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Showing papers on "Complementary DNA published in 1977"


Journal ArticleDOI
17 Jun 1977-Science
TL;DR: Recombinant bacterial plasmids have been constructed that contain complementary DNA prepared from rat islets of Langerhans messenger RNA that contain cloned sequences representing the complete coding region of rat proinsulin I, part of the preproinsulin II prepeptide, and the untranslated 3' terminal region of the mRNA.
Abstract: Recombinant bacterial plasmids have been constructed that contain complementary DNA prepared from rat islets of Langerhans messenger RNA. Three plasmids contain cloned sequences representing the complete coding region of rat proinsulin I, part of the preproinsulin I prepeptide, and the untranslated 3' terminal region of the mRNA. A fourth plasmid contains sequences derived from the A chain region of rat preproinsulin II.

1,135 citations


Journal ArticleDOI
TL;DR: Sequences of human beta-globin mRNA were determined by analysis of complementary DNA by constructing a sequence for the translated and 3'-terminal untranslated regions of humanbeta-mRNA.

218 citations


Journal ArticleDOI
01 Dec 1977-Cell
TL;DR: Using a 3H-cDNA for RNA sequences specifically associated with murine intracisternal type A particles, the presence of reiterated sequence transcripts in poly(A) RNA from a neuroblastoma A particle fraction was confirmed by direct hybridization of the RNA with cellular DNA.

135 citations


Journal ArticleDOI
TL;DR: DNA related to the genome of mouse mammary tumor virus is measured in normal organs from laboratory, feral and Asian mice, and in mammary tumors from laboratory mice, by annealing MMT § viral cDNA to cellular DNA to suggest that MMT viral DNA appears to evolve at least as fast as cellular unique-sequence DNA.

125 citations


Journal ArticleDOI
TL;DR: Control experiments support the hypothesis that globin mRNA sequences are degraded after infection by herpes simplex virus.
Abstract: The fate of preexisting mRNA sequences was examined after infection by herpes simplex virus. Murine erythroid cells transformed by Friend leukemia virus were used as the host. Such cells, when exposed to 2% dimethyl sulfoxide, produce large amounts of globin and globin mRNA. The protein and its mRNA are easily recognized at 4 days by electrophoresis in high percentage acrylamide gels and by hybridization to cDNA, respectively. Herpes simplex virus replicates in these cells. By 2 hr after infection the rate of protein synthesis decreases to 30% of the level in mock-infected cells and only 49+/-8% (SEM) of the globin mRNA sequences present prior to infection could be detected by hybridization to cDNA. At 4 hr after infection, when the rate of protein synthesis in infected cells is at a maximum, only about 15% of the globin mRNA sequences remained. Control experiments support the hypothesis that globin mRNA sequences are degraded after infection by herpes simplex virus.

115 citations



Journal ArticleDOI
TL;DR: The results indicate that the ovalbumin genes appear to be organized by chromatin proteins in such a way that they are rendered exceedingly sensitive to digestion by DNase I, and suggest that the maintenance of an active conformation about specific genes does not reflect the polymerase distribution about these genes.
Abstract: We have analyzed the DNA generated upon treatment of oviduct nuclei with pancreatic DNase I (deoxyribonucleate 3′-oligonucleotidohydrolase; EC 3146), with cDNA copies of specific mRNA sequences to study the structure and organization of transcriptionally active genes in chromatin In this report we examine the kinetics of digestion of three classes of genes in the oviduct which are transcribed at significantly different rates Our results indicate that the ovalbumin genes appear to be organized by chromatin proteins in such a way that they are rendered exceedingly sensitive to digestion by DNase I This sensitivity is not observed in the liver, a tissue in which these genes are transcriptionally inert Furthermore, the transcriptionally inactive globin genes in the oviduct are not selectively sensitive to nuclease attack and are digested 5 times more slowly in the ovalbumin genes in this tissue In addition, we have examined the accessibility of a complex subset of genes that are rarely represented in the mRNA and are likely to be transcribed at a frequency orders of magnitude below that of the ovalbumin gene Comparison of the accessibility of these sequences with that of the ovalbumin gene indicates that these two subsets of genes are recognized and cleaved by DNase I at similar rates These results suggest that the maintenance of an active conformation about specific genes does not reflect the polymerase distribution about these genes This active conformation is therefore not confined to sequences actively engaged in the transcription process and may reflect the structure about a subpopulation of the genome which represents the transcriptional potential of a given cell type

