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Biomedical subjects

R A Young

Publications and source records attributed to R A Young.

At least 19 recordsLinked to original sources
PubMed1992

A novel transcription factor reveals a functional link between the RNA polymerase II CTD and TFIID.

The RNA polymerase II large subunit carboxy-terminal domain (CTD) plays a role in transcription initiation, but its mechanism of action is not well understood. We have investigated the function of the SRB2 gene, which was isolated as a dominant suppressor of CTD truncation mutations. The allele specificity of this suppressor indicates that SRB2 and the CTD are involved in the same function. Indeed, cells lacking SRB2 and cells lacking a large portion of the CTD exhibit the same set of conditional growth phenotypes and exhibit very similar defects in gene expression in vivo. The SRB2 protein is a novel transcription factor that has an important role in basal and activated transcription in vitro and is essential for efficient establishment of the transcription initiation apparatus. Template commitment experiments suggest that SRB2 becomes physically associated with the transcription initiation complex. We find that SRB2 binds specifically to TFIID. As SRB2 and the RNA polymerase II CTD are involved in the same function, these results reveal a functional link between the CTD and the TATA-binding factor. This study implicates the CTD in recruitment of RNA polymerase II to the transcription initiation complex.

Amino Acid Sequence↗
PubMed1992

Genes encoding transcription factor IIIA and the RNA polymerase common subunit RPB6 are divergently transcribed in Saccharomyces cerevisiae.

The gene encoding Saccharomyces cerevisiae transcription factor TFIIIA has been found adjacent to RPB6, a gene that specifies a subunit shared by nuclear RNA polymerases. Analysis of DNA upstream of the RPB6 gene revealed an open reading frame that predicts a protein, designated PZF1, with nine C2H2 zinc fingers. The presence of nine C2H2 zinc fingers in PZF1 protein, a hallmark of amphibian TFIIIA proteins, suggested that PZF1 might be a TFIIIA homologue. We found that purified recombinant PZF1 specifically binds the internal control region (ICR) of the 5S rRNA gene in S. cerevisiae. The presence of nine C2H2 zinc fingers, the specific binding to ICR DNA, and the similarity of the predicted molecular mass of PZF1 with that determined for purified yeast TFIIIA, together indicate that PZF1 is TFIIIA. The yeast and amphibian TFIIIA proteins share only a limited number of residues outside of those normally conserved in C2H2 zinc fingers; these conserved residues may provide clues to the sequence specificity of these proteins. The PZF1 gene was found to be single copy, transcribed into a 1.5-kilobase mRNA, and essential for yeast cell viability. Interestingly, the yeast RPB6 and TFIIIA coding sequences are divergently transcribed and are separated by only 233 base pairs, providing the potential for coregulated expression of components of RNA polymerases and the 5S rRNA component of ribosomes.

Amino Acid Sequence↗
PubMed1992

Effects of Duvernoy's gland secretions from the eastern hognose snake, Heterodon platirhinos, on smooth muscle and neuromuscular junction.

Duvernoy's gland secretions (100 micrograms/ml) from the eastern hognose snake, Heterodon platirhinos, induced a neuromuscular blockade in the isolated frog sciatic nerve-gastrocnemius muscle preparation and effectively antagonized acetylcholine and histamine responses of the rat duodenum preparation. The Duvernoy's secretions (100 micrograms/ml) produced a reversible, excitatory effect in the guinea-pig ileum in vitro. Mice administered the secretions (100 mg/kg, i.p.) exhibited minor, transient signs of toxicity. These preliminary observations provide evidence for toxicity of H. platirhinos Duvernoy's gland secretions but do not warrant categorizing the potential toxicity of this species with that of proteroglyphous, solenoglyphous, or the more venomous opisthoglyphous species.

Animals↗
PubMed1992

Seroprevalence of human immunodeficiency virus among adolescent attendees of Mississippi sexually transmitted disease clinics: a rural epidemic.

