PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “DNA, Complementary”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1976

Gene specific priming of complementary DNA synthesis.

DNA, complementary to chicken globin mRNA was synthesized using either Avian Myeloblastosis virus reverse transcriptase, or E. coli DNA polymerase I. Transcriptase cDNA sediments at 9 S on sucrose gradients, and is 620 nucleotides in length, representing a complete copy of globin mRNA template. In contrast, Polymerase I cDNA sediments at 4 S, is 100 to 200 nucleotides in length, and is a copy of a small region at the 3'(poly A) end of globin mRNA. Similarly, Transcriptase cDNA and Polymerase I cDNA hybridize to globin mRNA template with characteristic, individual Crot1/2 values. The Crot1/2 value for Transcriptase cDNA hybridization is 7 X 10(-4) mol s 1(-1), and that for Polymerase I cDNA is 5 X 10(-3). This work shows that Avian Myeloblastosis virus reverse transcriptase can use Polymerase I cDNA to prime further cDNA synthesis along the mRNA template. The product of extended cDNA synthesis is identical in length and hybridization properties to oligo (dT) primed transcriptase cDNA.

Avian Myeloblastosis Virus↗
PubMed1977

Human placental lactogen mRNA and its structural genes during pregnancy: quantitation with a complementary DNA.

A complementary DNA (cDNA) strand was transcribed from human placental lactogen (hPL) mRNA. Based on alkaline sucrose gradient centrifugation, the size of the cDNA was about 8 S, which would represent at least 80% of the hPL mRNA. Previously we showed that four to five times more hPL was synthesized in cell-free extracts derived from term as compared to first trimester placentas. Hybridization of the cDNA with RNA derived from placental tissue revealed that there was about four times more hPL mRNA sequences in total RNA from term placenta than in a comparable quantity of total first trimester RNA. Only background hybridization was observed when the cDNA was incubated with RNA prepared from human kidney. To test if this differential accumulation of hPL mRNA was the result of an amplification of hPL genes, we hybridized the labeled cDNA with cellular DNA from first trimester and term placentas and with DNA isolated from human brain. In all cases, the amount of hPL sequences was approximately two copies per haploid genome. Thus, the enhanced synthesis of hPL mRNA appears to result from a transcriptional activation rather than an amplification of the hPL gene. The increase likely reflects placental differentiation in which the proportion of syncytial trophoblast increases at term.

DNA↗
PubMed1976

Fractionation of DNA nucleotide transcripts from Moloney sarcoma virus and isolation of sarcoma virus-specific complementary DNA.

Radioactive DNA complementary to nucleotide sequences in Moloney murine sarcoma virus (MSV) and Moloney leukemia virus (M-MuLV) complex was made by the endogenous reverse transcriptase reaction. These virus stocks contained a threefold excess of MSV over M-MuLV as measured by biological assay. The complementary DNA was an accurate copy of the viral RNA in that 86% of 35S viral RNA hybridized with complementary (cDNA) DNA at a 1.5 to 1 cDNA-RNA molar ratio. The complementary DNA, of a 4-6S size, was fractionated by sequential absorptions with MulV and the feline leukemia virus pseudotype of MSV, [MSV(FeLV)] RNA. In this manner three sets of nucleotide sequences whichrepresent different portions of the MSV viral complex were obtained: a sarcoma virus-specific fraction (cDNAsarc) with sequences that had no homology to M-MuLV RNA but which hybridized to MSV (FeLV) RNA, a sarcoma-leukemia fraction (cDNA common) with sequences common to MSV as well as M-MuLV viral RNA, and a cDNAleuk representing those nucleotide sequences found only in M-MuLV. Hybridization of MSV-MuLV viral 35S RNA with a threefold molar excess of cDNA's revealed that approximately 20% was hybridized with cDNAsarc, whereas approximately 75% was hybridized with cDNAcommon. M-MuLV 35S RNA alone did not hybridize with cDNAsarc but did hybridize 40 and 50% with cDNAleuk and cDNAcommon, respectively. The cDNAsarc represents about 25% of the total MSV sequences, whereas the cDNAcommon represents the remainder of the MSV virus genome. Some cDNAcommon sequences were shared by two other sarcoma viruses and several distinctly different isolates of MulV. In contrast, the MSV "sarc" sequences had little or no homology with two other murine sarcoma virus isolates.

Base Sequence↗
PubMed1975

Complexity of cytoplasmic RNA in different mouse tissues measured by hybridization of polyadenylated RNA to complementary DNA.

The kinetics of hybridization of polyadenylated RNA from mouse L-cells with complementary DNA (cDNA) synthesized with reverse transcriptase revealed three classes of differing abundance. The simplest interpretation requires three frequency classes representing polyadenylated RNA; 5, 45, and 50 percent of the total polyadenylated RNA and about 3, 300, and 7600 different RNA sequences of 6 times 10-5 daltons, respectively. The complementary DNA synthesized with L-cell polyadenylated RNA as template hybridized efficiently with RNA from different mouse tissues, indicating that most species of the L-cell RNA in the highand middle frequency class are present in all mouse tissues. Kinetics of hybridization of complementary DNA synthesized with cytoplasmic polyadenylated brain RNA as template suggested a higher complexity for brain RNA. Thirty-five percent of this brain cDNA failed to hybridize with L-cell RNA. This complementary DNA fraction, isolated by hydroxylapatite chromatography, represented approximately 11,000 RNA sequences specific for the brain. On the other hand, hybridization of complementary DNA synthesized on polyadenylated mouse liver RNA with L-cell RNA failed to demonstrate differences between these two groups of polyadenylated RNA.