110 citations


Journal ArticleDOI
15 Dec 1977-Nature
TL;DR: The Rous sarcoma virus (RSV) is a 30-40S RNA of 10,000 nucleotides coding for four known genes1 which map in the order 5′-gag, pol, env, src, poly(A)-3′.
Abstract: THE genome of Rous sarcoma virus (RSV) is a 30-40S RNA of 10,000 nucleotides coding for four known genes1 which map in the order 5′-gag, pol, env, src, poly(A)-3′ (refs 2, 3). This 30-40S RNA serves as mRNA for a gag precursor protein and perhaps a gag-pol precursor-polyprotein when translated in vitro4,5. The env gene, however, may be translated from a smaller intracellular messenger. Poly(A)-containing 20-24S cellular RNA from infected cells can, upon microinjection, complement an env-defective virus6 and may also direct in vitro synthesis of a protein that is serologically related to the env gene product7. Moreover, hybridisation with DNA complementary (cDNA) to env and src sequences of virion RNA detects discrete classes of subgenomic poly(A)-containing RNAs present in infected cells as steady state species, while gag and pol-specific cDNAs hybridise only to full sized RNA8,9.

102 citations


Journal ArticleDOI
01 Mar 1977-Cell
TL;DR: At the early protamine stage of testis development, polysomal and postribosomal supernatant fractions contain almost equal quantities ofpoly(A)+ protamine mRNA, but poly(A)- protamine RNA was found almost entirely in the polysomes.

92 citations


Journal ArticleDOI
01 Mar 1977-Cell
TL;DR: It is suggested that the gag genes (internal structural proteins) lie in the 2.25 kb region of homology near the 5' ends of M-MSV and M-MLV RNAs, and that the beta S segment contains a sarcoma (src) gene.

89 citations


Journal ArticleDOI
TL;DR: The hormonal control of hepatic alpha(2u)-globulin synthesis by sex steroids and thyroid hormone occurs through modulation of the cellular level of alpha(3)H-labeled complementary DNA (cDNA) specific for the mRNA coding for alpha( 2u)- globulin, a male rat liver protein under multihormonal control.
Abstract: A procedure is presented for the preparation of a 3H-labeled complementary DNA (cDNA) specific for the mRNA coding for α2u-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 α2u-globulin, which binds to the nascent α2u-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% α2u-globulin mRNA. A labeled cDNA is made to this α2u-globulin-enriched mRNA preparation by using RNA-dependent DNA polymerase (reverse transcriptase). To remove the non-α2u-globulin sequences, this cDNA preparation is hybridized to an RNA concentration × incubation time (R0t) of 1000 mol of ribonucleotide per liter × sec with female rat liver mRNA, which, though it shares the vast majority of mRNA sequences with male liver, contains no α2u-globulin mRNA sequences. The cDNA remaining single-stranded is isolated by hydroxylapatite chromatography and is shown to be specific for α2u-globulin mRNA by several criteria. Good correlation was found in all endocrine states studied between the hepatic level of α2u-globulin, the level of functional α2u-globulin mRNA as assayed in a wheat germ cell-free translational system, and the level of α2u-globulin mRNA sequences as measured by hybridization to the α2u-globulin cDNA. Thus, the hormonal control of hepatic α2u-globulin synthesis by sex steroids and thyroid hormone occurs through modulation of the cellular level of α2u-globulin mRNA sequences, presumably by hormonal control of transcriptive synthesis.

Journal ArticleDOI
01 Sep 1977-Cell
TL;DR: Comparison of the nucleotide sequence of these fragments with that predicted from the amino acid sequence of HCS demonstrated that the fragments are transcripts of H CS mRNA, containing most of the translated and 3′ untranslated regions.

Journal ArticleDOI
TL;DR: Analysis of the kinetics of cDNA synthesis and the size of c DNA synthesized as a function of time under different conditions indicates that the mechanism of action of reverse transcriptase is partially distributive, which accounts for the necessity of a high enzyme concentration to obtain high yields of full length cDNA.
Abstract: Conditions have been determined under which reverse transcriptase catalyzes the synthesis of the high yields of full length complementary deoxyribonucleic acid (cDNA). These conditions depend not only on the cencentration of deoxynucleoside triphosphates (1) but also on the concentration of reverse transcriptase. An analysis of the kinetics of cDNA synthesis and the size of cDNA synthesized as a function of time under different conditions indicates that the mechanism of action of reverse transcriptase is partially distributive. This accounts for the necessity of a high enzyme concentration to obtain high yields of full length cDNA. Additional experiments indicate that the yield of cDNA is limited by the fact that the template mRNA is rapidly inactivated. This is most likely due to the fact that the product cDNA is hydrogen bonded to the template mRNA during synthesis.

Journal ArticleDOI
01 Dec 1977-Gene
TL;DR: It is demonstrated that sites present in the double-stranded ovalbumin DNA are preserved in the chimeric plasmid and allowed us to determine the orientation of the inserted ovalbum in DNA.

Journal ArticleDOI
01 Apr 1977-Cell
TL;DR: The known sequence at the 3′ end of rabbit 18S ribosomal RNA cannot base-pair extensively with the 5′ noncoding region of β-globin mRNA; however, it does form 6 base pairs around the initiation codon.