Human immunodeficiency virus (HIV) infection among adolescents is causing increasing concern, and teenagers attending sexually transmitted disease (STD) clinics run a high risk of contracting it. To determine the status of HIV infection in a Mississippi adolescent population, we evaluated seroprevalence rates for adolescents attending Mississippi State Department of Health STD clinics from 1988 to 1990. During this 2-year period, 9855 adolescents (aged 13 to 20 years) attended STD clinics, and HIV antibody was confirmed in 39 (seroprevalence rate 4.0/1000; 95% confidence interval [CI] 2.7 to 5.2). Seropositive rates were almost equal for male and female subjects (4.1/1000 and 3.8/1000, respectively), suggesting predominantly heterosexual transmission. Rates among blacks were 3.5 times higher than among whites. Adolescents with HIV infection were identified throughout the state, irrespective of urban centers. Rates among the smallest counties (ie, population less than 25,000) were not significantly different from those of the largest counties (ie, population greater than 100,000). Mississippi's rank in the top 10 states for other STDs and the state's high teenage pregnancy rate make it an epicenter of the HIV epidemic among adolescents.

Adolescent↗
PubMed1992

Vaccinia virus infection induces a stress response that leads to association of Hsp70 with viral proteins.

We studied the impact of vaccinia virus infection on stress protein gene expression in human cells and investigated the possibility that eukaryotic heat shock proteins interact with viral components during assembly. Infection of human monocyte-macrophages by vaccinia virus caused a dramatic decrease in levels of cellular mRNAs such as those encoding actin and tubulin. In contrast, infection did not cause a significant reduction in the levels of Hsp90 and Hsp60 mRNAs and led to substantially increased levels of Hsp70 mRNAs. The accumulation of these stress protein mRNAs was due both to increases in their transcription rate and to their stability relative to other cellular mRNAs. The relative levels of the heat shock proteins and the other cellular proteins reflected the relative levels of their mRNAs. These results indicate that stress protein gene expression is relatively refractory to the generally deleterious effects of vaccinia virus infection on host cell gene expression. The continued expression of some of these stress proteins may be beneficial to the virus; the observations that the levels of Hsp70 are greatest at the peak of viral gene expression and that a large fraction of cellular Hsp70 is associated with vaccinia virus proteins suggest that Hsp70 is involved in vaccinia virus assembly.

Cell Line↗
PubMed1992

Cefotaxime dosage in infants and children. Pharmacokinetic and clinical rationale for an extended dosage interval.

Cefotaxime is a third generation cephalosporin antimicrobial agent which has received wide acceptance as a first-line antibiotic for many infections in neonates, infants and children. With an average elimination half-life of about 1 h, cefotaxime is not considered to be a 'long half-life cephalosporin' like ceftriaxone. For this reason, currently accepted dosage regimens for cefotaxime in infants and children employ a dosage of 50 mg/kg every 6 h. Re-examination of the paediatric pharmacokinetic data for cefotaxime and use of simple multiple-dose pharmacokinetic simulation of alternative dosage regimens was performed. From this analysis, regimens administering 75 mg/kg of the drug every 8 h or every 12 h were projected to produce serum cefotaxime concentrations adequate to effectively kill many of the common pathogens against which the drug is currently indicated for use in children. The clinical utility of these alternative dosage regimens was supported by a review of the medical literature and examination of the clinical results from studies in neonates, infants and children where cefotaxime was administered in 2 to 3 divided doses daily. It would appear, therefore, that increasing the cefotaxime dosage to 75 mg/kg administered at 8 h intervals would result in less frequent drug administration which would not be expected to compromise safety and efficacy. Alternative dosage regimens for cefotaxime merit further consideration and clinical evaluation before they become commonly used in paediatric therapeutics.

Cefotaxime↗
PubMed1991

Yeast RNA polymerase II subunit RPB9 is essential for growth at temperature extremes.