Adenine Nucleotides↗
PubMed1979

Elongation of DNA complementary to the 5' end of the avian sarcoma virus genome by the virion-associated RNA-dependent DNA polymerase.

RNA-dependent DNA synthesis in a virion-associated reaction has been described as being dependent upon the detergent concentration used for disruption of the virion. In this study, the Triton X-100 concentration was found to affect the elongation of the initially synthesized DNA complementary to the last approximately 100 nucleotides at the 5' end of the RNA (cDNA100). Whereas elongation of cDNA100 increased with time of incubation at the optimal detergent concentration, this process was retarded at higher detergent concentrations. At the optimal detergent concentration, elongated DNA was of low chemical complexity, indicating that extension of cDNA100 occurred at a unique site on the RNA. Higher than optimal detergent concentrations resulted in nonspecific elongation and in DNA of high chemical complexity. This was shown by oligopyrimidine tract analysis. Furthermore, actinomycin D was observed to inhibit the elongation of cDNA100 at the optimal detergent concentration. The nature of the elongation process was elucidated by analysis of DNA synthesized in a virion-associated reaction in the presence of bacteriophage Qbeta RNA. At the optimal detergent concentration DNA complementary only to avian sarcoma virus RNA was synthesized, whereas at higher concentrations DNA was copied from both avian sarcoma virus and Qbeta RNA. We conclude that the elongation mechanism of cDNA100 is affected by the detergent concentration and elongation is unspecific at higher than optimal detergent concentrations. The mechanism by which the nonionic detergent stimulates DNA synthesis has not yet been resolve. We assume that other factors in addition to DNA polymerase are involved in elongation of cDNA100.

Alpharetrovirus↗
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↗
PubMed1976

The synthesis and properties of the complete complementary DNA transcript of ovalbumin mRNA.

The synthesis of a complementary DNA copy (cDNA) of hen ovalbumin mRNA using AMV RNA-directed DNA polymerase was studied under different conditions of salt, deoxyribonucleotide concentrations, temperature, and time. It was observed that in the absence of monovalent cation at 46 degrees C a complete transcript of ovalbumin mRNA could be effected by the enzyme. The minimum deoxyribonucleotide requirement for complete synthesis was 35 muM for dATP, dGTP, and dCTP and 200 muM dTTP. By a number of different experimental criteria which included sedimentation on alkaline sucrose gradients and electrophoresis in polyacrylamide gels containing 98% formamide, direct electron microscope visualization, and protection of ovalbumin [25I]mRNA from nuclease digestion it could be demonstrated that a considerable fraction of a complete mRNA transcript was indeed synthesized. The cDNA/ovalbumin mRNA hybrid had a Tm on hydroxylapatite of 92 degrees C, indicating the synthesis of a RNA transcript with a high fidelity. When such a complete ovalbumin [3H]cDNA was synthesized with a specific activity of 10(8) cpm/mug and hyfridized to an excess of chick DNA, the kinetics of hybridization indicated that the cDNA was comprised of a nonrepetitive sequence.

Animals↗
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↗
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↗
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

[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↗
PubMed1975

Quantitative and qualitative differences in DNA complementary to avian myeloblastosis virus between normal and leukemic chicken cells.

Hybridization of avian myeloblastosis virus (AMV) RNA with DNA immobilized on filters or in liquid with a vast DNA excess was used to measure the viral specific DNA sequences in chicken cells. Newly synthesized viral DNA (v-DNA) appears within an hour after infection of chicken embryo fibroblasts (CEF) with avian oncornaviruses. A fraction of newly synthesized v-DNA becomes integrated into the cellular genome and the remainder gradually disappears. A covalent linkage between v-DNA and cellular DNA was demonstrated to exist in CEF and in leukemic myeloblasts by alkaline sucrose velocity sedimentation. Hybridization of AMV RNA in DNA excess has revealed that there are 2 clases of viral specific sequences within normal as well as in leukemic cells. The 2 types of sequences differ in their rate of hybridization. The amount of both types of DNA sequences is about 2 times higher in leukemic cells than in normal cells. Both the fast- and slowly reacting sequences in leukemic cells exhibit a higher Tm (2 degrees C) than the respective DNA sequences in normal cells. Furthermore, when nucleotide sequences in AMV RNA complementary to normal DNA are removed first by exhaustive hybridization with normal DNA, the residual RNA only hybridizes with leukemic DNA but not with normal DNA. These results suggest that leukemic cells contain viral specific DNA sequences which are absent in normal cells. Endogenous v-DNA has been shown to be integrated in cellular DNA region(s) with a reiteration frequency of approximately 1,200 copies per cell and each integration unit appears to have a size approximately equivalent to the 35S RNA subunit of the viral genome. Viral sequences acquired after infection appear to be integrated in the unique region of cell DNA, or in tandem with the endogenous viral sequences.

Animals↗