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At least 127 records · Page 7Linked to original sources
PubMed1988

Molecular cloning of human and rat complementary DNA encoding androgen receptors.

Complementary DNAs (cDNAs) encoding androgen receptors were obtained from human testis and rat ventral prostate cDNA libraries. The amino acid sequence deduced from the nucleotide sequences of the cDNAs indicated the presence of a cysteine-rich DNA-binding domain that is highly conserved in all steroid receptors. The human cDNA was transcribed and the RNA product was translated in cell-free systems to yield a 76-kilodalton protein. The protein was immunoprecipitable by human autoimmune antibodies to the androgen receptor. The protein bound androgens specifically and with high affinity.

Amino Acid Sequence↗
PubMed1987

Expression of functional cell-cell channels from cloned rat liver gap junction complementary DNA.

An oocyte expression system was used to test the relation between a complementary DNA (cDNA) clone encoding the liver gap junction protein and cell-cell channels. Total liver polyadenylated messenger RNA injected into oocytes induced cell-cell channels between paired oocytes. This induction was blocked by simultaneous injection of antisense RNA transcribed from the gap junction cDNA. Messenger RNA selected by hybridization to the cDNA clone and translated in oocyte pairs yielded a higher junctional conductance than unselected liver messenger RNA. Cell-cell channels between oocytes were also formed when the cloned cDNA was expressed under the control of a heat-shock promoter. A concentration-dependent induction of channels was observed in response to injection with in vitro transcribed gap junction messenger RNA. Thus, the liver gap junction cDNA encodes a protein that is essential for the formation of functional cell-cell channels.

Animals↗
PubMed1991

A new growth-regulated complementary DNA with the sequence of a putative trans-activating factor.

A new complementary DNA (cDNA) clone has been isolated by differential screening of a cDNA library. The cognate RNA of this clone, called SC1, is growth regulated in human, mouse, and hamster cell lines. Its kinetics of growth regulation (time of increase in mRNA levels, sensitivity to cycloheximide, behavior in G1-specific temperature-sensitive mutants) classify the SC1 gene as a late growth-regulated gene, like the histone genes and the genes coding for the proteins of the DNA synthesis apparatus. By run-on assay, there is a modest increase in transcriptional rates after serum stimulation, which is not sufficient to explain the sharp increase in mRNA levels. The SC1 gene localizes to human chromosome 6p21-22. In bacteria, the SC1 cDNA clone makes a protein of Mr 39,000, in agreement with the putative reading frame. The amino acid sequence derived from the cDNA sequence indicates a previously unknown gene with a domain strongly suggestive of a trans-activating domain. The SC1 gene can be considered as coding for a possible new trans-activating factor that could play an important role in the transcription of genes required for the later stages of cell cycle progression.

Amino Acid Sequence↗
PubMed1977

Human beta-globin messenger RNA. III. Nucleotide sequences derived from complementary DNA.

Sequences of human beta-globin mRNA were determined by analysis of complementary DNA. beta-mRNA was transcribed into double-stranded cDNA by RNA-dependent DNA polymerase. cDNA was cut by restriction endonucleases and the fragments were terminally labeled by means of polynucleotide kinase and [gamma-32P]ATP. After purification, fragments were degraded by snake venom phosphodiesterase. Alternatively single-stranded [32P]cDNA was prepared by transcription in the presence of [alpha-32P]dCTP and actinomycin D; the product was digested by endonuclease IV and degraded by snake venom phosphodiesterase. cDNA tracts obtained by both labeling methods enabled us to construct a sequence for the translated and 3'-terminal untranslated regions of human beta-mRNA.

Anemia, Sickle Cell↗
PubMed1979

Androgenic regulation of messenger RNA sequence complexity in accessory sexual tissues of the male rat studied with fractionated complementary DNA.