The Saccharomyces cerevisiae RNA polymerase II subunit gene RPB9 was isolated and sequenced. RPB9 is a single copy gene on chromosome VII. The RPB9 sequence predicts a protein of 122 amino acids with a molecular mass of 14,200 Da. The yeast RPB9 subunit is similar in size and sequence to a protein encoded by DNA adjacent to the suppressor of the Hairy Wing gene in Drosophila melanogaster. Deletion of the RPB9 gene produced cells that were heat- and cold-sensitive. The RPB9 subunit, like the previously described RNA polymerase II subunit RPB4, is not essential for synthesis of mRNA, but is required for normal cell growth over a wide temperature range.

Amino Acid Sequence↗
PubMed1991

Humoral and cell-mediated immune responses to live recombinant BCG-HIV vaccines.

Several viral and bacterial live recombinant vaccine vehicles are being developed to produce a new generation of vaccines against a broad spectrum of infectious diseases. The human tuberculosis vaccine Mycobacterium bovis bacillus Calmette-Guerin (BCG) has features that make it a particularly attractive live recombinant vaccine vehicle. BCG and other mycobacteria are highly effective adjuvants, and the immune response to mycobacteria has been studied extensively. With nearly two billion immunizations, BCG has a long record of safe use in man. It is one of the few vaccines that can be given at birth, it engenders long-lived immune responses with only a single dose, and there is a worldwide distribution network with experience in BCG vaccination. Recently developed molecular genetic tools and methods for mycobacteria have provided the means to introduce foreign genes into BCG. Here we report that a variety of human immunodeficiency virus type 1 polypeptides can be expressed in BCG recombinants under the control of the mycobacterial hsp70 promoter and that the foreign polypeptides produced in BCG can induce antibody and T-cell responses. These results demonstrate that BCG can be used as a live recombinant vaccine vehicle to induce immune responses to pathogen proteins produced by the bacillus.

Animals↗
PubMed1991

Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro.

RNA polymerase II lacking the fourth and seventh largest subunits (pol II delta 4/7) was purified from Saccharomyces cerevisiae strain rpb-4, in which the gene for the fourth largest subunit is deleted. pol II delta 4/7 was indistinguishable from wild-type pol II (holoenzyme) in promoter-independent initiation/chain elongation activity (400-800 nmol of nucleotide incorporated/10 min/mg of protein at 22 degrees C), in rate of chain elongation (20-25 nucleotides/s), and in the recognition of pause sites in the DNA template. In contrast to pol II holoenzyme, pol II delta 4/7 was inactive in promoter-directed initiation of transcription in vitro. The addition of an equimolar complex of the fourth and seventh largest subunits, purified from pol II holoenzyme by ion-exchange chromatography in the presence of urea, restored promoter-directed initiation activity to pol II delta 4/7. The transcriptional activator protein Gal4-VP16 could also elicit promoter-directed initiation by pol II delta 4/7 from a promoter with a Gal4 binding site. Complementation was observed between extracts of strain rpb-4, lacking the fourth largest subunit, and strain Y260-1, with a defect in the largest subunit. These extracts were individually inactive, but a mixture would support promoter-directed initiation. The fourth and seventh largest subunits may, therefore, shuttle between polymerase molecules.

Cell Nucleus↗
PubMed1991

Epitope tagging and protein surveillance.

The epitope tagging approach offers advantages of economy, universality, and precision over the use of antibodies raised directly against a protein of interest. The latter strategy promises a potentially greater diversity of reagents and obviates the need to modify the protein, but it may not yield sufficiently high-affinity, abundant, or specific antibodies. The major uncertainty in an epitope-tagging strategy, namely, the ability of the altered protein to function in vivo, is readily resolved in yeast by testing complementation of a null allele by the modified gene. Modification of the protein is easily accomplished by addition of the epitope coding sequence to the gene via oligonucleotide-mediated site-directed mutagenesis. The uniqueness of the epitope in the genome and the use of the monoclonal antibody assure a high-affinity, specific, and abundant antibody. Unrelated but identically modified proteins can be immunoprecipitated and affinity purified under the same conditions. Only extraction conditions and possibly a simple initial fractionation step need vary. Moreover, otherwise identical but differentially tagged proteins can be separated. Even proteins completely defective in an essential in vivo function can be purified and studied. Finally, polypeptides coprecipitating with the protein of interest are normally difficult to distinguish from those merely cross-reactive with the antibody used. As an alternative to defining a complex of proteins using a battery of antibodies, complexes are defined as a set of immunoprecipitable polypeptides present only in extracts containing the modified protein.

