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PubMed1997

Studying functional significance of the sequence 980-1061 in the central domain of human 18S rRNA using complementary DNA probes.

Region 980-1061 in human 18S rRNA has been chosen on the basis of our previous results, indicating that cross-linking sites of the alkylating mRNA analogs are located within this region. In the present study, we have used 10 DNA 15-mers complementary to various overlapping sequences within the 18S rRNA positions 980-1061. Their abilities to bind selectively to the target rRNA sequences were proved by hydrolysis of 18S rRNA within heteroduplexes with the corresponding probes by RNase H. Four sequences (980-994, 987-1001, 1025-1039 and 1032-1046) were found to be well accessible for binding of the respective cDNA probes within 40S subunits. None of the oligomers inhibited tRNA(Phe)-dependent binding of oligo(U) messenger to 40S subunits and binding of Met-tRNA(imet) to 40S subunits in the presence of eIF-2 and nonhydrolysable GTP analog. Nevertheless, two probes (complementary to the 18S rRNA sequences 987-1001 and 1025-1039) being covalently attached to 40S subunits, inhibited translation of poly(U) by human 80S ribosomes in a cell-free system. The same oligomers revealed the most pronounced inhibitory action on the binding of messenger trinucleotide in the complex pAUG.40S.Met-tRNA(imet).eIF-2.GTP. Results of these functional assays demonstrate the importance of the 18S rRNA sequences 987-1001 and 1025-1039 for translation process on human ribosomes, most probably at the initiation step.

Alkylation↗

PubMed1995

In vivo transfection of hepatitis C virus complementary DNA into rodent liver by asialoglycoprotein receptor mediated gene delivery.

An in vivo model of hepatitis C virus (HCV) infection is needed to enable investigation of the mechanism of the liver injury that it causes. In this study, we used asialoglycoprotein receptor mediated gene delivery to obtain expression of the complementary DNA (cDNA) coding the core and part of the envelope 1 protein of HCV because selective delivery to the hepatocytes has been reported to be attained with this method. The optimum carrier-DNA ratio was examined using in vitro transfection and found to be important for the efficiency of this method. In transfection in vivo, microautoradiographical examination showed that the transfected plasmids were delivered selectively to the liver parenchymal cells. To obtain an immunohistochemically detectable level of protein expression in rodent liver, some modifications for increasing the in vivo transfection efficiency were performed; a lysosomal enzyme inhibitor, chloroquine, was used and the administration route of the carrier-DNA complex was changed from the tail vein to the portal vein. On the bases of these results, in vivo transfection with expression vector of HCV core/E1 region was performed. In rat liver transfected by intraportal injection with chloroquine, the transcript RNA and the core protein were detected. These results indicated that the HCV core/E1 expression vector was not merely delivered but also successfully expressed in the liver using asialoglycoprotein receptor mediated gene delivery. The number of the HCV core expressing cells in the transfected liver was similar to that in patients with hepatitis C. These in vivo transfected animals should be useful for investigating the role of this region in the liver injury caused by HCV.

Animals↗

PubMed1998

Full-length complementary DNA of hepatitis C virus genome from an infectious blood sample.

We constructed a full-length complementary DNA (cDNA) clone of hepatitis C virus (HCV) from a blood sample of an HCV carrier. The blood from the carrier was eventually transfused to a patient who later developed typical posttransfusion hepatitis C. It was also shown to be infectious to chimpanzees. We obtained 12 overlapping cDNA fragments altogether, covering the entire HCV genome. By subcloning and sequencing, clones considered to constitute the major population were selected. We could also detect 98 base pairs of extra sequences at the 3' end of the genome. After confirming the overlapping sequences, we combined the fragments to make a full-length cDNA. The HCV population in the donor was heterogeneous, as determined by their nucleotide sequences of the hypervariable region in envelope protein, but a few virus clones were selected in the recipient after transmission. The similar convergence of the virus population was previously observed when the same blood sample was injected into a chimpanzee. Interestingly, virus clones isolated during the acute phase in the recipient and the chimpanzee had sequences in the hypervariable region identical to that of the full-length cDNA clone. The full-length cDNA clone of HCV constructed in this study may originate from infectious virus clones.