Journal ArticleDOI
TL;DR: The majority of G-negative or lightly staining bands were found to be preferential sites of hybridization in the human lymphocyte cell line Wil2.
Abstract: Total cytoplasmic polyadenylated RNA was isolated from the human lymphocyte cell line Wil2 by oligo(dT)-cellulose chromatography. Tritiated complementary DNA (cDNA) was transcribed from the RNA and used as a probe for in situ hybridization to metaphase chromosomes. The majority of G-negative or lightly staining bands were found to be preferential sites of hybridization.

Journal ArticleDOI
01 Oct 1977-Cell
TL;DR: Heterologous hybridization experiments suggest that there is significant sequence homology in the RNA sequences present in the prostate irrespective of the hormonal status of the animals, and appears to be the regulation of the abundance of specific RNA sequences.

Journal ArticleDOI
TL;DR: It is concluded that nucleated erythroid cells obtained from the spleens of anemic mice have a 15S RNA which contains globin messenger RNA sequences and is a good candidate for a globin mRNA precursor.
Abstract: Nucleated erythroid cells were incubated for 10 min in the presence of [5-3H]uridine, and the total RNA was isolated by three different extraction procedures. RNA containing globin messenger RNA sequences was purified from other cellular RNAs by selective hybridization to globin complementary DNA cellulose. Depending upon the extraction procedure employed, 0.4-0.6% of the radioactively-labeled total cellular RNA applied to the column annealed to globin complementary DNA cellulose. The annealed RNA was treated with formaldehyde and analyzed by formaldehyde/polyacrylamide gel electrophoresis. Mature globin mRNA and an RNA migrating at approximately 15 S were observed. No globin mRNA containing sequences larger than 20 S were present. The 15S RNA was partially resolved from mature globin mRNA by neutral sucrose density gradient centrifugation. The RNA isolated from the heavy region of this gradient migrated as 15 S in the formaldehyde/polyacrylamide gels and retained its ability to quantitatively anneal to globin complementary DNA cellulose. On the basis of these observations, we conclude that nucleated erythroid cells obtained from the spleens of anemic mice have a 15S RNA which contains globin mRNA sequences. The 15S RNA is not an aggregate and is a good candidate for a globin mRNA precursor.

Journal ArticleDOI
01 Aug 1977-Cell
TL;DR: Treatment of a nontumorigenic clone of AKR mouse embryo cells in culture with a variety of polycyclic aromatic hydrocarbons has resulted in the development of derivative clones which are highly tumorigenic and exhibit other characteristics of the transformed phenotype.

Journal ArticleDOI
TL;DR: The known 75-residue sequence adjacent to poly(A) in chicken ovalbumin messenger RNA was used as a model sequence to develop a generally applicable rapid gel sequencing method for RNA based on the “plus and minus” method of Sanger & Coulson (1975).

Journal ArticleDOI
TL;DR: In hybridizations using cDNA, which had been fractionated into sequences representing abundant and scarce polysomal mRNA molecules, it was found that although abundant cytoplasmic messenger sequences are also abundant in the nucleus, they exist in a significantly lower frequency range in the nuclear compartment.
Abstract: DNA complementary to polysomal poly(A)-containing mRNA (cDNA) of male rat liver was used to study the diversity of messenger sequences in the nucleus and in polysomes. 1. Hybridization of cDNA against an excess of its own polysomal mRNA template revealed that about 10000 different mRNA species are expressed in the liver tissue. They are distributed in a wide frequency range derived from approximately 0.5% of the total genome. 2. Hybridization of the cDNA against total nuclear RNA shows that messenger sequences comprise less than 1% of the mass of total nuclear RNA. Messenger sequences have a different frequency distribution in nucleus and cytoplasm. 3. In hybridizations using cDNA, which had been fractionated into sequences representing abundant and scarce polysomal mRNA molecules, it was found that although abundant cytoplasmic messenger sequences are also abundant in the nucleus, they exist in a significantly lower frequency range in the nuclear compartment.

Journal ArticleDOI
TL;DR: It is concluded that, in trout testis, the most abundant polyadenylated mRNAs are not preferentially transcribed from repetitive DNA, as it has shown to be the case in two eukaryotic cell lines.
Abstract: We have determined the fraction of polyadenylated cytoplasmic RNA from trout testis complementary to unique and repetitive DNA. Some 21% of the cDNA probe representative of this RNA population renatures with rapid kinetics, characteristics of repetitive sequences. The major proportion of the cDNA renatures with unique sequence DNA. Experiments with fractionated cDNA probes allow us to conclude that, in trout testis, the most abundant polyadenylated mRNAs are not preferentially transcribed from repetitive DNA, as it has shown to be the case in two eukaryotic cell lines. Treatment of trout testis nuclei with DNase I, under conditions in which 10% of the total DNA is digested, preferentially depletes the DNA of sequences being transcribed into polyadenylated mRNA. These data confirm the results of H. Weintraub and M. Groundine [(1976) Science 193, 848-856] and those of A. Garel and R. Axel [(1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3966-3970] and suggest that the conformation of DNA in the active genes of chromatin is such that it is more susceptible to digestion by DNaseI.

