An almost identical sequence had been previously described as the 5' untranslated region (5' UTR) of A. thaliana calmodulin cDNA (ACaM-2). Sequence comparison with three additional A. thaliana truncated cDNA clones which appear in a database (GenBank) supports the conclusion that pAUK1 is identical to the 5' UTR of ACaM-2 and that the 5' UTR of ACaM-2 is an independent cDNA artificially linked to A. thaliana calmodulin cDNA.

Amino Acid Sequence↗
PubMed1987

Construction of a human ventricular cDNA library and characterization of a beta myosin heavy chain cDNA clone.

We have constructed and characterized for the first time a complementary DNA (cDNA) clone, pHMC3, which codes for a cardiac myosin heavy chain mRNA from human heart. This clone contains a 1.7 kb DNA segment and specifies 543 amino acids of the carboxyl portion of the myosin heavy chain. The DNA sequence and encoded amino acid sequence were compared to the hamster alpha (pVHC1) and beta (pVHC2/pVHC3) cardiac myosin heavy chain cDNA and amino acid sequences and the rat cardiac myosin heavy chain sequences as well. The myosin heavy chain mRNAs are highly conserved and this is reflected in our cDNA clone. The pHMC3 clone is 87.9% homologous to the hamster alpha cDNA and 92.2% homologous to the hamster beta cDNA clones. The 3' untranslated region of pHMC3 is 64.1% homologous to the hamster beta clone while the hamster alpha myosin heavy chain shows only 25% homology to pHMC3 and exhibits extensive diversity. Similar results were obtained when pHMC3 was compared to the rat cardiac myosin heavy chain cDNA sequences. The comparisons showed that pHMC3 is a beta cardiac myosin heavy chain cDNA clone.

Amino Acid Sequence↗
PubMed1992

Hybridization fingerprinting of high-density cDNA-library arrays with cDNA pools derived from whole tissues.

As part of an integrated mapping and sequencing analysis of genomes, we have developed an approach allowing the characterization of large numbers of cDNA library clones with a minimal number of experiments. Three basic elements used in the analysis of cDNA libraries are responsible for the high efficiency of this new approach: (1) high-density library arrays allowing thousands of clones to be screened simultaneously; (2) hybridization fingerprinting techniques to identify clones abundantly expressed in specific tissues (by hybridizations with labeled tissue cDNA pools) and to avoid the repeated selection of identical clones and of clones containing noncoding inserts; and (3) a computerized system for the evaluation of hybridization data. To demonstrate the feasibility of this approach, we hybridized high-density cDNA library arrays of human fetal brain and embryonal Drosophila with radiolabeled cDNA pools derived from whole mouse tissues. Fingerprints of the library arrays were generated, localizing clones containing cDNA sequences from mRNAs expressed at middle to high abundance (> 0.1-0.15%) in the respective tissue. Partial sequencing data from a number of clones abundantly expressed in several tissues were generated to demonstrate the value of the approach, especially for the selection of cDNA clones for the analyses of genomes based on expressed sequence tagged sites. Data obtained by the technique described will ultimately be correlated with additional transcriptional and sequence information for the same library clones and with genomic mapping information in a relational database.

Animals↗
PubMed1997

cDNA-mediated Ty recombination can take place in the absence of plus-strand cDNA synthesis, but not in the absence of the integrase protein.

Ty elements belong to the family of LTR-containing retrotransposons. Ty RNA is reverse transcribed by Ty-encoded proteins. The cDNA then transposes to new locations in the genome by a process that involves the integrase protein encoded by the element. We have previously shown that the Ty cDNA molecule can participate in recombination events with genomic Tys. In this study we have analyzed the role of the integrase protein in cDNA-mediated Ty recombination. We found that this process involves the integrase protein in a temperature-dependent manner. In addition we have investigated whether double-stranded DNA is the only molecule that can participate in cDNA-mediated Ty recombination. We have shown that mutations in the polypurine tract that abolish plus-strand synthesis do not prevent cDNA-mediated recombination, implying that other types of intermediates from the reverse-transcription process (e.g. single-stranded DNA or a hybrid RNA-cDNA molecule) can participate in cDNA-mediated Ty recombination.

DNA, Complementary↗
PubMed1989

Differential screening of a cDNA library with cDNA probes amplified in a heterologous host: isolation of murine GRP78 (BiP) and other serum-regulated low-abundance mRNAs.

A novel method was used to screen differentially a cDNA library for clones representing serum-regulated mRNA species of low abundance. To increase the amount of probe available for screening, the cDNA probe was cloned and amplified. Two separate cDNA 'probe' libraries were constructed in the Escherichia coli plasmid vector pDE613, using poly(A)+mRNA from murine cells at 0 and 16 h after stimulation of a G0 population. Radiolabelled plasmid DNA from each library was hybridized sequentially to colony blots of the third 'target' library, constructed with mRNA from serum-stimulated cells in the Bacillus subtilis vector pBD214. Differential screening of the target cDNA library with the two probe libraries identified novel murine cDNA clones, some representing cytoplasmic poly(A)+RNA species of low (0.01%) abundance, accumulating after serum stimulation of a quiescent mouse embryo fibroblast population. One cDNA clone was found to correspond to mitochondrial 16S rRNA and a second was identified as the murine equivalent of previously described cDNA clones for the hamster 78-kDa glucose-regulated protein (GRP78) and the rat immunoglobulin heavy-chain-binding protein. GRP78 mRNA has not previously been recognized as a serum-inducible message.