Amino Acid Sequence↗

PubMed1987

Complementary DNA for human T-cell cyclophilin.

Complementary DNA encoding human cyclophilin, a specific cyclosporin A-binding protein, has been isolated from the leukemic T-cell line Jurkat and sequenced. Comparison of the deduced amino acid sequence with the previously determined sequence of bovine thymus cyclophilin reveals only three differences: an additional amino acid at the carboxy terminus end and two internal changes. RNA transfer blot analysis indicates an mRNA size of approximately 1 kb for human T-cell cyclophilin. Phytohaemagglutinin and phorbol myristate acetate induction of T cells treated or not with cyclosporin A affects only marginally the level of cyclophilin mRNA. Southern blot analysis of human genomic DNA digested with different restriction enzymes strongly suggests the existence of a multigene family for cyclophilin.

Amino Acid Sequence↗

PubMed1991

Cloning and expression of complementary DNA encoding an antigen of Trichinella spiralis.

Complementary DNA (cDNA) encoding a 49-kDa antigen of Trichinella spiralis was cloned, characterized, and expressed in Escherichia coli by recombinant DNA methods. As a first step, 29 residues of N-terminal amino acid sequence were determined by direct analysis of highly purified antigen. Mixed-sequence primers based on the amino acid sequence were then synthesized and used as primers to generate a partial cDNA by the polymerase chain reaction (PCR). A nearly full-length cDNA was then obtained by PCR using a specific 5'-end primer and a non-specific 3'-end primer. Complete sequence of the 1133-bp cDNA was determined. Translation of this sequence predicts an N-glycosylated polypeptide of 322 amino acids. The cDNA was expressed as a fusion protein in bacteria which could be recognized in Western blots both by sera from mice immunized with purified native 49-kDa antigen and by sera from swine infected with the parasite. These findings suggest that recombinant P49 is a potentially valuable antigen both for vaccine development and immunodiagnosis.

Amino Acid Sequence↗

PubMed1988

Role of the potential secondary structures in phage G4 origin of complementary DNA strand synthesis.

Phage G4 origin of complementary DNA strand synthesis (oric) consists of three stable stem-loop structures (I, II, and III). Mutant oric sequences with alterations in the structure of stem-loop II, stem-loop III, and the stem-loop II-III spacer region have been constructed and cloned into the filamentous phage vectors to assay their functional activity. Changes in the lowermost GC base pair in the stem of stem-loop III, in the 9-bp spacer region between the stems of stem-loops II and III, and in the loop of stem-loop II, impair or abolish in vivo oric function. The results suggest that recognition sequences for dnaG primase must be present in the loop of stem-loop II, and in the spacer region between the stems of stem-loops II and III.

Bacteriophages↗

PubMedDate not supplied

Amino-acid sequence of the catalytic subunit of the (Na+ + K+)ATPase deduced from a complementary DNA.

We have isolated and characterized a complementary DNA for the catalytic subunit of the sheep kidney sodium/potassium-dependent ATPase. The 1,016-amino-acid protein seems to have eight transmembrane domains. The apparent ouabain binding site is located at the extracellular junction of two transmembrane domains and is linked to the phosphorylation site by a 60-amino-acid conserved sequence that may be a major channel for energy transduction.

Amino Acid Sequence↗

PubMedDate not supplied

Amino-acid sequence of a Ca2+ + Mg2+-dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence.

We have cloned and sequenced complementary DNA encoding a Ca2+-ATPase of rabbit muscle sarcoplasmic reticulum. We propose a model of the protein which has 3 cytoplasmic domains joined to a set of 10 transmembrane helices by a narrow, penta-helical stalk. In this model, ATP bound to one cytoplasmic domain would phosphorylate an aspartate in an adjoining cytoplasmic domain, inducing translocation of Ca2+ from binding sites on the stalk.

Amino Acid Sequence↗

PubMed1987

Isolation and sequence of complementary DNA encoding human extracellular superoxide dismutase.