Effects of androgens on mRNA sequence complexity in the rat seminal vesicle have been investigated using complementary DNA fractionated on the basis of sequence abundance. Total cDNA complementary to poly(A)-rich RNA from normal rats was hybridised with an excess of the same RNA to controlled rot values and then the free cDNA was separated from cDNA . RNA hybrids by hydroxyapatite chromatography. Three cDNA fractions were obtained with very different hybridisation characteristics. Abundant cDNA hybridised to an excess of its parental RNA with an rot 1/2 of 2.46 x 10(-3) mol 1(-1) s and is complementary to about six or seven average-sized sequences. Use of hybrid-arrested translation in a cell-free protein-synthesising system has shown that this class of mRNA includes mRNAs coding for major androgen-dependent secretory proteins. Moderate and scarce cDNA fractions each showed more complex hybridization kinetics; computer analysis suggested each is complementary to two groups of average-sized sequences. Each cDNA fraction was hybridised to excess poly(A)-rich RNA from normal or castrated rats and the kinetics compared. Castration had no effect on the total number of sequences present in any class and did not alter the relative concentration of the scarce sequences. A small (threefold) decrease was seen in the concentration of abundant sequences with a larger (tenfold) decrease in the moderate class. Both de-reases were reversed by testosterone in vivo. The results are consistent with earlier studies where the effects of testosterone on seminal vesicle mRNA were followed using a translation assay and confirm that no gross differential effects are exerted on abundant mRNA coding for major secretory proteins. The cDNA fractions were also used to investigate the overlap in genetic expression between seminal vesicle and ventral prostate. Both tissues share all the scarce sequences in the same relative abundance. Less than 0.0015% and 0.004% of prostatic mRNA is complementary to seminal vesicle abundant and moderate sequences respectively. Similarly prostatic abundant sequences account for less than 0.004% of seminal vesicle mRNA.

Animals↗
PubMed1977

The use of a complementary DNA probe to detect accumulation of mengo RNA in infected cells pretreated with interferon.

Complementary DNA (cDNA) from Mengo virus RNA has been synthesized and used as a probe to measure the synthesis and accumulation of viral RNA in Mengo infected L cell cultures, treated or untreated with interferon. Under experimental conditions used (200 units interferon/ml and 50 virus plaque-forming units/cell) results show that there is some synthesis of Mengo virus RNA in cells treated with interferon. One hour after infection, treated cells contain three times less viral RNA than untreated cells; five hours after infection, this difference has increased to ten fold. As in the control, no fragmented Mengo virus RNA molecules were found in interferon treated cells. The smaller recovery of infectious particles from interferon treated cells as compared to RNA accumulation suggests that not only RNA accumulation is inhibited but also a step posterior in viral maturation.

Cytoplasm↗
PubMed1977

Multihormonal induction of hepatic alpha2u-globulin mRNA as measured by hybridization to complementary DNA.

A procedure is presented for the preparation of a (3)H-labeled complementary DNA (cDNA) specific for the mRNA coding for alpha(2u)-globulin, a male rat liver protein under multihormonal control that represents approximately 1% of hepatic protein synthesis. Rat liver polysomes are incubated with monospecific rabbit antiserum to alpha(2u)-globulin, which binds to the nascent alpha(2u)-globulin chains on the polysomes. These antibody-polysome complexes are then adsorbed to goat antiserum to rabbit IgG that is covalently linked to p-aminobenzylcellulose. mRNA preparations are thus obtained that contain 30-40% alpha(2u)-globulin mRNA. A labeled cDNA is made to this alpha(2u)-globulin-enriched mRNA preparation by using RNA-dependent DNA polymerase (reverse transcriptase). To remove the non-alpha(2u)-globulin sequences, this cDNA preparation is hybridized to an RNA concentration x incubation time (R(0)t) of 1000 mol of ribonucleotide per liter x sec with female rat liver mRNA, which, though it shares the vast majority of mRNA sequences with male liver, contains no alpha(2u)-globulin mRNA sequences. The cDNA remaining single-stranded is isolated by hydroxylapatite chromatography and is shown to be specific for alpha(2u)-globulin mRNA by several criteria. Good correlation was found in all endocrine states studied between the hepatic level of alpha(2u)-globulin, the level of functional alpha(2u)-globulin mRNA as assayed in a wheat germ cell-free translational system, and the level of alpha(2u)-globulin mRNA sequences as measured by hybridization to the alpha(2u)-globulin cDNA. Thus, the hormonal control of hepatic alpha(2u)-globulin synthesis by sex steroids and thyroid hormone occurs through modulation of the cellular level of alpha(2u)-globulin mRNA sequences, presumably by hormonal control of transcriptive synthesis.

