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At least 37 records · Page 2Linked to original sources
PubMed1978

Synthesis of Sindbis virus complementary DNA by avian myeloblastosis virus RNA-directed DNA polymerase.

Sindbis virus 42 S RNA was efficiently transcribed into complementary DNA (CDNA) by avian myeloblastosis virus alphabeta DNA polymerase using oligo- (dT) or single-stranded calf thymus DNA as primers. Both of the Sindbis virus cDNA products were able to protect 60% of 125I-labeled Sindbis virus RNA, at near equal weight ratios, from RNAase A and T1 digestion. Using hybridization kinetics, the Crt 1/2 value for hybridization of the calf thymus-primed cDNA product with excess Sindbis RNA was determined to be 1.8 9 10-2 mol . s . 1-1. Thes data demonstrate that the Sindbis virus cDNA products are relatively uniform representations of Sindbis virus RNA sequences.

Avian Myeloblastosis Virus↗
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↗
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↗
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↗
PubMed1979

Sequence homology of nucleic acids from human breast cancer cells and complementary DNA's from murine mammary tumor virus and Mason-Pfizer monkey virus.

Simultaneous presence of murine mammary tumor virus- and Mason-Pfizer monkey virus-specific sequences has been detected in nucleic acids isolated from some human breast tumors and from MCF-7 cells, a well-characterized human breast cancer cell line. Carefully characterized long complementary DNA transcripts were used in the molecular hybridization experiments. From the data that are presently available, it would appear that when homology is detected with one of the mammary tumor probes the other also generally shows shows homology. Among all the complementary DNA-RNA hybrids only three, all murine mammary tumor virus hybrids, show Tm values close to 80 degrees. The rest of the hybrids are low melting with shallow slopes for their Crt curves, indicating partial and imperfect hybrids in the majority of cases. Low levels of weak hybrid formation are also detectable with the tumor DNA's. The present experiments cannot ascertain whether the hybridizing sequences from Mason-Pfizer monkey virus and murine mammary tumor virus code for specific viral functions in their natural hosts. Annealing experiments using gene specific cDNA's would be required for fully characterizing these sequences.

Base Sequence↗
PubMed1975

Region of immunoglobulin light-chain mRNA transcribed into complementary DNA by RNA-dependent DNA polymerase of avian myeloblastosis virus.

The mRNA coding for a kappa-type immunoglobulin light (L)-chain and its complementary DNA (cDNA) hybridize with a Crt1/2 of 2.6 x 10(-4) moles of ribonucleotide x liter-1 x sec, forming well-matched duplexes (melting temperature Tm equals 89 degrees). The molecular weight of the cDNA is about 280,000 (840 nucleotides) as determined by alkaline sucrose gradient centrifugation and from the extent of protection of the mRNA by the cDNA from ribonuclease digestion. The cDNA anneals with kappa-type mRNAs of the same and different subgroups with comparable Crt1/2 values, but not with a lambda-type mRNA. Thus, one kappa-type cDNA can be used to quantify the mRNAs coding for all kappa-type L-chains. The values of cDNA hybridized at saturation with various kappa-type mRNAs indicate that: (1) the cDNA is complementary to the entire constant region and to about half of the variable (V)-region; (2) V-regions of similar amino-acid sequence are coded by a similar nucleotide sequence; (3) the nucleic acid probe to one V-region may anneal and quantify V-region genes of members of the same subgroup.

Avian Leukosis Virus↗
PubMed1979

Synthesis of long complementary DNA in the endogenous reaction by equine infectious anemia virus.

In the endogenous reverse transcriptase reaction, equine infectious anemia virus is able to synthesize complementary DNA (cDNA) of 8,000 nucleotides in high yield. After 2 h in 50 muM dNTP, about 2.8 mug of cDNA per mg of protein is produced, almost 30% of which is long cDNA. The system thus compares favorably with the other two well-characterized endogenous reaction systems, Moloney murine leukemia virus and avian sarcoma virus. Elongation rates of 100 to 150 nucleotides per min have been observed; these rates are comparable to those seen with purified avian myeloblastosis virus reverse transcriptase and significantly higher than those observed in vivo. In the absence of actinomycin D, equine infectious anemia virus does not require high dNTP levels for either optimal incorporation or long cDNA synthesis. The amount of long cDNA synthesized is maximal at 2 h in 50 muM dNTP; neither longer time nor higher dNTP levels (through 1.8 mM) increased this yield. Half-maximum yield in 2 h was achieved at about 15 muM dNTP, which is very similar to the published K(M)'s for isolated avian and murine reverse transcriptases. Total incorporation, on the other hand, continues to rise slowly through 1 mM dNTP; the half-maximum was 30 to 50 muM dNTP. In the presence of 100 mug of actinomycin D per ml, however, higher dNTP levels are required for long cDNA synthesis. We conclude that equine infectious anemia virus is exceptionally well-suited to studies of the physical organization of the retrovirus genome and to investigations of the mechanism of synthesis of the double-standard cDNA endogenous reaction product.