A complementary DNA (cDNA) clone from a human placenta cDNA library encoding extracellular superoxide dismutase (EC-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) has been isolated and the nucleotide sequence determined. The cDNA has a very high G+C content. EC-SOD is synthesized with a putative 18-amino acid signal peptide, preceding the 222 amino acids in the mature enzyme, indicating that the enzyme is a secretory protein. The first 95 amino acids of the mature enzyme show no sequence homology with other sequenced proteins and there is one possible N-glycosylation site (Asn-89). The amino acid sequence from residues 96-193 shows strong homology (approximately 50%) with the final two-thirds of the sequences of all known eukaryotic CuZn SODs, whereas the homology with the P. leiognathi CuZn SOD is clearly lower. The ligands to Cu and Zn, the cysteines forming the intrasubunit disulfide bridge in the CuZn SODs, and the arginine found in all CuZn SODs in the entrance to the active site can all be identified in EC-SOD. A comparison with bovine CuZn SOD, the three-dimensional structure of which is known, reveals that the homologies occur in the active site and the divergences are in the part constituting the subunit contact area in CuZn SOD. Amino acid sequence 194-222 in the carboxyl-terminal end of EC-SOD is strongly hydrophilic and contains nine amino acids with a positive charge. This sequence probably confers the affinity of EC-SOD for heparin and heparan sulfate. An analysis of the amino acid sequence homologies with CuZn SODs from various species indicates that the EC-SODs may have evolved from the CuZn SODs before the evolution of fungi and plants.

Amino Acid Sequence↗

PubMed1992

Increased growth of NIH/3T3 cells by transfection with human p120 complementary DNA and inhibition by a p120 antisense construct.

The human nucleolar antigen p120 was detected with an anti-p120 monoclonal antibody in most human malignant tumors but not in most resting human tissues (J. W. Freeman et al., Cancer Res., 48: 1244-1251, 1988) and has been used as a prognostic tumor marker in breast cancer patients (J. W. Freeman et al., Cancer Res., 51: 1973-1978, 1991). After the complementary DNA and gene for the human p120 protein were isolated and sequenced (review: H. Busch, Cancer Res., 50: 4830-4838, 1990), constructs were prepared to study the expression of the sense p120 and its antisense, p021 message. NIH/3T3 cells were transfected by electroporation with pSVX plasmids containing either the p120 complementary DNA (pSVX120) or the antisense, p021 DNA (pSVX021), and clones containing these constructs were selected. The expression of p120 or p021 in these constructs was regulated by Moloney murine leukemia virus long terminal repeats. In pSVX120-transfected NIH/3T3 cells, the expressed human p120 protein was localized to the nucleoli as shown by anti-p120 monoclonal antibody immunofluorescence. Expression of the p120 message and protein was confirmed by Northern (mRNA) and Western (protein) blots. Transfection of the p120 complementary DNA in sense orientation caused malignant transformation of NIH/3T3 cells in vitro and produced rapidly growing tumors in nude mice. Transfection of the antisense p120 constructs markedly delayed the growth of these tumors in vitro and in vivo (L. Perlaky et al., Proc. Am. Assoc. Cancer Res., 32: 1682, 1991). When transformed 3T3/pSVX120 cells were transfected with an inducible antisense p120 construct (pMSG021), dexamethasone induction decreased the growth rate by 62%, and the cell line returned to its normal phenotype. Northern blot analysis showed a decreased level of p120 mRNA, and the immunofluorescence was also markedly reduced.

3T3 Cells↗

PubMed1989

ATP synthase from bovine mitochondria: complementary DNA sequence of the import precursor of a heart isoform of the alpha subunit.

