Biomedical subjects
S D Hanes
Publications and source records attributed to S D Hanes.
Specific DNA recognition and intersite spacing are critical for action of the bicoid morphogen.
We examined DNA site recognition by Bicoid and its importance for pattern formation in developing Drosophila embryos. Using altered DNA specificity Bicoid mutants and appropriate reporter genes, we show that Bicoid distinguishes among related DNA-binding sites in vivo by a specific contact between amino acid 9 of its recognition alpha-helix (lysine 50 of the homeodomain) and bp 7 of the site. This result is consistent with our earlier results using Saccharomyces cerevisiae but differs from that predicted by crystallographic analysis of another homeodomain-DNA interaction. Our results also demonstrate that Bicoid binds directly to those genes whose transcription it regulates and that the amino acid 9 contact is necessary for Bicoid to direct anterior pattern formation. In both Drosophila embryos and yeast cells, Bicoid requires multiple binding sites to activate transcription of target genes. We find that the distance between binding sites is critical for Bicoid activation but that, unexpectedly, this critical distance differs between Drosophila and S. cerevisiae. This result suggests that Bicoid activation in Drosophila might require an ancillary protein(s) not present in S. cerevisiae.
A genetic model for interaction of the homeodomain recognition helix with DNA.
The Bicoid homeodomain protein controls anterior development in the Drosophila embryo by binding to DNA and regulating gene expression. With the use of genetic assays in yeast, the interaction between the Bicoid homeodomain and a series of mutated DNA sites was studied. These experiments defined important features of homeodomain binding sites, identified specific amino acid-base pair contacts, and suggested a model for interaction of the recognition alpha-helices of Bicoid and Antennapedia-class homeodomain proteins with DNA. The model is in general agreement with results of crystallographic and magnetic resonance studies, but differs in important details. It is likely that genetic studies of protein-DNA interaction will continue to complement conventional structural approaches.
DNA specificity of the bicoid activator protein is determined by homeodomain recognition helix residue 9.
Formation of anterior structures in the Drosophila embryo requires the product of the gene bicoid. The bicoid protein contains a homeodomain and may exert its effects in early development by regulating transcription of the gap gene, hunchback (hb). Consistent with this view, we have demonstrated that DNA-bound Bicoid fusion proteins stimulate gene expression. We used the gene activation phenotype in yeast to study DNA recognition by the Bicoid homeodomain. We found that a single amino acid replacement at position 9 of the recognition helix was sufficient to switch the DNA specificity of the Bicoid protein. The altered specificity Bicoid mutants recognized DNA sites bound by Ultrabithorax, fushi tarazu, and other related homeo-domain proteins. Our results suggest that DNA specificity in Bicoid and Antennapedia class proteins is determined by recognition helix residue 9.
Expression of a cDNA derived from the yeast killer preprotoxin gene: implications for processing and immunity.
The type I killer strains of Saccharomyces cerevisiae secrete a dimeric 19-kDa protein that kills sensitive cells by disrupting cytoplasmic membrane function. This toxin is encoded by the double-stranded RNA plasmid M1-dsRNA, which also determines specific immunity to toxin. A preprotoxin, the 35-kDA in vitro translation product of denatured M1-dsRNA, is presumed to be the primary in vivo gene product. To facilitate studies on preprotoxin structure and maturation, we have inserted a partial cDNA copy of M1-dsRNA into the yeast vector p1A1, bringing it under control of the phosphate-repressible PHO5 promoter. This in-frame gene fusion encodes all of the preprotoxin sequence except for its N-terminal secretion leader, which is replaced by the leader sequence of PHO5. Transformation of sensitive yeast strains lacking M1-dsRNA with such fusion plasmids converts them to phosphate-repressible, immune killers, demonstrating that both toxin and immunity determinants are contained within the preprotoxin molecule. L-1-Tosylamido-2-phenylethyl chloromethyl ketone retards glycosylation of preprotoxin to toxin, facilitating size comparisons and indicating that processing of the normal precursor involves three glycosylation events but does not involve cotranslational leader peptidase action. In contrast, the PHO5 leader is apparently removed from the fusion preprotoxin.
Control of cell growth and division in Saccharomyces cerevisiae.
Considerable advances have been made in recent years in our understanding of the biochemistry of protein and nucleic acid synthesis and, particularly, the molecular biology of gene expression in eukaryotes. The yeast Saccharomyces cerevisiae, and to a lesser extent Schizosaccharomyces pombe, has had a preeminent role as a focus for these studies, principally because of the facility with which these organisms can be experimentally manipulated biochemically and genetically. This review will be designed to critically examine and integrate recent advances in several vital areas of regulatory control of enzyme synthesis in yeast: structure and organization of DNA, transcriptional regulation, post-transcriptional modification, control of translation, post-translational modification and secretion, and cell-cycle modulation. It will attempt to emphasize and illustrate, where detailed information is available, principal underlying molecular mechanisms, and it will attempt to make relevant comparisons of this material to inferred and demonstrated facets of regulatory control of enzyme and protein synthesis in higher eukaryotes.
5-bromodeoxyuridine-induced amplification of prolactin gene in GH cells is an extrachromosomal event.