Journal ArticleDOI
01 Oct 1977-Cell
TL;DR: Homologous and heterologous hybridization with total and fractionated Friend cell cDNA probes revealed that all Friend cell polysomal poly(A) + RNA sequences are common to embryonal carcinoma cellpolysomes—apart from a small group of sequences drawn from the abundant class, corresponding to about 10% of Friendcell cDNA.

Journal ArticleDOI
01 Aug 1977-Cell
TL;DR: Rabbit and human α-globin complementary DNA was synthesized using the primer (dT 10 )dGdCdC hybridized to globin mRNA with reverse transcriptase, and sequenced using the plus and minus gel sequencing procedure (Sanger and Coulson, 1975; Brownlee and Cartwright, 1977).

Journal ArticleDOI
01 Jun 1977-Virology
TL;DR: The use of this DNA to analyze homologies among retrovirus genomes is of limited value because there is little or no complementarity between cDNA 5 , and the genomes of golden pheasant virus, Moloney murine leukemia virus, and Moloney Murine sarcoma virus.

Journal ArticleDOI
TL;DR: In a patient with homozygous betaO-thalassemia in whom studies of reticulocyte hemoglobin synthesis showed no beta-globin chain synthesis in vivo and in vitro, molecular hybridization studies revealed RNA sequences complementary to beta- globin cDNA.
Abstract: In a patient with homozygous betaO-thalassemia in whom studies of reticulocyte hemoglobin synthesis showed no beta-globin chain synthesis in vivo and in vitro, molecular hybridization studies revealed RNA sequences complementary to beta-globin cDNA The fact that these sequences were authentic beta-globin mRNA was shown by fingerprint analysis of T1 ribonuclease-digested mRNA and by sequencing of oligonucleotides unique to beta-globin mRNA The beta-mRNA that failed to direct beta-globin chain synthesis was not detectably shortened or degraded and contained poly(A) sequences

Journal ArticleDOI
TL;DR: The lack of hybridization of the cDNA probe to total rat liver RNA demonstrates that there is no pool of unprocessed message in the cell and indicates that the absence of AFP synthesis in normal liver is due to the lack of synthesis of stable mRN&v.

Journal ArticleDOI
TL;DR: Double-stranded ovalbumin DNA was amplified and purified by the cloning of bacterial transformants and used to transform the E. coli strain X1849, finding that the amount of inserted DNA in clones tested varied from 680 to 1090 base pairs.

Journal ArticleDOI
TL;DR: The results suggest acquisition of at least three types of type-C viral sequences in the human population, which are not endogenous elements but are acquired after fertilization.
Abstract: A [3H[cDNA probe synthesized from the RNA genome of Rauscher murine leukemia virus (MuLVR) and purified by hybridization to MuLVR70S RNA was hybridized to DNA from human normal and hemotopoietic neoplasia tissues. This cDNA hybridized completely to its homologous 70S RNA and was free of self-complementary sequences. Sequences complementary to MuLVR cDNA were found in DNA from tissues of some patients with leukemia (2 of 8), Hodgkin's disease (3 of 10), and one patient with multiple myeloma. DNA from spleen and kidney of a patient with nonneoplastic disease did not contain detectable MuLVR-related sequences. These virus-related sequences in the DNA from these neoplastic tissues were related but not identical to MuLVR sequences because differences of approximately 6 degrees in the midpoints of thermal elution profiles were found between the heterologous and homologous duplexes. These nucleotide sequences are not the same as the proviral sequences of baboon type-C virus previously found from some other patients with leukemia [Reitz et al. (1976) Proc. Natl. Acad. Sci. USA 73,2113-2117; Wong-Staal et al. (1976) Nature 262, 190-195], because there is no sequence homology between nucleic acids from MuLVR and baboon virus. The absence of these nucleic acid sequences in many tissues of patients with neoplasia and from the few tissues examined from people with nonneoplastic disease suggests that they are not endogenous elements but are acquired after fertilization. Taken together with the previous detection of baboon and woolly monkey type-C viral related components in some human tumors, the results suggest acquisition of at least three types of type-C viral sequences in the human population.

Journal ArticleDOI
TL;DR: Experiments were performed which demonstrated that the globin mRNA isolated by hybridization to cDNA-cellulose retained its biological activity when assayed in a wheat germ cell-free lysate.