The alpha-subunit of ATP synthase from mitochondria is a major component of the extrinsic membrane sector of the enzyme. It is encoded in nuclear DNA. A family of overlapping complementary DNA clones encoding its precursor has been isolated from a bovine library by using in the first instance a mixture of 128 synthetic oligonucleotides designed on the basis of the known protein sequence, and the sequence of the full-length cDNA has been determined. The deduced protein sequence shows that the alpha-subunit of ATP synthase has a presequence of 43 amino acids that is not present in the mature protein. Presumably it directs the protein into the mitochondrial matrix and is removed during the import process. The encoded protein sequence is also longer by one amino acid at its C-terminal end than the protein isolated from F1-ATPase, but this alanine residue may have been removed artifactually during release of the F1-ATPase particle from the inner mitochondrial membrane. With the exception of one uncertainty caused by an ambiguity at one position in the nucleotide sequence, the mature protein sequence encoded in the cDNA is exactly the same as the sequence determined previously by direct analysis of the protein isolated from bovine heart mitochondria [Walker et al. (1985) J. Mol. Biol. 184, 677-701]. The cDNA sequence differs in 158 nucleotides over a region of alignment of 1097 nucleotides from a partial cDNA for the alpha-subunit that has been isolated from a bovine cDNA derived from liver RNA [Breen (1988) Biochem. Biophys. Res. Commun. 152, 264-269].(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Complementary DNA cloning, messenger RNA expression, and induction of alpha-class glutathione S-transferases in mouse tissues.

Glutathione S-transferases (EC 2.5.1.18) are a multigene family of related proteins divided into four classes. Each class has multiple isoforms that exhibit tissue-specific expression, which may be an important determinant of susceptibility of that tissue to toxic injury or cancer. Recent studies have suggested that alpha-class glutathione S-transferase isoforms may play an important role in the development of cancers. Several alpha-class glutathione S-transferase isozymes have been characterized, purified, and cloned from a number of species, including rats, mice, and humans. Here we report on the cloning, sequencing, and mRNA expression of two alpha-class glutathione S-transferases from mouse liver, termed mYa and mYc. While mYa was shown to be identical to the known alpha-class glutathione S-transferase complementary DNA clone pGT41 (W. R. Pearson et al., J. Biol. Chem., 263: 13324-13332, 1988), the other clone, mYc, was demonstrated to be a novel complementary DNA clone encoding a glutathione S-transferase homologous to rat Yc (subunit 2). The mRNA for this novel complementary DNA is expressed constitutively in mouse liver. It also is the major alpha-class glutathione S-transferase isoform expressed in lung. The levels of expression of the butylated hydroxyanisole-inducible form (mYa) are highest in kidney and intestine. Treatment of mice with butylated hydroxyanisole had little effect on the expression levels of mYc but strongly induced mYa expression in liver. Butylated hydroxyanisole treatment increased expression levels for both mYa and mYc to varying degrees in kidney, lung, and intestine. The importance of the novel mouse liver alpha-class glutathione S-transferase isoform (mYc) in the metabolism of aflatoxin B1 and other carcinogens is discussed.

Amino Acid Sequence↗

PubMed1989

Viral infectious complementary-DNA studies may identify nonviral genes critical to central nervous system disease.

A major interest of modern science and medicine is the delineation of genes that cause disease. In the case of cancer, the study of viral oncogenic genes led to the recognition of similar human genes that play an important role in this disease. In an analogous fashion, the identification of viral genes important in central nervous system disease may lead to the recognition of related cellular genes that are important in nonviral central nervous system disease. New molecular techniques now provide tools for identification of pathogenic viral genes and elucidation of mechanisms of disease production. Positive-strand RNA viruses such as picornaviruses provide an especially attractive model system for studies of central nervous system disease-producing genes. A limitation in molecular studies of these viruses has resulted from an inability to use restriction enzymes, since these enzymes are active against DNA and not RNA. This limitation has recently been overcome with the preparation of infectious picornavirus complementary-DNA. This review highlights the importance of infectious complementary-DNA in pathogenesis studies and provides a glimpse of the impact of such studies on neurology.

DNA↗

PubMed1990

Cloning and sequencing of a complementary DNA encoding a Mr 70,000 human breast epithelial mucin-associated antigen.