Amino Acid Sequence↗
PubMed1993

Isolation of a tomato alcohol dehydrogenase 2-encoding cDNA using phage-promoted antibody screening of a plasmid cDNA library.

We describe the cloning of a cDNA encoding tomato alcohol dehydrogenase 2 (Adh2) by screening plasmid cDNA clones in phage plaques. A cDNA library constructed in a plasmid vector containing a unique SstI site at the 5' end of the cDNA insert was transferred into the SstI site of the lacZ gene of phage lambda Charon16, and screened by anti-Adh2 antibody to identify reactive plaques. Plasmid cDNA clones were recovered by SstI digestion, ligation, and transformation from phage minipreps for subsequent characterization. This system preserves the original plasmid library for subsequent screening with nucleic acid probes to identify full-length, multiple independent, or related cDNA clones not subject to the selection pressure of phage growth or lysogeny, or negative antibody reactivity. Thirty-two cDNA clones were identified with polyclonal antiserum to Adh2. Three of these reacted with monoclonal anti-Adh2 and only those three hybridized to maize adh1 sequence. One of these cDNAs, Adh31, was further characterized as encoding Adh2 by hybrid-selected translation and high sequence homology with the maize adh1 gene.

Alcohol Dehydrogenase↗
PubMed1999

Rapid cloning of cDNA ends polymerase chain reaction of G-protein-coupled receptor kinase 6: an improved method to determine 5'- and 3'-cDNA ends.

Rapid cloning of 5'- and 3'-cDNA ends polymerase chain reaction (5'-/3'-RACE-PCR) is useful to determine unknown 5'- and 3'-cDNA termini. Even if the method can yield complete cDNA sequences within a couple of days, the RACE procedure bears some characteristic traps and often results in amplification of unspecific PCR-products. Here we used improved 5'- and 3'-RACE-PCR protocols to obtain the complete cDNA sequence of the G-protein-coupled receptor kinase 6 (GRK6) from a rat brain cDNA library. The use of an anchored oligo-(dT)16-V-primer in the cDNA synthesis, the addition of single-sided PCR steps prior to the RACE-PCRs and the optimization of the dA-tailing reaction conditions in 5'-RACE enhanced RACE-PCR efficiency. Taken together, the method is a tool to determine unknown 5' and 3'-cDNA ends and enables the detection of different transcription initiation sites and mRNA splice variants even from small tissue samples like distinct brain regions. The extensive troubleshooting section discusses typical problems of each substep and contains additional references for support protocols.

Animals↗
PubMed1990

cDNA cloning of cytochrome P-450 related to P-450p-2 from the cDNA library of human placenta. Gene structure and expression.

We have isolated and analyzed cDNA (designated P-450HP cDNA) clones from a human placenta cDNA library, using the cDNA for rabbit pulmonary cytochrome P-450p-2, a prostaglandin omega-hydroxylase, as a hybridization probe. The cDNA obtained encoded a polypeptide comprising 511 amino acids with a calculated molecular mass of 58987 Da, and the amino acid sequence similarity with P-450p-2 and rat liver laurate omega-hydroxylase (P-450LA omega) was only about 50%. RNA blot analysis showed that the mRNA hybridizable with the human P-450HP cDNA was inducibly expressed 3-5-fold in rabbit small intestine and lung by gestation, but the expression remained constant in rabbit liver and kidney. This mode of expression was quite different from that of P-450p-2 and P-450LA omega. Interestingly, the mRNA hybridized with the cDNA of P-450HP was found to be expressed in all the human tumor tissues so far examined, in sharp contrast with the facts that almost all the other species of P-450s are known to disappear in the tumor tissues. Taken together, the deduced hemoprotein termed P-450HP dose not seem to be the human counterpart of rabbit P-450p-2 or rat P-450LA omega, and is presumably a new member of the P-450 family including P-450p-2 and P-450LA omega. Furthermore, the corresponding genomic DNA was also cloned and analyzed. The gene of P-450HP spanned 18.8 kb and was separated into 11 exons by 10 introns whose locations were completely different from those of P-450 genes so far determined.

Amino Acid Sequence↗
PubMed1996

Full-length cDNA cloning and determination of mRNA 5' and 3' ends by amplification of adaptor-ligated cDNA.

