Structure and in Vitro Transcription of Human Globin Genes

Proudfoot, Nick J.; Shander, Monica H. M.; Manley, J L; Gefter, Malcolm L.; Maniatis, Tom

doi:10.1126/science.6158093

Cited by 226 publications

(76 citation statements)

References 82 publications

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“…The DNA was gel-purified before experimental use. For mapping, the primer extension technique originally described by Proudfoot et al (1980) was modified. Synthetic DNA was labelled at the 5' end with [),-3zp]dATP and T4 polynucleotide kinase according to standard procedures.…”

Section: Methodsmentioning

confidence: 99%

Abundant 5 kb RNA of Human Cytomegalovirus without a Major Translational Reading Frame

Plachter

Traupe²,

Albrecht³

et al. 1988

Journal of General Virology

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SUMMARYAlthough all herpesviruses are similar in their temporal regulation of gene expression, the organization of the immediate early (IE) genes varies markedly between the different members of the group. Most of the IE transcripts of human cytomegalovirus originate from a restricted region within the long unique segment of its linear dsDNA genome of 235 kb. One of the predominant transcripts from the IE region is a 5 kb RNA. Northern blot analyses revealed that this class of RNA is continuously present in infected cells. It was detected at high levels in IE and late RNA preparations, and in low amounts in early RNA preparations. It was not confined to the poly(A) + fraction upon oligo(dT) selection, but also appeared in similar amounts in poly(A)-fractions. Fine mapping of this transcript was done by nuclease protection and primer extension. The RNA appeared to be unspliced, and no signals such as TATA or CCAAT, known to be important elements in eukaryotic RNA polymerase II promoters, were found close to the 5' end. Sequence analysis revealed multiple stop codons throughout the AT-rich potential coding region. Since no splicing was found to occur, the largest protein deduced from the DNA sequence would be of not more than 12000 Mr. However, a computer program designed to detect protein-coding DNA sequences by codon usage did not reveal significant evidence for a protein encoded in this region. Therefore this RNA is likely to represent an unprecedented case of a large non-coding transcript present in cells that are lytically infected by an animal virus.

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Section: Methodsmentioning

confidence: 99%

Abundant 5 kb RNA of Human Cytomegalovirus without a Major Translational Reading Frame

Plachter

Traupe²,

Albrecht³

et al. 1988

Journal of General Virology

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“…The 5' termini of specific f u r transcripts were determined by primer extension analysis [19,20]. RNA was isolated from growing E. coli cells as described [21].…”

Section: Analysis Of In Vivo Transcriptional Initiation Sitesmentioning

confidence: 99%

Fur (ferric uptake regulation) protein and CAP (catabolite‐activator protein) modulate transcription of fur gene in Escherichia coli

Lorenzo

Herrero

Giovannini

et al. 1988

European Journal of Biochemistry

194

166

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A fusion between the fur (ferric uptake regulation) gene, known to mediate negative regulation of iron absorption in Escherichia coli, and lacZ was constructed in vitro. P-Galactosidase levels of cells harboring this fusion were under the control of sequences contained in a 185-bp DNA fragment located upstream of the fur structural gene. The fusion was prepared in multicopy (pVLN102 plasmid) and low-copy-number states, the latter constructed as a I phage lysogen carrying a fur'-'lacZ insert. DNase I footprinting experiments with purified Fur protein, performed on a 250-bp restriction fragment carrying the promoter region of the fusion, showed the presence of a single Fur-protected site overlapping the -10 region of a potential promoter sequence. Examination of the DNA sequences located upstream of the fur gene revealed a possible binding site for the catabolite-activator protein (CAP). P-Galactosidase synthesis of E. coli cells harboring the fusion were measured in fur, crp and cya genetic backgrounds and compared with the corresponding levels in wild-type strains. The data obtained indicate a moderate autoregulation of fur expression by its gene product and also a significant stimulation by the CAMP-CAP system. Transcription start sites were mapped by primer-extension experiments with total RNA obtained in vivo from cells harboring pVLN102. The results show that transcription of thefur gene is initiated from at least two different sites separated by 6 bp, which appear to originate from two overlapping promoters sensitive to catabolic activation.