Alpha-Globulins↗
PubMed1982

A reaction rate constant for avocado sunblotch viroid and its complementary DNA with application to viroid detection.

Molecular hybridization between avocado sunblotch viroid and its complementary DNA (cDNA) proceeds as if it were a bimolecular chemical reaction with a rate constant of 0.21 liters per nanomole of nucleotide per day. Computer simulations show the minimum incubation times required to detect the viroid with cDNA probes of varying sensitivity with respect to viroid concentration, and estimate the concentration of one of the reactants when the other is known.

Chemical Phenomena↗
PubMed1982

Cloning and characterization of DNA complementary to the measles virus mRNA encoding hemagglutinin and matrix protein.

Since cloning and characterization of DNA complementary to measles virus mRNA encoding for the nucleocapsid protein (M. Gorecki and S. Rozenblatt, Proc, Natl. Acad, Sci, U.S.A. 77:3686--3690, 1980), two additional measles-specific clones containing different classes of sequences have been characterized. The cloned plasmids contain inserts of 480 and 530 base pairs as shown by agarose gel electrophoresis and electron microscopy. The sizes of the mRNA species complementary to these inserts are 1,700 and 1,550 nucleotides, respectively as determined by the Northern technique. The cloned DNA fragments were further identified as reverse transcripts of the mRNA coding for the glycoprotein and matrix protein of measles virus. The major cell-free translation products of mRNA selected by hybridization to the individual cloned DNAs comigrated with the 70K in vitro products and matrix proteins. One of the cell-free translation products (70K) was also immunoprecipitated specifically with monoclonal antibodies against measles virus glycoprotein.

Base Sequence↗
PubMed1988

New BK virus episomal vector for complementary DNA expression in human cells.

The properties of pRP-c, a new vector for complementary DNA (cDNA) expression, are described. The vector contains the early region and replication origin of BK virus (BKV), a human papovavirus. Due to the presence of these BKV sequences, pRP-c replicates in human cells allowing amplification of inserted cDNAs. The promoter, intron and polyadenylation region for cDNA expression are separated by unique restriction sites and can therefore be individually excised and substituted with different transcription signals. Coding sequences of the bacterial genes for chloramphenicol-acetyl transferase (CAT) or neomycin phosphotransferase (neo) were inserted into the cDNA cloning site of pRP-c and expressed in human cells in transient assays or stable clones. In both cases expression of the inserted sequences was significantly more efficient than by using the integration vectors pSV2CAT and pSV2neo, demonstrating the advantages of episomal expression vectors in human cells. Possible uses of pRP-c to express viral and cellular cDNAs in human cells are discussed.

BK Virus↗
PubMed1987

Replication origin (oric) on the complementary DNA strand of Escherichia coli phage G4: biological properties of mutants.

Phage G4 origin of complementary DNA strand synthesis (oric) consists of three stable secondary loop structures. In a cloned 274-bp DNA fragment that is active as an ori in the filamentous phage cloning vector R199, insertion mutants have been constructed by introducing EcoRI and HindIII linkers at the base of loop III. The in vivo activity of these oric mutants (conversion of single-strand form to replicative form in the presence of rifampicin) was significantly reduced (50-70%) but not completely abolished. Nucleotide sequences and/or potential secondary structure of loop III centered at the AvaII site are therefore an important functional part of oric.

Base Sequence↗
PubMed1990

Isolation of complementary DNA clones encoding pathogenesis-related proteins P and Q, two acidic chitinases from tobacco.

