Molecular cloning of ovomucoid gene sequences from partially purified ovomucoid messenger RNA

Stein, Jason; Catterall, James F.; Woo, Savio L. C.; Means, Anthony R.; O’Malley, Bert W.

doi:10.1021/bi00619a025

Cited by 49 publications

(39 citation statements)

References 54 publications

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“…The blunt-ended DNAs were ligated to BamHI linkers and cloned as BamHI/ EcoRI inserts in pBR322. Transformation and identification of the recombinant plasmids were carried out according to Stein et al (15). Exact endpoints of the deletion were determined by DNA sequence analysis by the procedure of Maxam 2) were used as templates.…”

Section: Ing False Hogness Boxes (T-a-t-a-a-a-a and T-a-t-a-t-a-t) Nomentioning

confidence: 99%

Specific 5′ flanking sequences are required for faithful initiation of in vitro transcription of the ovalbumin gene

Tsai

O’Malley

1981

Proc. Natl. Acad. Sci. U.S.A.

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] was adapted for studying initiation of transcription of the ovalbumin gene. The DNA template was a cloned ovalbumin gene fragment that contained 5' flanking sequences, the first structural sequence region, and a portion of the first intervening sequence. A HeLa cell crude extract was used as the source of RNA polymerase and initiation factors. Correct initiation was judged by the sizes of the transcription products generated from ovalbumin templates truncated at various positions before the 3' end of the gene. Transcription of the specific product was carried out by RNA polymerase II, as judged from a-amanitin sensitivity. A series of deletion mutants was constructed by trimming 5' flanking sequences of the ovalbumin DNA template by using exonuclease HI. The DNAs generated were then cloned in pBR322 and used as templates to determine which sequences were necessary for initiation of transcription. Specific initiation of the ovalbumin gene was unaffected by deletion of all but 61 nucleotides of the 5' flanking sequence but completely abolished by deletion of all but 26 nucleotides of 5' flanking sequence. Thus, a region between 61 and 26 nucleotides upstream from the cap site, which includes the Hogness box (T-A-T-A-T-A-T) at position 32-26, is essential for the correct initiation of the ovalbumin gene. Nevertheless, natural DNA fragments containing false Hogness boxes (T-A-T-A-A-A-A and T-A-T-A-T-A-T) not normally located in the immediate 5' flanking region of an authentic gene did not serve as promoters for initiation of transcription. These results suggest that the Hogness box is essential, but not sufficient, for specific initiation of RNA synthesis.The mechanisms that permit the transcription of eukaryotic genes are poorly understood. The development of DNA-dependent, cell-free, in vitro transcription systems would facilitate the elucidation of the steps involved in these processes. In 1978, Wu showed that accurate synthesis of a 5.5S RNA can be carried out by using a soluble extract from KB cells containing polymerase III and adenovirus 2 (Ad-2) DNA (1). Subsequently, specific transcription of Xenopus 5S RNA and yeast tRNA was reported in an oocyte cell-free system (2-4) and a KB cell reconstituted system (5). By the construction of 5' and 3' deletion mutants and an in vitro assay system, Bogenhagen et al. (6) (9) showed the selective initiation of transcription at a major late promoter of Ad-2 DNA by using crude extracts from KB cells supplemented with purified RNA polymerase II. Similarly, Manley et al. (10) showed the specific initiation of transcription at various late promoters of Ad-2 DNA by using a whole-cell axtract from HeLa cells. We have adapted the HeLa crude enzyme system of Manley et al (10) to study in vitro transcription of the ovalbumin gene in the hope of defining the specific features in the DNA sequences that are essential for accurate initiation. We have previously identified the transcription unit of the ovalbumin gene (11,12), the sequences of the entire ovalbumin natural gene, and t...

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Section: Ing False Hogness Boxes (T-a-t-a-a-a-a and T-a-t-a-t-a-t) Nomentioning

confidence: 99%

Specific 5′ flanking sequences are required for faithful initiation of in vitro transcription of the ovalbumin gene

Tsai

O’Malley

1981

Proc. Natl. Acad. Sci. U.S.A.