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“…However, extensive evidence against phosphorylation of DRB beyond the monophosphate level in vivo has been presented (13)(14)(15). Because isolated nuclei are not very efficient in in vitro initiation of transcription (16,17), the development of new cell-free transcription systems (18,19) (20,21) The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.…”

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confidence: 99%

“…The RNAs synthesized in vitro are analyzed on polyacrylamide/urea gels as sizespecific run-offproducts from DNA templates cut with restriction enzymes. With a whole-cell extract as a source of RNA polymerase II and factors and human e-globin DNA cut with BamHI (20, 21) as template, RNA initiated at the cap site produces a run-off product 460 nucleotides long (20,21) (Fig. 2, lane 1).…”

mentioning

confidence: 99%

Mechanism of action of dichloro-beta-D-ribofuranosylbenzimidazole: effect on in vitro transcription.

Zandomeni¹,

Mittleman²,

Bunick³

et al. 1982

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

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The adenosine analog 5,6-dichloro-1-3-D-ribofuranosylbenzimidazole (DRB) and its mono-and triphosphate derivatives inhibit RNA polymerase II-specific transcription in an extract ofwhole HeLa cells. The analog does not inhibit RNA polymerase m-specific adenovirus VA RNA transcription in the whole cell extract. With purified RNA polymerase II under nonspecific transcription conditions, no effect ofDRB could be detected. DRB is equally effective in inhibiting in vitro transcription from several of the adenovirus promoters and the human e-globin gene. The inhibitory effects are in the order DRB > DRB monophosphate > DRB triphosphate. Thus DRB acts in vitro presumably on systems in which specific RNA polymerase H initiation of transcription occurs and with no detectable effect on premature termination. This will provide a suitable model for study of the molecular mechanism of action of DRB on transcription.The nucleotide analog 5,6-dichloro-1-,B3D-ribofuranosylbenzimidazole (DRB) is a widely used inhibitor of mRNA synthesis in eukaryotic cells (for review, see ref. 1). The action of DRB is selective for RNA polymerase II transcripts; RNA polymerase III 5S and tRNA-specific transcripts and the rates of RNA polymerase I-mediated rRNA synthesis are all not affected (1). Initial reports (2, 3) indicated that DRB acted at or very close to the site of initiation of transcription, on the basis of incorporation of [3H]uridine into specific-size mRNAs of Balbiani rings in Chironomus (2) and of the labeling kinetics of HeLa cell mRNA precursors (3). The effect of adding DRB to HeLa cells is very rapid (<5 min). Even in the presence of DRB, however, 30% of the HeLa cell large nuclear RNA can be labeled (3-5), but the role of this RNA, which is not transported to the cytoplasm, remains unclear. By contrast, short, capped RNAs transcribed by RNA polymerase II continue to be made in DRBtreated HeLa cells (4, 6).Synthesis of specific low molecular weight DRB-resistant RNAs was also described for the globin gene of Friend erythroleukemia cells (7), adenovirus-specific RNA in infected HeLa cells (8, 9), and simian virus 40-specific RNA in infected monkey cells (10). In the case of the adenovirus major late promoter RNA, these short RNA species are initiated at the same site as the viral mRNA precursor transcripts of normal length and are capped (11). Some of these low molecular weight RNAs therefore could represent prematurely terminated mRNA precursors.DRB has also been shown to inhibit the methylation of 5'-end caps of mRNAs larger than 18 S without affecting the methylation of shorter mRNA-like molecules (5). Transcription of the nuclear low molecular weight RNAs with trimethylated caps shows DRB sensitivity identical to that of the large mRNA precursor RNAs (5). Thus, transcription of short or prematurely terminated capped mRNA-like molecules seems to be resistant to DRB. It has been suggested (6) that DRB acts to enhance premature termination, but from the evidence presented it is difficult to establish whether this phen...

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Structure and in Vitro Transcription of Human Globin Genes

Cited by 226 publications

References 82 publications

Abundant 5 kb RNA of Human Cytomegalovirus without a Major Translational Reading Frame

Abundant 5 kb RNA of Human Cytomegalovirus without a Major Translational Reading Frame

Fur (ferric uptake regulation) protein and CAP (catabolite‐activator protein) modulate transcription of fur gene in Escherichia coli

Mechanism of action of dichloro-beta-D-ribofuranosylbenzimidazole: effect on in vitro transcription.

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