Complementary DNA clones encoding two isoforms of the acidic endochitinase (chitinase, EC 3.2.1.14) from tobacco were isolated. Comparison of amino acid sequences deduced from the cDNA clones and the sequence of peptides derived from purified proteins show that these clones encode the pathogenesis-related proteins PR-P and PR-Q. The cDNA inserts were not homologous to either the bacterial form of chitinase or the form from cucumber but shared significant homology to the basic form of chitinase from tobacco and bean. The acidic isoforms of tobacco chitinase did not contain the amino-terminal, cysteine-rich "hevein" domain found in the basic isoforms, indicating that this domain, which binds chitin, is not essential for chitinolytic activity. The accumulation of mRNA for the pathogenesis-related proteins PR-1, PR-R, PR-P, and PR-Q in Xanthi.nc tobacco leaves following infection with tobacco mosaic virus was measured by primer extension. The results indicate that the induction of these proteins during the local necrotic lesion response to the virus is coordinated at the mRNA level.

Amino Acid Sequence↗
PubMed1992

Molecular cloning of rat prostate transglutaminase complementary DNA. The major androgen-regulated protein DP1 of rat dorsal prostate and coagulating gland.

Complementary DNA (cDNA) that codes for a major androgen-dependent secretory protein of rat coagulating gland and dorsal prostate, dorsal protein 1 (DP1), was isolated by molecular cloning. Recombinant DP1 cDNA clones were identified from a bacteriophage lambda gt11 rat coagulating gland expression library using an affinity purified polyclonal antibody. Amino acid sequence deduced from DNA contained sequences identical with several DP1 cyanogen bromide cleavage fragments. Northern blot hybridization of poly(A) RNA isolated from intact rat dorsal prostate and coagulating gland revealed a predominant messenger RNA (mRNA) species of approximately 3200 nucleotides. Tissue-specific expression of DP1 mRNA was indicated by the absence of DP1 mRNA in ventral prostate and other tissues of the rat. Expression of DP1 mRNA was androgen-dependent, decreasing approximately 80% 7 days after castration and increasing rapidly following androgen replacement. Southern blot analysis of restriction enzyme-digested rat DNA indicated that DP1 is encoded by a single gene and that no major genomic rearrangements accounted for its lack of expression in the dorsal prostate-derived rat Dunning tumor. Sequence comparisons revealed that rat prostate DP1 shares sequence identity with Factor XIIIa and tissue transglutaminase, including the active center, GQCWVF, indicating that DP1 is a member of the transglutaminase gene family.

Amino Acid Sequence↗
PubMed1990

Isolation of novel complementary DNA clones from T lymphoma cells: one encodes a putative multiple membrane-spanning protein.

Five novel complementary DNA (cDNA) clones which are differentially expressed between two closely related T lymphoma cell clones were isolated using subtraction-enriched differential screening. SL12.4 cells, from which the cDNAs were isolated, have characteristics of thymocytes at an intermediate stage in development and cause prominent extranodal ovarian tumors in syngeneic animals. A sister cell clone, SL12.3, derived from the same tumor, has a distinct phenotype and causes more aggressive, diffuse lymphomas. Four of the five novel genes are expressed in normal thymus, activated spleen cells, or gut-associated lymphoid tissue. The DNA sequence and predicted protein sequence are presented for one of the novel cDNA clones. This novel cDNA clone detects mRNA in normal thymus, gut-associated lymphoid tissue, and ovarian tissue. The predicted protein has four putative transmembrane-spanning regions. The expression of the transcript is repressed in somatic cell hybrids formed from SL12.4 cells fused with three different T lymphoma cell lines which lack detectable mRNA complementary to the novel cDNA clone. This trans-negative regulation suggests that the expression of the gene is regulated by repressional mechanisms.

Animals↗
PubMed1976

Quantitation of casein messenger ribonucleic acid sequences using a specific complementary DNA hybridization probe.

