In vitro transcription of cloned 5S RNA genes of the newt Notophthalmus

Recombinant plasmids that carried genes coding for 5S ribosomal RNA of the newt, Notophthalmus viridescens, were transcribed in vitro with extracts of Xenopus laevis oocyte nuclei. Plasmids containing multiple repeats of the 5S gene and spacer directed accurate transcription of 5S RNA (120 bases). Individual repeat units were recloned by inserting Sau 3A restriction fragments into the Bam HI site of plasmid pBR322. Because each repeat was cut by the enzyme within the coding region, the inserts had incomplete coding regions at their ends and spacer sequences in the middle. The DNA of these subclones directed synthesis of a 5S-size RNA that contained both plasmid and 5S RNA sequences. Transcription initiated in the vector, proceeded through the gene segment coding for nucleotides 41-120, and terminated at the end of the gene. The initiation of in vitro transcription required neither the original 5' flanking sequences of the spacer nor the first third of the gene. We conclude that intragenic DNA sequences control the initiation of transcription. Other subclones that include pseudogenes gave rise to some transcripts 156 nucleotides long. These long transcripts represented continuation of transcription through the 36-base-pair pseudogene that is located immediately downstream from the 5S gene. However, most transcripts of these subclones terminated at the end of the normal gene before the beginning of the pseudogene. It is probable that a run of four or more Ts serves as part of the termination signal.

[1]  D. Fowlkes,et al.  Transcriptional control regions of the adenovirus VAI RNA gene , 1980, Cell.

[2]  D. Söll,et al.  Two control regions for eukaryotic tRNA gene transcription. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[3]  I. Tinoco,et al.  DNA-RNA hybrid duplexes containing oligo(dA:rU) sequences are exceptionally unstable and may facilitate termination of transcription. , 1980, Nucleic acids research.

[4]  D. Engelke,et al.  Specific interaction of a purified transcription factor with an internal control region of 5S RNA genes , 1980, Cell.

[5]  D. Bogenhagen,et al.  A control region in the center of the 5S RNA gene directs specific initiation of transcription: I. The 5′ border of the region , 1980, Cell.

[6]  D. Bogenhagen,et al.  A control region in the center of the 5S RNA gene directs specific initiation of transcription: II. The 3′ border of the region , 1980, Cell.

[7]  G. Wu.  Faithful transcription of adenovirus 5.5 S RNA gene by RNA polymerase III in a human KB cell-free extract. , 1980, The Journal of biological chemistry.

[8]  R. Grosschedl,et al.  A tRNA gene of Xenopus laevis contains at least two sites promoting transcription. , 1979, Nucleic acids research.

[9]  O. Hagenbüchle,et al.  The primary transcription product of a silkworm alanine tRNA gene: Identification of in vitro sites of initiation, termination and processing , 1979, Cell.

[10]  N. Jones,et al.  A mutation which alters initiation of transcription by RNA polymerase III on the Ad5 chromosome , 1979, Cell.

[11]  R. Garber,et al.  Transcription of a cloned bombyx mori trna2 Ala gene: Nucleotide sequence of the tRNA precursor and its processing in vitro , 1979, Cell.

[12]  L. J. Korn,et al.  Transcription initiation of Xenopus 5S ribosomal RNA genes in vitro. , 1979, Nucleic acids research.

[13]  D. Söll,et al.  The nucleotide sequence of a cloned Drosophila arginine tRNA gene and its in vitro transcription in Xenopus germinal vesicle extracts. , 1979, The Journal of biological chemistry.

[14]  R. Reeder,et al.  The nucleotide sequence of the initiation and termination sites for ribosomal rna transcription in x. laevis , 1979, Cell.

[15]  R. Roeder,et al.  Faithful transcription of eukaryotic genes by RNA polymerase III in systems reconstituted with purified DNA templates. , 1979, The Journal of biological chemistry.

[16]  H. Kang,et al.  In vitro transcription and processing of a yeast tRNA gene containing an intervening sequence , 1979, Cell.

[17]  R. Koski,et al.  Delimitation of a promoter for RNA polymerase III by means of a functional test. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[18]  H. Hamada,et al.  In vitro synthesis of a 5S RNA precursor by isolated nuclei of rat liver and HeLa cells , 1979, Cell.

[19]  L. J. Korn,et al.  Nucleotide sequence of xenopus borealis oocyte 5S DNA: Comparison of sequences that flank several related eucaryotic genes , 1978, Cell.

[20]  E. Birkenmeier,et al.  A nuclear extract of xenopus laevis oocytes that accurately transcribes 5S RNA genes , 1978, Cell.

[21]  J. Mao,et al.  Specific transcription of eukaryotic tRNA genes in Xenopus germinal vesicle extracts. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[22]  R. Levis.  Kinetic analysis of 5 S RNA formation in Drosophila cells. , 1978, Journal of molecular biology.

[23]  J. Sutcliffe,et al.  pBR322 restriction map derived from the DNA sequence: accurate DNA size markers up to 4361 nucleotide pairs long. , 1978, Nucleic acids research.

[24]  J. Gurdon,et al.  Cloned single repeating units of 5S DNA direct accurate transcription of 5S RNA when injected into Xenopus oocytes. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[25]  G. Wu.  Adenovirus DNA-directed transcription of 5.5S RNA in vitro. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[26]  B. Jacq,et al.  Structure and processing of precursor 5 S RNA in Drosophila melanogaster. , 1977, Journal of molecular biology.

[27]  S. Falkow,et al.  Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. , 1977, Gene.

[28]  G. Rubin,et al.  Effect of heat shock on the synthesis of low molecular weight RNAs in drosophila: Accumulation of a novel form of 5S RNA , 1975, Cell.

[29]  T. Maniatis,et al.  Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis. , 1975, Biochemistry.

[30]  D. Hogness,et al.  A system for mapping DNA sequences in the chromosomes of Drosophila melanogaster. , 1974, Cell.

[31]  R. Roeder,et al.  Role of DNA-dependent RNA polymerase 3 in the transcription of the tRNA and 5S RNA genes. , 1974, Proceedings of the National Academy of Sciences of the United States of America.

[32]  M. Wegnez,et al.  Sequence heterogeneity of 5 S RNA in Xenopus laevis , 1972, FEBS letters.

[33]  Fritz Eckstein,et al.  Thiophosphate analogs of nucleoside di- and triphosphates , 1971 .

[34]  D. Helinski,et al.  Properties of a supercoiled deoxyribonucleic acid-protein relaxation complex and strand specificity of the relaxation event. , 1970, Biochemistry.

[35]  S. Osawa,et al.  Evolutionary change in 5S RNA secondary structure and a phylogenic tree of 54 5S RNA species. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[36]  C. S. Parker,et al.  Transcription of cloned Xenopus 5S RNA genes by X. laevis RNA polymerase III in reconstituted systems. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[37]  D Court,et al.  Regulatory sequences involved in the promotion and termination of RNA transcription. , 1979, Annual review of genetics.

[38]  J. Sutcliffe.  Complete nucleotide sequence of the Escherichia coli plasmid pBR322. , 1979, Cold Spring Harbor symposia on quantitative biology.