The human milk fat globule (HMFG) membrane contains several glycoproteins that have been referred to as breast differentiation antigens and that are expressed in normal breast, breast tumors, breast tumor-derived cell lines, and are found in breast cancer patient serum. These antigens include a high molecular weight mucin and several smaller components including Mr 150,000; 70,000; and 46,000 glycoproteins. We have used 2 monoclonal antibodies (McR2 and Mc13) that bind the Mr 70,000 component of HMFG to immunoscreen a lambda gt11 expression library prepared from human lactating breast tissue. We report here the sequence of a complementary DNA clone (BA70-1) that codes for a peptide that binds both McR2 and Mc13 but not monoclonal antibodies to the breast mucin or other components of HMFG.A 1.8-kilobase RNA was detected in 9 of 9 breast tumor cell lines using 32P-labeled BA70-1 as probe. The BA70-1 RNA was highly expressed in 6 of 9 cells lines of breast and several other carcinomas lines compared with a lymphoblastoid cell line (Raji). The BA70-1 complementary DNA sequence has no extensive homology with previously reported sequences including the high-molecular weight mucin complementary DNA. Since the Mr 70,000 molecule appears to be associated with the breast mucin by disulfide bonds, its study could help elucidate the structure of this latter complex and how it is organized in the cell membrane, and prove useful in diagnosis and therapy of breast cancer.

Antibodies, Monoclonal↗

PubMed1995

Expression cloning of gastric mucin complementary DNA and localization of mucin gene expression.

BACKGROUND & AIMS: Secretory mucins play an important role in gastric cytoprotection and are derived from a heterogeneous family of genes. The aim of this study was to determine the specific type and location of mucin gene expression in the human stomach. METHODS: Expression cloning was performed by screening a human gastric complementary DNA expression library with antisera against deglycosylated gastric mucin. RNA analysis and immunohistochemistry were used to quantify and localize mucin gene expression. RESULTS: Sequencing of positive clones revealed two clones containing tandem repeats. The first contained a 169-amino acid repeat and was named MUC6 (as previously described). The second contained the same 8-amino acid repeat consensus sequence (APTTSTTS) as complementary DNAs previously isolated from a tracheobronchial complementary DNA library and was labeled MUC5 (or MUC5AC). RNA analysis indicated that the gastric epithelium contains high levels of MUC5 and MUC6 messenger RNA with little or no MUC2, MUC3, and MUC4 messenger RNA. Immunohistochemical analysis showed that surface mucous cells of the cardia, fundus, and antrum expressed MUC5 peptide. In contrast, MUC6 peptide expression was limited to mucous neck cells of the fundus, antral-type glands of the antrum and cardia, and Brunner's glands of the duodenum. CONCLUSIONS: MUC5 and MUC6 represent major secretory mucins in the stomach and are localized to distinct cell types.

Amino Acid Sequence↗

PubMed1976

Importance of full size complementary DNA in nucleic acid hybridization.

The size of the DNA product synthesized by RNA-directed DNA polymerase (isolated from avian myeloblastosis virus) was found to be important for complementary DNA (cDNA)-mRNA hybridization reactions. Incomplete cDNA to rabbit reticulocyte globin mRNA formed poor hybrids and presumably lacked sequences needed for hybridization. The size of the cDNA synthesized was influenced by the reaction conditions used. The complementary DNA product contained 10 S material when synthesis was done at high deoxynucleoside triphosphate concentrations (greater than 50 muM) while the product was smaller than the template when synthesis was at lower concentrations. The concentration and size (oligo(dT)6 to (dT)10) of primer had little or no effect on the product size. Increasing the concentration of 10 S globin mRNA caused the cDNA product to contain more small material. The cDNA synthesized at high deoxynucleoside triphosphate concentrations was fractionated into heavy, medium, and light fractions by alkaline sucrose density centrifugation. All hybridized to globin mRNA. The larger cDNAs had a higher TM when hybridized to globin mRNA, a lower dTMP/dCMP ratio (indicating that the poly(dT) region constituted a smaller fraction of the molecule), and gave increased protection of 125I-labeled mRNA from nuclease digestion. The full size cDNA was especially useful for studying the RNA transcribed from chromatin by RNA polymerase. The complement of the 5' end of the mRNA is contained only in full size cDNA; the 5' end is the part of the mRNA first transcribed by the RNA polymerase assuming correct transcription. Thus, full size cDNA can hybridize more effectively to the short RNA transcripts that are obtained than partial cDNA. RNA transcribed from rabbit bone marrow chromatin by Escherichia coli RNA polymerase hybridized twice as efficiently to complete cDNA as it did to partial cDNA demonstrating the usefulness of full size cDNA.