Cell-Free System↗
PubMed1979

Optimal conditions for synthesis of long complementary DNA product with Moloney murine leukemia virus.

Studies are described on the interrelationships between divalent metals, dNTP's and PPi in determining the properties of complementary DNA (cDNA) product from the in vitro reverse transcriptase reaction with detergent-treated Moloney murine leukemia virus. In spite of the several-fold greater amount of cDNA product with Mn2+ than with Mg2+, net yield of high-molecular-weight cDNA was much greater with Mg2+ thant with Mn2+. This held true, as well, for the reactions containing excess dNTP or dNTP plus PPi, both of which (as has been reported for Mg2+) promote synthesis of high-molecular-weight cDNA product. Hif total dNTP concentration remained important for maximum high-molecular-weight product with Mg2+ and was not replaced by simply providing dNTP in excess over Mg2+. Under the conditions tested here, addition of PPi did not further increase cDNA product size with Mg2+ when compared with dNTP in excess over Mg2+. Extent of degradation of the RNA template during the incubations was correlated with the size of cDNA product.

Cell-Free System↗
PubMed1978

Thyroglobulin complementary DNA as a means to investigate congenital goiters with impaired thyroglobulin synthesis.

A congenital goiter with impaired thyroglobulin synthesis has been investigated for the presence of messenger RNA coding for thyroglobulin. Using complementary DNA to beef 33 S thyroglobulin mRNA as a probe, we have found that thyroglobulin related sequences are present in the goiter but at a concentration lower than in normal human thyroids. We conclude that the disease is not due to a complete deletion of thyroglobulin gene.

Animals↗
PubMed1978

[Degradation of ribosomal RNA from Escherichia coli by ribonuclease U2 and in vitro transcription of "RNA-fragments" into complementary DNA].

Under well defined conditions ribosomal RNAs purified from Escherichia coli can be degraded by ribonuclease U2 giving rise to RNA fragments of 60--70 nucleotides. In vitro, these fragments are efficiently transcribed into a complementary DNA by DNA polymerase RNA dependent, partially purified from extracts of E. coli. In vivo, "RNA-fragments-U2" inhibit the development of plant tumors.

Animals↗
PubMed1978

Procollagen complementary DNA, a probe for messenger RNA purification and the number of type I collagen genes.

Type I procollagen mRNAs were separated from contaminating low-abundance messenger and nuclear RNAs by chromatography over Sepharose 4B in 0.65 M NaCl at room temperature. All of 27S rRNA and four-fifths of procollagen mRNAs bind to Sepharose under these conditions, while 18S rRNA and about three-fourths of other poly(A)-containing RNAs do not bind. AMV reverse transcriptase was used to prepare complementary DNA to procollagen mRNA at each purification step. Hybridization studies, in RNA excess, were carried out to establish the enrichment at each step both with respect to total RNA and to poly(A)-containing RNA. While "purified" procollagen mRNA preparations still consist of about 50% 27S rRNA, over 80% of cDNA prepared from it back hybridizes to its template at a log of cr0t1/2 of -1.9. This type I procollagen cDNA hybridizes in DNA excess to DNA isolated from chicken erythrocytes and from embryonic chick calvaria at a log c0t1/2 of 3.1, demonstrating that procollagen cDNA is complementary to unique gene sequences in both tissues and that procollagen genes are not reiterated.

Animals↗
PubMed1976

Estimation of the number of nucleotide sequences in mouse DNA complementary to messenger RNAs specifying a complete mouse immunoglobulin.

A fraction of ribonucleic acid (RNA) enriched in messenger RNA (mRNA) coding for immunoglobulin G (IgG) was isolated from cells of mouse myeloma RPC5 using specifically purified antibodies to immunoprecipitate polyribosomes engaged in IgG psi heavy and K light chain synthesis. More than 85% of the RNA present consisted of IgG mRNA as determined by an analysis of the products translated in its presence in the wheat germ system. IgG mRNA labeled with 125I was hybridized with mouse liver DNA. Approximately 95% of the RNA hybridized with mouse a Cot 1/2 of 4.0 X 103, indicating that the complementary DNA sequences were present less than five times per haploid genome. In contrast, approximately 75% of poly(adenylic acid) containing RNA prepared from unfractionated polyribosomes of RPC5 cells hybridized with a Cot 1/2 of 3.3 X 103; 25% of such RNA formed hybrids at lower Cot values.

Animals↗
PubMed1979

Detection of virus-specific RNA in simian sarcoma-leukemia virus-infected cells in in situ hybridization to viral complementary DNA.

An in situ molecular hybridization system which will detect retrovirus RNA in the cytoplasm of individual virus-infected cells has been developed. The technique was applied to cells infected with simian sarcoma-leukemia virus, where the virus-specific RNA was detected by hybridization to simian sarcoma-leukemia virus 3H-labeled complementary DNA. The system is useful for detecting viral RNA-containing cells in the presence of an excess of virus-negative cells and for determining which type of cell in a heterogenous population is expressing viral RNA.

Cell Line↗