Biomedical subjects

R W Carthew

Publications and source records attributed to R W Carthew.

27 records · Page 2Linked to original sources

PubMed1991

Evolution of gene position: chromosomal arrangement and sequence comparison of the Drosophila melanogaster and Drosophila virilis sina and Rh4 genes.

The seven in absentia (sina) gene of Drosophila encodes a nuclear protein required for normal eye development. In Drosophila melanogaster, the sina gene is located within an intron of the Rh4 opsin gene. We examine here the nucleotide sequences and chromosomal arrangements of these genes in Drosophila virilis. An interspecies comparison between D. melanogaster and D. virilis reveals that the protein-coding sequences of the sina and Rh4 genes are highly conserved, but the relative chromosomal position and structural arrangement of these genes differ between the two species. In particular, the sina and Rh4 genes are widely separated in D. virilis, and there is no intron in the Rh4 gene. Our results suggest that the Rh4 gene was translocated to another chromosomal location by a retrotransposition event.

Animals↗

PubMed1990

seven in absentia, a gene required for specification of R7 cell fate in the Drosophila eye.

Signals from directly adjacent cells are critical for the development of the R7 photoreceptor cell in the Drosophila eye. We have identified a gene, seven in absentia (sina), for which loss of function results in the R7 precursor cell adopting the fate of a nonneuronal cell type. Function of the sina gene is required only in R7 for correct R7 cell development. The sina transcription unit is located within the intron of a gene that encodes an R7 cell-specific opsin. The sina protein, which has a potential metal binding domain, is localized in the nuclei of several ommatidial precursor cells including R7, and sina expression in R7 appears before R7 overtly begins to differentiate. These data indicate that the sina gene product is necessary at a stage in the determination of R7 cell fate when R7 receives and interprets developmental signals from neighboring cells, and possibly acts by regulating gene expression.

Amino Acid Sequence↗

PubMed1988

Formation of transcription preinitiation complexes with an amanitin-resistant RNA polymerase II.

Accurate transcription by RNA polymerase II has been shown to require multiple factors which participate in a number of intermediate steps prior to initiation. The last detectable step before initiation is the formation of an activated or rapid start complex which is template-associated. In this study we used two phenotypically distinguishable forms of mammalian RNA polymerase II to examine the requirement for specific factors in the formation of the activated complex. RNA polymerase II was purified from a mutant cell line which was resistant to levels of alpha-amanitin that are toxic for normal cells. When added to a polymerase-dependent transcription system consisting of HeLa factors and adenovirus DNA, the mutant polymerase accurately transcribed the template in an amanitin-resistant fashion. This amanitin-resistant transcription was competitively inhibited when wild-type polymerase was also added to the system. Preincubation of amanitin-resistant polymerase with DNA and factors produced a polymerase-associated complex, defined by its resistance to exchange for the amanitin-sensitive polymerase. Complex formation required the presence of polymerase, DNA, and the HeLa factors during the preincubation but did not require the presence of nucleotides. Complexes were template-associated, as shown by their inability to exchange onto a second template. Thus, prior to initiation, RNA polymerase II forms a stable association with the DNA template in an activated complex.

Amanitins↗

PubMed1988

Human CCAAT-binding proteins have heterologous subunits.

We have characterized three distinct proteins present in HeLa cell extracts that specifically recognize different subsets of transcriptional elements containing the pentanucleotide sequence CCAAT. One of these CCAAT-binding proteins, CP1, binds with high affinity to CCAAT elements present in the human alpha-globin promoter and the adenovirus major late promoter (MLP). A second protein, CP2, binds with high affinity to a CCAAT element present in the rat gamma-fibrinogen promoter. Finally, the third CCAAT-binding protein is nuclear factor I (NF-I), a cellular DNA-binding protein that binds to the adenovirus origin of replication and is required for the initiation of adenoviral replication. CP1, CP2, and NF-I are distinct activities in that each binds to its own recognition site with an affinity that is at least three orders of magnitude higher than that with which it binds to the recognition sites of the other two proteins. Surprisingly, CP1, CP2, and NF-I each appear to recognize their binding site with highest affinity as a multisubunit complex composed of heterologous subunits. In the case of CP1, two different types of subunits form a stable complex in the absence of a DNA-binding site. Moreover, both subunits are present in the CP1-DNA complex. We thus propose the existence of a family of related multisubunit CCAAT-binding proteins that are composed of heterologous subunits.

Animals↗

PubMed1987

The adenovirus major late transcription factor activates the rat gamma-fibrinogen promoter.

The major late transcription factor (MLTF) is a 46-kilodalton polypeptide that specifically binds to and activates transcription from the major late promoter of adenovirus. The presence of this promoter-specific transcription factor in uninfected HeLa cell extracts suggests that MLTF is also involved in the transcription of cellular genes. This report demonstrates that MLTF specifically stimulates transcription of the rat gamma-fibrinogen gene through a high-affinity binding site. Stimulation of transcription by MLTF was not dependent on the exact position of the MLTF binding site with respect either to the transcription initiation site or to adjacent promoter elements. These results suggest that one of the cellular functions of MLTF is to control gamma-fibrinogen gene expression.

