We have developed a procedure for preparing extracts from nuclei of human tissue culture cells that directs accurate transcription initiation in vitro from class II promoters. Conditions of extraction and assay have been optimized for maximum activity using the major late promoter of adenovirus 2. The extract also directs accurate transcription initiation from other adenovirus promoters and cellular promoters. The extract also directs accurate transcription initiation from class III promoters (tRNA and Ad 2 VA).

/pdf/accurate-transcription-initiation-by-rna-polymerase-ii-in-a-3j9dln38ha.pdf

Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei

https://www.cell.com/cell/pdf/0092-8674(83)90210-6.pdf

The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter

To investigate the sequences necessary for proper initiation of transcription of SV40 early genes, we have constructed several deletion mutants in the promoter region. The TATA box region is apparently involved in fixing initiation precisely within a narrow area, but is dispensable for gene expression, while the sequences located more than 150 base pairs upstream are indispensable.

In vivo sequence requirements of the SV40 early promoter region

The structure and evolution of the human β-globin gene family

Argiris Efstratiadis Department of Biological Chemistry Harvard Medical School Boston, Massachusetts 02115 James W. Posakony, Tom Maniatis, Richard M. Lawn* and Catherine O’Connell+ Division of Biology California Institute of Technology Pasadena, California 91125 Richard A. Spritz, Jon K. DeRiel,# Bernard G. Forget and Sherman M. Weissman Departments of Genetics and Internal Medicine Yale University School of Medicine New Haven, Connecticut 06510 Jerry L. Slightom, Ann E. Blechl and Oliver Smithies Laboratory of Genetics University of Wisconsin Madison, Wisconsin 53706 Francisco E. Baralle, Carol C. Shoulders and Nicholas J. ProudfootQ MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 2QH, England Summary We present the results of a detailed comparison of the primary structure of human p-like globin genes and their flanking sequences. Among the se- quences located 5’ to these genes are two highly conserved regions which include the sequences ATA and CCAAT located 31 2 1 and 77 + 10 bp, respectively, 5’ to the mRNA capping site. Similar sequences are found in the corresponding locations in most other eucaryotic structural genes. Calcula- tion of the divergence times of individual @like globin gene pairs provides the first description of the evolutionary relationships within a gene family based entirely on direct nucleotide sequence com- parisons. In addition, the evolutionary relationship of the embryonic e-globin gene to the other human P-like globin genes is defined for the first time. Finally, we describe a model for the involvement of short direct repeat sequences in the generation of deletions in the noncoding and coding regions of B-like globin genes during evolution.

https://www.cell.com/cell/pdf/0092-8674(80)90429-8.pdf

The Structure and Evolution of the Human /?-Globin Gene Family

Specific initiation of transcription of cloned conalbumin and ovalbumin genes has been obtained in a cell-free system in the presence of purified calf thymus RNA polymerase B. Comparison with the adenovirus-2 early (E1 A) and major late genes reveals marked variations in transcriptional efficiencies. Specific fragmentation of the conalbumin gene DNA indicates that a short DNA segment 5′ to the initiation site is involved in specific transcription.

Specific in vitro initiation of transcription on conalbumin and ovalbumin genes and comparison with adenovirus-2 early and late genes

HeLa cell nuclei, isolated 17 h after infection with human adenovirus type 2 (Ad2), were treated with 200 mM ammonium sulfate The extract (S200 fraction) contained 50 to 70% of the nonintegrated Ad2 DNA, which was in the form of nucleoprotein complexes These complexes contained native, intact Ad2 DNA (with the exception of replicative intermediates) and could be partially purified and resolved by velocity gradient centrifugation Using high-salt (200 mM ammonium sulfate) incubation conditions, more than 95% of the nuclear RNA polymerase activity belonged to class B About 45% of the class B enzyme molecules bound to DNA in the nuclei (those "engaged" in RNA synthesis) were released from the nuclei in the form of Ad2 transcriptional complexes by treatment with 200 mM ammonium sulfate At least 90% of the RNA synthesized in high salt in the nuclei or in the S200 fraction was Ad2 specific, and essentially all of this RNA was complementary to the l strand of Ad2 DNA These findings are compatible with what is known about Ad2-specific RNA synthesis in vivo The analysis of the RNA synthesized from partially purified transcriptional complexes supports the contention that its transcription is almost entirely asymmetric, and that the asymmetry observed in vivo is not a consequence of the rapid degradation of h-strand transcripts The RNA synthesized in vitro in the absence of detectable RNase activity sedimented with a maximum size of 35 to 40S Less than 5% of the nuclear or the S200 fraction RNA polymerase activity was class C when assayed under non-reinitiating conditions Although much of the RNA synthesized by the class C enzyme was Ad2 specific, 55S virus-associated RNA was not the predominant product The isolation of Ad2 DNA transcriptional complexes provides an attractive system for further characterizing the Ad2 DNA template used for transcription and for studying the regulation of the expression of the Ad2 genome during the productive infection cycle

Characterization of adenovirus type 2 transcriptional complexes isolated from infected HeLa cell nuclei.

