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Biomedical subjects

D D Brown

Publications and source records attributed to D D Brown.

At least 109 records · Page 6Linked to original sources
PubMed1984

The role of stable complexes that repress and activate eukaryotic genes.

The state of activity or repression of 5S RNA genes is built into the chromatin in the form of stable complexes. The characteristics and composition of these complexes will be described. Stable transcription complexes that activate 5S RNA genes consist of multiple factors bound tightly to a control region of the gene. Repressed 5S RNA genes do not have these factors bound to them; their repressed state is maintained by nucleosomes.

Animals↗
PubMed1984

Prehospital defibrillation performed by emergency medical technicians in rural communities.

Survival after out-of-hospital cardiac arrest is poor in communities served only by basic ambulance services, but conventional advanced prehospital care is not an option for most rural communities. Ambulance technicians in 18 small communities (average population, 10,400) were trained to recognize and defibrillate ventricular fibrillation. Neither endotracheal intubation nor medication was used. Twelve additional communities of similar size where such early defibrillation was not attempted provided control data. In the communities where early defibrillation was available, 12 of 64 patients (19 per cent) who were found in ventricular fibrillation were resuscitated and discharged alive from the hospital; this was true of only 1 of 31 such patients (3 per cent) in the control communities, where only basic life support was available (P less than 0.05). Ten (83 per cent) of the long-term survivors received electrical shocks administered solely by the technicians. Early defibrillation by minimally trained ambulance technicians is an effective approach to emergency cardiac care in rural communities.

Adolescent↗
PubMed1984

Domains of the positive transcription factor specific for the Xenopus 5S RNA gene.

The 40 kd positive transcription factor that interacts with the 50 nucleotide internal control region of 5S ribosomal RNA genes in Xenopus can be subdivided into three functional domains by proteolytic cleavage. At one end of the protein is a 10 kd domain that is required for efficient RNA transcription but not for binding to the DNA. This adjoins a second domain that binds to the 5' end of the internal control region, a region of the DNA known to contribute only weakly to binding the protein but one that is essential for RNA transcription. The removal of both of these protein domains leaves a half of the protein that binds only to the 3' side of the control region and is inactive in promoting transcription. Quantitative DNA binding and in vitro transcription experiments show that only a single molecule of 40 kd factor binds to the internal control region, and that this stoichiometry is sufficient to give maximal stimulation of 5S RNA transcription in a reconstituted system. Consideration of the essential DNA contact points of the factor, taken together with the constraints imposed by a 1:1 protein to DNA stoichiometry permit the linear projection of the extended protein molecule onto its binding site along the internal control region of the 5S RNA gene.

Animals↗
PubMed1984

The transcriptional regulation of Xenopus 5s RNA genes in chromatin: the roles of active stable transcription complexes and histone H1.

The properties of active and repressed 5S rna genes in somatic cell chromatin from Xenopus laevis cultured cells were studied by transcription in vitro. The somatic 5S RNA genes, which are active in vivo, are packaged in chromatin as active stable transcription complexes lacking only RNA polymerase III for transcription activity. The oocyte 5S RNA genes, which are inactive in somatic tissues, do not have transcription factors bound to them in purified chromatin and are prevented from binding these factors by a structure dependent on histone H1. In chromatin active stable transcription complexes protect 5S RNA genes from histone H1-mediated repression and H1 binding prevents the formation of stable active transcription complexes.

Animals↗
PubMed1984

Association of fertility, fitness and longevity with the murine Ah locus among (C57BL/6N) (C3H/HeN) recombinant inbred lines.

The Ah locus encodes a cytosolic receptor which controls the induction of enzymes that metabolize drugs, chemical carcinogens, and other environmental pollutants. B6NXC3N recombinant inbred lines have been developed from the progenitors C57BL/6N and C3H/HeN inbred mouse strains. Ah phenotyping at each generation has resulted in the establishment of some lines containing high levels of the high-affinity Ah receptor; other lines, very low levels. A genetic model involving two unlinked loci is offered to explain the distribution of Ah receptor levels among (C57BL/6N) (C3H/HeN)F2 individuals. Between generations 7 and 13, individual females and males from the B6NXC3N recombinant inbred lines were crossed with DBA/2N males and females. Presence of high levels of the high-affinity Ah receptor in both female and male B6NXC3N mice was found to be associated with greater fertility, fitness, and longer life span. The data suggest that these parameters are correlated with the Ah locus or a closely segregating gene.

Animals↗
PubMed1984

Sequence homology between the structural proteins of Kilham rat virus.