Animals↗

PubMed1992

Fluorescent-labeled oligonucleotides that exhibit a measurable signal in the presence of complementary DNA.

Oligonucleotide derivatives with a fluorescent dye were designed for exhibiting a measurable signal only when they bind to complementary DNA in aqueous solution. The oligonucleotide with a dansyl group at the specific 2'-sugar residue was synthesized by using the protected 2'-dansylaminouridine phosphorobisamidite. The dansyl-oligonucleotide conjugate binds to its complementary DNA to form duplex with a normal stability and exhibits enhanced fluorescence together with a blue-shift in emission maxima after the hybridization. Another possible candidate involved the use of pyrene-excimer emission upon forming ternary complex between two pyrene-labeled oligonucleotide probes with target DNA. A new and general method for introduction of a pyrene fluorophore into the 3'- or 5'-terminal hydroxyl group of oligonucleotides via different linkers was developed.

DNA↗

PubMed1997

p53 abnormalities in primary prostate cancer: single-strand conformation polymorphism analysis of complementary DNA in comparison with genomic DNA. The Cooperative Prostate Network.

BACKGROUND: The reported frequency of mutation of the p53 tumor suppressor gene (also known as TP53) in human carcinomas of the prostate has varied widely, ranging from 3% to 42%. This variability may be a consequence of tumor heterogeneity and/or the use of different methods of analysis. Since p53 mutation has been associated with clinical outcome for a number of cancer types, determination of its true frequency in primary carcinomas of the prostate is important. PURPOSE: The principal aims of this study were as follows: 1) to validate the utility of detecting p53 gene mutations by means of polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis of complementary DNA (cDNA) (synthesized from prostate tissue RNA and 2) to study the concordance of RNA- and DNA-based PCR-SSCP assays in detecting p53 mutations in individual tumor fragments. METHODS: RNA and genomic DNA were isolated by means of standard techniques from specimens of 19 carcinomas of the prostate, selected on the basis of p53 data obtained in a previous analysis of cDNA (indicating that 14 were mutant and five were wild-type). RNA was converted into cDNA by means of reverse transcription (RT); the cDNA was then amplified by means of nonisotopic (i.e., nonradioactive) PCR, and the PCR products were subjected to SSCP analysis in polyacrylamide gels (RT-PCR-SSCP analysis). Genomic DNA was examined by means of SSCP analysis of isotopically labeled (32PO4) PCR products (DNA-PCR-SSCP analysis). In both approaches, the protein coding region of the p53 gene was divided into multiple, smaller fragments for study. PCR products exhibiting abnormal migration in SSCP gels were subjected to direct nucleotide sequencing or to cloning and sequencing of multiple clones. RESULTS: RT-PCR-SSCP and DNA-PCR-SSCP identified p53 gene abnormalities in 15 of the 19 selected carcinomas, including one previously reported to be wild-type for p53. Overall, PCR-SSCP analysis identified 18 p53 fragments with abnormalities; three carcinomas showed two abnormalities each. Six (33%) of the 18 abnormalities were detected by both RT-PCR-SSCP and DNA-PCR-SSCP, 10 (56%) were detected by RT-PCR-SSCP alone, and two (11%) were detected by DNA-PCR-SSCP alone. The 18 abnormalities were caused by 20 changes in the sequence of the p53 gene; in one carcinoma, double mutations in two individual p53 exons were identified. CONCLUSIONS AND IMPLICATIONS: PCR-SSCP analysis of both RNA and DNA allows the detection of more mutations than the analysis of either alone. Some primary carcinomas of the prostate contain more than one altered p53 gene, consistent with the possibility of intratumoral heterogeneity of mutation of this gene. For comprehensive analysis of p53 mutations in carcinomas of the prostate, and perhaps in other tumor tissues, SSCP analysis of cDNA should be used in combination with SSCP analysis of genomic DNA.

DNA, Complementary↗