Deoxyribonucleoprotein complexes released 17 h postinfection from adenovirus type 1 (Ad2)-infected HeLa cell nuclei were shown by electron microscopy to contain filaments much thicker (about 200 A [20 nm]) than double-stranded DNA (about 20 A [2 nm]). The complexes were partially purified through a linear sucrose gradient, concentrated, and further purified in a metrizamide gradient. The major protein present in the complexes was identified as the 72,000-dalton (72K), adenovirus-coded single-stranded DNA-binding protein (72K DBP). Three types of complexes have been visualized by electron microscopy. Some linear complexes were uniformly thick, and their length corresponded roughly to that of the adenovirus genome. Other linear genome-length complexes appeared to consist of a thick filament connected to a thinner filament with the diameter of double-stranded DNA. Forked complexes consisting of one thick filament connected to a genome-length, thinner double-stranded DNA filament were also visualized. Both thick and thin filaments were sensitive to DNase and not to RNase, but only the thick filaments were digested by the single-strand-specific Neurospora crassa nuclease, indicating that they correspond to a complex of 72K DBP and Ad2 single-stranded DNA. Experiments with anti-72K DBP immunoglobulins indicated that these nucleoprotein complexes, containing the 72K DBP, correspond to replicative intermediates. Both strands of the Ad2 genome were found associated to the 72K DBP. Altogether, our results establish the in vivo association of the 72K DBP with adenovirus single-stranded DNA, as previously suggested from in vitro studies, and support a strand displacement mechanism for Ad2 DNA replication, in which both strands can be displaced. In addition, our results indicate that, late in infection, histones are not bound to adenovirus DNA in the form of a nucleosomal chromatine-like structure.

Structural analysis of viral replicative intermediates isolated from adenovirus type 2-infected HeLa cell nuclei.

When HeLa cell nuclei, isolated 17 h after infection with adenovirus type 2 (Ad2), were extracted with 200 mM ammonium sulfate, Ad2 nucleoprotein complexes were selectively released These complexes contained a DNA polymerase activity that corresponded to DNA polymerase molecules actively engaged in Ad2 DNA replication Under our high-salt (200 mM ammonium sulfate) incubation conditions, where no reinitiation occurred, full-length Ad2 DNA chains were synthesized by elongation of chains that had been initiated in vivo This conclusion was further supported by density labeling experiments indicating that the in vitro DNA synthesis was semiconservative Evidence is presented suggesting that at least part of the DNA polymerase molecules engaged in Ad2 DNA replication belong to the gamma class

Enzymatic properties of viral replication complexes isolated from adenovirus type 2-infected HeLa cell nuclei.

The relationship between adenovirus replication and late transcription has been investigated using viral replication and transcription complexes isolated from infected HeLa cell nuclei. These two types of complexes extracted from adenovirus type 2-infected cell nuclei did not sediment at the same rate on sucrose gradients. Viral replicative intermediates were quantitatively precipitated by immunoglobulins raised against purified 72,000-dalton DNA-binding protein, whereas viral transcription complexes remained in the supernatant. These results show that late transcription does not occur on active replication complexes or on 72,000-dalton DNA-binding protein-containing replicative intermediates inactive in DNA synthesis. Additional evidence is presented indicating that it is very unlikely that replicative intermediates lacking the 72,000-dalton DNA-binding protein could be the template for late transcription.

O Brison

Papers

Specific in vitro initiation of transcription on conalbumin and ovalbumin genes and comparison with adenovirus-2 early and late genes

Characterization of adenovirus type 2 transcriptional complexes isolated from infected HeLa cell nuclei.

Structural analysis of viral replicative intermediates isolated from adenovirus type 2-infected HeLa cell nuclei.

Enzymatic properties of viral replication complexes isolated from adenovirus type 2-infected HeLa cell nuclei.

Adenovirus DNA template for late transcription is not a replicative intermediate.