The capsid proteins of the autonomous parvovirus Kilham rat virus were purified and analyzed for peptide composition. Partial proteolysis mapping and two-dimensional thin-layer chromatography of tryptic peptide digests revealed extensive amino acid sequence homology between the two major Kilham rat virus capsid proteins.

Amino Acid Sequence↗
PubMed1983

Onset of 5 S RNA gene regulation during Xenopus embryogenesis.

The transcription of 5 S RNA genes during oogenesis results in the storage of sufficient 5 S RNA in ribosomes to support subsequent embryogenesis. Xenopus oocytes of all stages synthesize oocyte-type 5 S RNA. A generalized repression of transcription occurs at meiosis and is maintained throughout early cleavage. The onset of 5 S RNA synthesis is detected at approximately the 4000-cell blastula stage (stage 9), concomitant with de novo synthesis of other species of RNA. At this developmental stage the level of 5 S RNA synthesis is low relative to the synthesis of tRNA and small nuclear RNAs. Analysis of this newly synthesized 5 S RNA reveals it to be a nearly equal mixture of oocyte and somatic 5 S RNA derived from both maternal and paternal genes. Given the 50:1 ratio of oocyte to somatic 5 S RNA genes in X. laevis, these results indicate that the majority of the oocyte 5 S RNA genes are inactivated at this time. This reflects differential transcription of the two families of 5 S RNA genes rather than post-transcriptional stability as demonstrated by the ability of a chromatin template isolated from stage 9 embryos to direct the same ratio of oocyte to somatic 5 S RNA synthesis in vitro as that observed in vivo. By completion of gastrulation, 5 S RNA synthesized in vivo and directed from chromatin in vitro is at least 90% somatic 5 S RNA. These results are consistent with a model in which the decrease in concentration of the 5 S-specific transcription factor relative to the number of 5 S RNA genes during embryogenesis contributes to the inactivation of the oocyte 5 S RNA genes.

Age Factors↗
PubMed1983

Purified RNA polymerase III accurately and efficiently terminates transcription of 5S RNA genes.

RNA polymerase III was purified to about 90% homogeneity from ovarian tissue of Xenopus laevis. The enzyme accurately initiates and terminates transcription of 5S RNA synthesis in isolated nuclei, but not when naked 5S DNA is used as a template. A sensitive hybridization technique was used to demonstrate that the purified polymerase, even when supplemented with a transcription factor that binds specifically to the 5S RNA gene internal control region, is unable to initiate synthesis at the start site of the 5S RNA gene. However, the polymerase alone terminates transcription at precisely the same site that is recognized in vivo and in complete transcription extracts. The purified polymerase distinguishes between weak and strong terminator sequences with the same relative efficiency as the enzyme in complete extracts. We conclude that the pure enzyme can recognize the simple consensus sequence found at the end of genes transcribed by RNA polymerase III.

Animals↗
PubMed1982

Contact points between a positive transcription factor and the Xenopus 5S RNA gene.

We have investigated the contact points of a positive transcription factor with the internal control region of the 5S ribosomal RNA genes of Xenopus. The methylation of any one of eight G residues clustered at the 3' end of the internal control region on the noncoding strand of the DNA or the ethylation of their surrounding phosphates interferes with the binding of this protein. The importance of the 5' half of the control region is shown by a binding affinity of the protein to oocyte 5S RNA genes that is one fourth that to somatic 5S RNA genes. This can be attributed to two base changes at residues 53 and 55 in the 5' part of the control region. The different affinities of the transcription factor for the oocyte and somatic 5S RNA genes may contribute to their differential expression in somatic cells. The almost exclusive location of strong contact points on the noncoding strand of the gene suggests how transcription events could proceed repeatedly along a gene that is assembled into a stable transcription complex. It is proposed that the complex is transiently shifted to the noncoding strand as each round of RNA synthesis occurs from the coding strand.

Animals↗
PubMed1982

Stable transcription complexes of Xenopus 5S RNA genes: a means to maintain the differentiated state.

Cloned 5S RNA genes added to Xenopus oocyte nuclear extract assemble into stable active transcription complexes that persist for many rounds of 5S RNA synthesis. This stability of the complex has been demonstrated by its resistance to dilution and to competitor DNA. A stable complex is formed within minutes and lasts for at least 40 rounds of transcription per template over several hours. Stable, transcriptionally inactive complexes can be formed by incubation of cloned 5S RNA genes in an oocyte nuclear extract depleted of a 5S-specific transcription factor and supplemented with histones. The stable, transcriptionally active and inactive states of 5S RNA gene complexes that can be formed in vitro are analogous to the states of the somatic and oocyte 5S RNA genes as they exist in somatic cell chromatin. Oocyte 5S RNa genes remain repressed in chromatin isolated from somatic cells, but can be activated by washing chromatin with high salt. Maintenance of the differentiated state of cell requires that selected genes remain stably active while others are stably repressed for long periods of time. We propose that stable transcription complexes may play an important role in the maintenance of the differentiated state in eucaryotic cells.