Two highly purified rat casein mRNA fractions were used as templates to synthesize complementary DNA (cDNA) hybridization probes using RNA-directed DNA polymerase isolated from avian myeloblastosis virus. Both of the probes selectively hybridized to RNA isolated from lactating mammary tissue, but not to poly(adenylic acid)-containing rat liver RNA. An analysis of the kinetics of hybridization of the cDNA derived from the 15S casein mRNA (cDNA12S) with their individual mRNA templates indicated that greater than 90% hybridization occurred over a R0t range of one and one-half logs with R0t 1/2 values of 0.0023 and 0.0032 mol s l.-1, respectively. Compared with the total RNA isolated from lactating mammary tissue, these values represented a 166- and 245-fold purification, respectively, of these individual mRNA fractions. Using the 15S casein mRNA as a template, two probes of different lengths and specific activities were synthesized. The deoxyribonucleotide and mRNA concentrations and the temperature of incubation were optimized to obtain either a high specific activity cDNA probe, 330 nucleotides long, which represented approximately 25% of the mRNA or a lower specific activity preparation containing some complete cDNA copies, 1300 nucleotides in length. The Tm of the longer cDNA15S-15S mRNA hybrid was 88.5 degrees C, while that of the short cDNA15S-RNA hybrid was 82.5 degrees C. Following this initial characterization, the cDNA15S probe was utilized for three separate determinations: (1) Analysis of the sequence divergence between mouse and rat casein mRNAs. It was observed that the rate of hybridization of heterologous rat cDNA15S-mouse casein mRNA was only 20% that of the homologous rat cDNA15S-rat casein mRNA hybridization. The resulting heterologous hybrid displayed approximately 17% mismatching compared with the homologous hybrid. (2) Determination of the gene dosage for casein mRNA in normal and malignant mammary cells. In this study, an analysis of the kinetics of hybridization of the high specific activity cDNA15S probe with an excess of DNA isolated from lactating mammary tissue, carcinogen-induced mammary tumors, or rat liver indicated that casein mRNA was transcribed from the nonlification or deletion was observed during tumor formation or the process of mammary differentiation. (3) Quantitation of casein mRNA sequences during normal mammary gland development. RNA excess hybridizations were performed using RNA extracted from either pregnant, lactating, or regressed rat mammary tissue. The concentration of casein mRNA molecules/alveolar cell was found to increase 12-fold from 5 days of pregnancy until 8 days of lactation and then declined to approximately 2% of the maximal level of 79 000 molecules/cell by 7 days after weaning. A coordinate increase was observed in casein mRNA sequences detected by cDNA hybridization and mRNA activity measured in a cell-free translation assay.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1990

Diagnostic complementary DNA probes for genome segments 2 and 3 of epizootic hemorrhagic disease virus serotype 1.

Potential diagnostic complementary DNA (cDNA) clones of gene segments 2 and 3 from epizootic hemorrhagic disease virus serotype 1 (EHDV-1) have been produced. Individual segments of EHDV-1 were isolated, denatured with methylmercury hydroxide, and polyadenylated. The polyadenylated RNA was reverse-transcribed and self-hybridized into duplex structures, and the incomplete ends were repaired. The resulting product was then cloned into the plasmid vector pBR322, using the complementary tailing method. Two clones, 1 from segment 2 (E1-2-10) and 1 from segment 3 (E1-3-16) were isolated, colony-purified, and characterized by cDNA/RNA blot hybridization and endonuclease restriction analysis. The cDNA clones of RNA segment 3 of EHDV-1 cross hybridized with the corresponding segment of EHDV serotype 2 by results of cDNA/RNA blot hybridization, but not with RNA of bluetongue virus serotypes isolated in the United States. After cDNA/RNA dot-blot hybridization analysis of 17 EHDV field strains, the segment-2 clone was found to be serotype-specific, whereas the segment-3 clone was serogroup-specific.

Animals↗
PubMed1977

DNA complementary to parathyroid mRNA directs synthesis of pre-proparathyroid hormone in a linked transcription-translation system.

DNA complementary in sequence to the messenger RNA for pre-proparathyroid hormone was synthesised using reverse transcriptase. In a linked transcription-translation system using RNA polymerase and cell-free extract from wheat germ, the DNA directed the synthesis of a protein identified as pre-proparathyroid hormone by N-terminal sequencing and by electrophoretic and immunologic criteria.

Amino Acid Sequence↗