Adenoviruses, Human↗

PubMed1987

The major late transcription factor binds to and activates the mouse metallothionein I promoter.

Human (HeLa) cells contain a protein, MLTF, which specifically binds to a DNA sequence in the adenovirus 2 major late promoter and activates transcription of that promoter. The presence of MLTF in uninfected cells suggests that this factor contributes to the transcription of some cellular genes. We find that MLTF binds in a sequence-specific manner to the 5'-flanking region of the mouse metallothionein I (mMTI) gene. Binding was localized between -101 and -94 (relative to the initiation site at +1) by DNA-binding gel electrophoresis assay and DNA methylation interference analysis. As in adenovirus, binding occurred in a region containing the sequence CPuCGTGAC. Deletion of this sequence both eliminated the binding of MLTF and produced a fourfold reduction in transcriptional efficiency in vitro. In contrast to the intact promoter, transcription from the deletion mutant promoter was not stimulated by addition of purified MLTF to an in vitro reconstituted reaction. These results suggest that MLTF contributes to the transcription of cellular genes.

Adenoviruses, Human↗

PubMed1986

A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor.

A simple approach has been developed for the unambiguous identification and purification of sequence-specific DNA-binding proteins solely on the basis of their ability to bind selectively to their target sequences. Four independent methods were used to identify the promoter-specific RNA polymerase II transcription factor MLTF as a 46-kilodalton (kDa) polypeptide. First, a 46-kDa protein was specifically cross-linked by UV irradiation to a body-labeled DNA fragment containing the MLTF binding site. Second, MLTF sedimented through glycerol gradients at a rate corresponding to a protein of native molecular weight 45,000 to 50,000. Third, a 46-kDa protein was specifically retained on a biotin-streptavidin matrix only when the DNA fragment coupled to the matrix contained the MLTF binding site. Finally, proteins from the most highly purified fraction which were eluted and renatured from the 44- to 48-kDa region of a sodium dodecyl sulfate-polyacrylamide gel exhibited both binding and transcription-stimulatory activities. The DNA-binding activity was purified 100,000-fold by chromatography through three conventional columns plus a DNA affinity column. Purified MLTF was characterized with respect to the kinetic and thermodynamic properties of DNA binding. These parameters indicate a high degree of occupancy of MLTF binding sites in vivo.

Adenoviruses, Human↗

PubMed1985

Isolation of three proteins that bind to mammalian RNA polymerase II.

We have used affinity chromatography on columns containing immobilized calf thymus RNA polymerase II to isolate three phosphoproteins (RAP72, RAP38, and RAP30) that bind directly to RNA polymerase II. All could be isolated from cell nuclei, and all three could be detected in mouse and human tissue culture cell lines, but only RAP38 and RAP30 have so far been isolated from calf thymus. RAP38 stimulates nonspecific transcription of native DNA templates by RNA polymerase II in the presence of Mn2+; it appears to be similar or identical to SII, a previously identified RNA polymerase II stimulatory factor (Nakanishi, Y., Mitsuhashi, Y., Sekimizu, K., Yokoi, H., Tanaka, Y., Horikoshi, M., and Natori, S. (1981) FEBS Lett. 130, 69-72). Unlike RAP38, RAP72 and RAP30 do not affect nonspecific transcription by RNA polymerase II. However, RAP30 may have a role in regulating some alterations of transcription that accompany cellular differentiation; RAP30 is partially dephosphorylated when murine erythroleukemia cells are induced with dimethyl sulfoxide to undergo terminal erythroid differentiation. We suggest that phosphate groups in RNA polymerase II-binding proteins may regulate transcription by modulating the interaction of RNA polymerase II with other regulatory proteins that possess sequence recognition specificity.

Adrenal Cortex Neoplasms↗

PubMed1985

An RNA polymerase II transcription factor binds to an upstream element in the adenovirus major late promoter.

A gel electrophoresis DNA binding assay has been used to identify proteins in HeLa cell extracts that specifically bind to the major late promoter of adenovirus. A major late promoter transcription factor MLTF has been detected as a discrete protein-DNA complex. MLTF binds specifically and with high affinity to sequences upstream of the TATA box of the major late promoter. This factor protects a 17 bp (-50 to -66) region in a DNAase I footprinting assay. The same region has been shown to be important for efficient transcription from the major late promoter both in vivo and in vitro. MLTF stimulates in vitro transcription only from a template containing this upstream region. The binding, footprinting, and transcription-stimulatory activities of MLTF cofractionate through two chromatographic steps. These results suggest that direct binding of MLTF to an upstream element activates transcription from the major late promoter.

Adenoviruses, Human↗