Animals↗
PubMed1982

Gene expression in eukaryotes.

Gene expression in eukaryotes is influenced by a wide variety of mechanisms, including the loss, amplification, and rearrangement of genes. Genes are differentially transcribed, and the RNA transcripts are variably utilized. Multigene families regulate the amount, the diversity, and the timing of gene expression. The present level of understanding of gene expression in eukaryotes is attributable mainly to biochemical methods rather than to traditional genetics. The new techniques that permit analysis and modification of purified genes of known function will identify both the control regions in eukaryotic genes and the molecules within cells that influence gene expression.

Cell Differentiation↗
PubMed1981

Gene expression in eukaryotes.

Gene expression in eukaryotes is influenced by a wide variety of mechanisms including the loss, amplification, and rearrangement of genes. Genes are differentially transcribed, and the RNA transcripts are variably utilized. Multigene families regulate the amount, the diversity, and the timing of gene expression. The present level of understanding of gene expression in eukaryotes is attributable mainly to biochemical methods rather than to traditional genetics. The new techniques that permit analysis and modification of purified genes of known function will identify both the control regions in eukaryotic genes as well as the molecules within cell that influence gene expression.

Animals↗
PubMed1981

A quantitative assay for Xenopus 5S RNA gene transcription in vitro.

The in vitro transcription of Xenopus 5S RNA genes and of deletion mutants of these genes has been quantitated by assays that measure the efficiency of transcription and the ability to compete with the transcription of a "wild-type" 5S RNA gene. The difference between the competition strength of one repeating unit of X. borealis somatic 5S DNA and its plasmid vector is fifteenfold. tRNA genes and the adenovirus VA RNA genes are weak competitors of 5S RNA synthesis (and vice versa). Deletion of the 5' flanking region reduces the competition strength of somatic but not oocyte 5S DNA. Except for the influence of the flanking sequence, the regions within the 5S RNA gene that determine competition strength are those that have been shown to interact with a specific transcription factor that is required for accurate initiation of 5S RNA transcription. The major oocyte (Xlo) and trace oocyte (Xlt) 5S RNA genes from X. laevis are transcribed as efficiently as somatic 5S DNAs but compete only one fourth as well. This fourfold difference in the competition strength is due to oocyte-specific base changes within the intragenic control region.

Animals↗
PubMed1981

The binding of a transcription factor to deletion mutants of a 5S ribosomal RNA gene.

A transcription factor binds specifically to a region within the 5S ribosomal RNA gene that is known to be required for accurate initiation of transcription of the gene. We have confirmed the significance of this interaction by comparing various deletion mutants for their ability to initiate transcription and to bind the protein. Loss of specific binding of the protein occurs when the gene is deleted beyond +83 from the 3' direction. This coincides exactly with the loss of ability to initiate transcription. In contrast, deletions entering the gene from the 5' direction and extending beyond the 5' border of the control region (+50/+55) give partial protection of the remainder of the control region. This observation can explain why deletions beyond the 5' border of the control region that are unable to initiate transcription can still compete for the transcription of the wild-type 5S RNA gene.

Animals↗
PubMed1981

Nucleotide sequences in Xenopus 5S DNA required for transcription termination.

We have analyzed the DNA sequences required for termination of Xenopus 5S RNA synthesis in vitro. Termination occurs within clusters of four or more consecutive T residues in the noncoding DNA strand sequence. The 3' flanking sequence following the gene is not required for termination. The distance between the T cluster and the site of transcription initiation as well as the exact nucleotide sequences preceding the T cluster can be varied without impairing termination. Thus sequence signals for accurate initiation and termination of transcription are separable and can function independently. No significant dyad symmetry near the end of the gene appears to be required, in contrast to termination sites in procaryotes. However, the nucleotide sequences adjacent to the T cluster influence the efficiency of transcription termination. Efficient termination is observed whenever GC-rich sequences surround the T cluster. The inefficient termination of transcription of X. laevis major oocyte type 5S RNA genes appears to result from groups of two or more consecutive A residues within three nucleotides preceding or following the T cluster. Other variables, such as the temperature, the nucleotide concentration and the addition of alpha-amanitin, also influence the efficiency of termination.

Animals↗