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“…(8). Tetr/Amps colonies were screened by colony filter hybridization mainly as described by GrUnstein-Hogness (9) with a 32P-cDNA probe to enriched mRNAIV [prepared by sucrose gradient centrifugation(5)].…”

Section: Introductionmentioning

confidence: 99%

Use of a cloned double stranded cDNA coding for a major androgen dependent protein in rat seminal vesicle secretion: the effect of testosterone in gene expression

1981

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Section: Methodsmentioning

confidence: 99%

“…Poly(A-RNA that had been enriched to about 8% prothrombin mRNA was used to synthesize a cDNA by using reverse transcriptase and oligo(dT) as primer (7). After base hydrolysis of the mRNA, double-stranded DNA was synthesized with reverse transcriptase and made blunt-ended with nuclease S1 (7). Poly(C) (approximately nine dCMP residues) was then added to the 3' ends of the double-stranded DNA by use of terminal deoxynucleotidyltransferase (7).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Cloning and analysis of a cDNA coding for bovine prothrombin.

MacGillivray¹,

Degen²,

Chandra³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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Poly(A)RNA enriched for prothrombin was isolated by specific immunoprecipitation ofbvine liver polysomes. Prothrombin consisted of about 8% of the cell-free translation products of this RNA. A double-stranded cDNA was synthesized by using reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) and made blunt-ended with nuclease SI. After tailing with dCTP and terminal transferase, the double-stranded cDNA was annealed to pBR322 DNA that had been cleaved previously at the single Pst I site and similarly tailed with dGTP. The resulting plasmids were used to transform Escherichia coli strain RRI under P3-EKI conditions. Sixtythree tetracycline-resistant clones were obtained that hybridized to nP-labeled cDNA synthesized from prothrombin-enriched mRNA. Recombinants containing cDNA to prothrombin mRNA sequences were screened by a solution hybridization assay with a[cDNA synthesized from mRNA. This enriched mRNA was 50% prothrombin mRNA, as determined by a reticulocyte lysate translation assay. Three positive clones were identified by this assay; they contained bovine DNA inserts of 700,500, and 400 base pairs. The DNA sequence of the 700-base-pair insert was then determined. This recombinant plasmid contained DNA coding for the carboxyl-terminal 160 residues of bovine prothrombin followed by a noncoding region of 119 base pairs and a poly(A) tail of 60 base pairs.The coagulation of blood is the result of a series of stepwise reactions in which many of the coagulation proteins in plasma are converted from zymogens to highly specific serine proteases (see ref. 1 for a recent review). In the final stages of the blood coagulation cascade, prothrombin is converted to thrombin by Factor Xa in the presence of phospholipid, calcium ions, and Factor Va. Thrombin then cleaves fibrinogen to form an insoluble fibrin clot. Although prothrombin is one of the most abundant coagulation proteins present in plasma, its mRNA constitutes only about 1% of the total poly(A)-RNA in bovine liver (2). This level of RNA makes it difficult to purify prothrombin mRNA to the degree of homogeneity required for many biochemical investigations. Recent developments in recombinant DNA techniques, however, permit the isolation of large amounts of homogeneous DNA corresponding to the specific mRNAs. Such cloned DNAs can be used for many different studies, such as the quantitation of cellular mRNA levels in response to various stimuli, the purification of specific mRNA by hybridization to immobilized DNA, and as a hybridization probe to isolate and study genomic DNA.In this paper, we describe the construction, cloning in Escherichia colh, and sequence of a recombinant plasmid containing DNA coding for the carboxyl-terminal portion of bovine prothrombin. Enrichment of mRNA. Polysomes were prepared from bovine liver as described (2). Polysomes synthesizing prothrombin were enriched by specific immunoprecipitation with antiprothrombin prepared in rabbits and either goat anti-rabbit immunoglobulin coupled to p-aminobenzyl-cellulose (3) or ...

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Molecular cloning of ovomucoid gene sequences from partially purified ovomucoid messenger RNA

Cited by 49 publications

References 54 publications

Specific 5′ flanking sequences are required for faithful initiation of in vitro transcription of the ovalbumin gene

Specific 5′ flanking sequences are required for faithful initiation of in vitro transcription of the ovalbumin gene

Use of a cloned double stranded cDNA coding for a major androgen dependent protein in rat seminal vesicle secretion: the effect of testosterone in gene expression

Cloning and analysis of a cDNA coding for bovine prothrombin.

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