An efficient cDNA amplification procedure is described for determining of the 5' and 3' ends of mRNAs and cloning full-length cDNAs. In this approach, a double-stranded (ds) adaptor is ligated to both ends of a library of ds cDNA by T4 DNA ligase. This adaptor-ligated ds cDNA is then used to selectively amplify 5'- or 3'-cDNA fragments by PCR with a combination of gene-specific and adaptor-specific primers. This is a unified method for 5' and 3' rapid amplification of cDNA ends (RACE) from the same adaptor-ligated ds cDNA template. A specially designed adaptor combines features of "vectorette PCR" and "suppression PCR" technologies that significantly reduce background during amplification. The application of "long and accurate PCR" (LA PCR) technology makes possible the amplification of large RACE products and full-length cDNAs with high fidelity to the original mRNA. We investigated efficacy and limitations of this PCR-based approach for cDNA cloning by amplification of 5'- and 3'-RACE fragments and full-length cDNAs of three members of the abundant human actin gene family (1.3-1.9 kb), the medium abundance transferrin receptor mRNA (5.0 kb) and the low-medium abundance insulin-like growth factor II receptor mRNA (9.1 kb).

Actins↗
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↗
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↗
PubMed1986

Isolation and characterization of expressible cDNA clones for mouse Thy-1: a model system for cDNA expression of cell surface proteins.

By using the mouse Thy-1 gene as a model, we have developed a procedure to distinguish functional vs nonfunctional cDNA of lymphocyte surface antigens by transfecting COS-7 monkey cells and testing for expression of cell surface products encoded by the cDNA inserts. By cross-hybridization with a mouse Thy-1 probe, we isolated cDNA clones from a pcD-expression library prepared from mRNA of C5 cells. Two functional clones were distinguished from the remainder by detection of Thy-1.2 on the surface of 0.5% of COS-7 cells transiently transfected by the DEAE-Dextran method. Inclusion of chloroquine in the transfection procedure greatly facilitated the detection of functional cDNA by raising the percentage of expressing cells to 30%. Nucleotide sequencing of one functional cDNA, about 1700 bp long, confirmed that the gene encodes a protein whose sequence agrees with the published Thy-1.2 protein sequence with the additional 31 amino acids attached at the COOH-terminus. A 75 bp 5' untranslated region preceding the coding region contains 50 bp not found in the genomic clones. Comparison indicates that one or more introns are present in the 5' untranslated region, but are not found in the mature mRNA. The first exon may be separated by at least 1 kb intron from the initiation codon. Because the expressible clones are approximately the size of the mRNA seen on Northern blots, we believe that these clones are nearly full-length cDNA. Dilution experiments indicate that this strategy should also be useful for identifying functional cDNA clones for cell surface proteins solely on the basis of their expression in mammalian cells.

Animals↗
PubMed1995

Expression cloning of the cDNA encoding a melanoma-associated Ag recognized by mAb HMB-45. Identification as melanocyte-specific Pmel 17 cDNA.

BACKGROUND: mAb HMB-45 recognizes a melanocyte lineage-associated Ag present in "activated melanocytes" and malignant melanoma cells. Despite its important practical significance in diagnostic pathology and its potential role as an "activation marker" of melanocytic cells, the HMB-45-reactive Ag remained undefined. Molecular characterization of the HMB-45-reactive Ag may help in analyzing underlying mechanisms of melanocyte activation and melanoma tumor progression. EXPERIMENTAL DESIGN: A cDNA library constructed from the HMB-45-immunoreactive human melanoma cell line SK-MEL-28 was screened for expression of the cDNA encoding the HMB-45-reactive protein. Screening was performed by expression in COS-7 cells and subsequent immunocytochemical screening. Correlation between HMB-45 immunoreactivity and mRNA expression of the cloned cDNA was analyzed by antisense mRNA expression in HMB-45-immunoreactive SK-Mel-28 cells, by Northern blot hybridization, and by comparative immunohistochemistry and in situ hybridization. RESULTS: A cDNA clone encoding the HMB-45-reactive Ag was isolated and shown to be homologous to the Pmel 17 cDNA. Expression of either HMB-45 or Pmel 17 cDNA in COS-7 cells induced cytoplasmic HMB-45 immunoreactivity as described for melanoma cells. Conversely, constitutional HMB-45 immunoreaction in SK-MEL-28 cells could be markedly reduced by expression of antisense Pmel 17 RNA. In several tissues, Pmel 17 mRNA content conformed to the known expression pattern of HMB-45-reactive Ag. Comparative in situ hybridization and immunohistochemistry demonstrated a consistent co-localization of Pmel 17 transcripts and HMB-45-reactive Ag at the cellular level, with strong expression in "activated" melanocytes and melanoma cells and significantly less expression in normal adult melanocytes. CONCLUSIONS: We conclude that Pmel 17 cDNA encodes the HMB-45-reactive Ag. Pmel 17 has been postulated to be involved in melanin synthesis and expression of melanoma-peptide epitopes recognized by CTLs. Differential expression of Pmel 17/HMB-45 may have considerable effects in the stepwise process of melanoma progression by aberrant pigment formation and expression of CTL-reactive epitopes.

Antibodies, Monoclonal↗