Base Sequence↗
PubMed1991

RNA polymerase II carboxy-terminal domain contributes to the response to multiple acidic activators in vitro.

The largest subunit of RNA polymerase II contains a unique carboxy-terminal domain (CTD) that consists of repeats of the heptapeptide YSPTSPS. RNA polymerase II CTD truncation mutations affect the ability to induce transcription of a subset of yeast genes in vivo, and the lack of response to induction maps to the upstream activating sequences of these genes. Here, we report that progressive truncation of the yeast RNA polymerase II CTD causes progressive loss of trans-activator-dependent transcription in nuclear extracts but has little effect on elongation or termination. Specific transcription, which is reduced by up to 50-fold in these assays, can be restored in the defective nuclear extracts by adding purified wild-type RNA polymerase II. The defects in factor-dependent transcription are observed with templates that are assembled into nucleosomes as well as with templates that are not so assembled. Defects in factor-independent transcription are also observed, but these are not as profound as those observed in the presence of trans-activators. These results indicate that the RNA polymerase II CTD functions during transcription initiation and is required for normal levels of activated transcription in vitro.

Amino Acid Sequence↗
PubMed1991

Mutations in a conserved region of RNA polymerase II influence the accuracy of mRNA start site selection.

A sensitive phenotypic assay has been used to identify mutations affecting transcription initiation in the genes encoding the two large subunits of Saccharomyces cerevisiae RNA polymerase II (RPB1 and RPB2). The rpb1 and rpb2 mutations alter the ratio of transcripts initiated at two adjacent start sites of a delta-insertion promoter. Of a large number of rpb1 and rpb2 mutations screened, only a few affect transcription initiation patterns at delta-insertion promoters, and these mutations are in close proximity to each other within both RPB1 and RPB2. The two rpb1 mutations alter amino acid residues within homology block G, a region conserved in the large subunits of all RNA polymerases. The three strong rpb2 mutations alter adjacent amino acids. At a wild-type promoter, the rpb1 mutations affect the accuracy of mRNA start site selection by producing a small but detectable increase in the 5'-end heterogeneity of transcripts. These RNA polymerase II mutations implicate specific portions of the enzyme in aspects of transcription initiation.

Alleles↗
PubMed1991

Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly.

Mutations in the three largest subunits of yeast RNA polymerase II (RPB1, RPB2, and RPB3) were investigated for their effects on RNA polymerase II structure and assembly. Among 23 temperature-sensitive mutations, 6 mutations affected enzyme assembly, as assayed by immunoprecipitation of epitope-tagged subunits. In all six assembly mutants, RNA polymerase II subunits synthesized at the permissive temperature were incorporated into stably assembled, immunoprecipitable enzyme and remained stably associated when cells were shifted to the nonpermissive temperature, whereas subunits synthesized at the nonpermissive temperature were not incorporated into a completely assembled enzyme. The observation that subunit subcomplexes accumulated in assembly-mutant cells at the nonpermissive temperature led us to investigate whether these subcomplexes were assembly intermediates or merely byproducts of mutant enzyme instability. The time course of assembly of RPB1, RPB2, and RPB3 was investigated in wild-type cells and subsequently in mutant cells. Glycerol gradient fractionation of extracts of cells pulse-labeled for various times revealed that a subcomplex of RPB2 and RPB3 appears soon after subunit synthesis and can be chased into fully assembled enzyme. The RPB2-plus-RPB3 subcomplexes accumulated in all RPB1 assembly mutants at the nonpermissive temperature but not in an RPB2 or RPB3 assembly mutant. These data indicate that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during the assembly of RNA polymerase II.

Amino Acid Sequence↗
PubMed1991

RNA polymerase II.

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Amino Acid Sequence↗