Treatment of a 5-bromodeoxyuridine-resistant (brdUrdr) and prolactin-nonproducing (Prl-) subclone of GH cells with this drug led to amplification of the prolactin (Prl) gene and induced Prl synthesis. Withdrawal of the drug treatment reversed both of these processes. In normal rats, the increased Prl synthesis observed during late pregnancy and lactation does not seem to be mediated via amplification of the gene. Amplification of the Prl gene and induction of Prl synthesis can also be observed in the Prl-, brdUrd-sensitive (brdUrds) GH cell strain. Prl gene amplification thus does not seem to be associated with the mechanism that confers the brdUrdr phenotype to these cells. brdUrd-induced amplification of the Prl gene can be identified with the low molecular weight, extrachromosomal, supernatant DNA fraction, isolated by Hirt's method. Southern blot analysis of Hirt's supernatant DNA (undigested) from brdUrd-treated cells generated a distinct band following hybridization with [32P]pDNAPrl-insert. The size of this band is greater than 23 kb but smaller than chromosomal DNA. Growth hormone (Gh) and albumin (Alb) gene sequences can be detected in the chromosomal DNA preparation but are absent in the extrachromosomal DNA prepared from Hirt's supernatant. The levels of Gh and Alb sequences are unaffected by brdUrd treatment of these cells. Results presented here suggest that in rat pituitary glands as well as in GH cells, hormonally controlled increased Prl synthesis is not caused by gene amplification. However, the brdUrd-induced expression of the Prl gene seems to be linked to the mechanism of drug-induced amplification of the Prl gene, mediated via an extrachromosomal event.
Mechanism of action of benzo(a)pyrene and nicotine on hormone production by rat pituitary tumor cells.
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Increased level of prolactin gene sequences in bromodeoxyuridine treated GH cells.
The 5-bromodeoxyuridine-resistant (BrdUrdr) derivative (F1BGH12C1) of prolactin nonproducing (PRL-) rat pituitary tumor cell-subclone GH12C1, synthesize prolactin (PRL) in the presence of the drug. Analysis of nuclear RNA isolated from BrdUrd treated F1BHG12C1 cells demonstrated several high molecular weight RNA PRL sequences, similar to those observed in the nuclear RNA fraction of PRL producing (PRL+) GH3 cells. No such RNAPRL sequences could be detected in nuclear RNA fraction of untreated F1 BGH12C1 cells. PRL sequences in the genome of GH3 (PRL+), GH12C1 (PRL-) and F1BGH12C1 (PRL-, BrdUrdr) GH cells could be identified by blot analysis in 4.8-5.2kb fragment of restriction endonuclease, Hind III digested DNA. Both PRL+ and PRL- cells seem to have approximately the same level of PRL gene sequences in total cell DNA. However Hind III digested DNA of BrdUrd treated F1BGH12C cells revealed the presence of significantly higher levels of PRL gene sequences, in comparison, to that observed in total DNA of untreated cells. The increased level of PRL gene sequences was dependent on the period of drug treatment and a parallel increase in the cytoplasmic RNAPRL sequences was also observed.
Thyrotropin releasing hormone receptors in regulation of prolactin gene expression in GH cells.
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Mechanism of induction of prolactin synthesis in GH cells.
Prolactin-specific RNA (RNA(PRL)) in total nuclear RNA and in cytoplasmic poly(A)(+)RNA isolated from GH (rat pituitary) cells was selectively hybridized to immobilized cloned cDNA(PRL). Agarose gel electrophoresis of the nuclear RNA(PRL) sequences eluted from the nitrocellulose filters revealed several RNA species of approximately 25-30, 18-19, and 12-13 S. Only the 12-13 S RNA species could be detected in the cytoplasmic poly(A)(+)RNA fraction. Comparative analysis of total nuclear RNA of control and thyrotropin-releasing hormone (thyroliberin)-treated cells by the reverse Southern blot technique demonstrated increased levels of all the nuclear RNA(PRL) species in hormone-treated cells. Nuclear and cytoplasmic RNA(PRL) sequences in control and treated cells were quantitated by molecular hybridization to cloned cDNA(PRL). The 2- to 3-fold stimulation of PRL production by thyrotropin-releasing hormone-treated GH(4)C(1) cells could be correlated to the corresponding increase of nuclear RNA(PRL) sequences. The hybrid strain, which produces 1/5th the amount of PRL that the parent GH(4)C(1) does, had 1/5th the amounts of nuclear RNA(PRL) sequences. Thyrotropin-releasing hormone affected neither prolactin production nor nuclear RNA(PRL) level in 928-9b cells. RNA(PRL) sequences could not be detected either in nuclei or in cytoplasm of prolactin nonproducing F(1)BGH(1)2C(1) cells. However, prolactin production could be induced and RNA(PRL) sequences could be detected in the total nuclear RNA and in cytoplasmic poly(A)(+)RNA fraction after treatment of this GH cell substrain with 5-bromodeoxyuridine. These results demonstrate that differential basal prolactin production and its modulation by thyrotropin-releasing hormone and by 5-bromodeoxyuridine can be correlated to the altered levels of nuclear RNA(PRL) sequences in the three GH cell strains.
An interactive computer program that accurately estimates the ED50, its standard error and other parameters related to the probit regression line.
A program suitable for terminal, conversational-mode usage was developed in the BASIC language to compute the median effective dose (LD50), various error terms, and confidence intervals. The overall algorithm used in the program was based on a method described by Finney. The accuracy, speed and flexibility of the program makes the terminal approach an efficient alternative to simple graphic methods.
Sequence and mutational analysis of ESS1, a gene essential for growth in Saccharomyces cerevisiae.
A newly isolated gene, ESS1, was shown to encode a protein required for vegetative growth in Saccharomyces cerevisiae. The nucleotide sequence of ESS1 revealed a 172 amino acid open reading frame predicting a highly basic, 19.5 kilodalton product. Although the gene was isolated by cross-hybridization with the vertebrate v-sis oncogene, the primary amino acid sequence bears only a slight resemblance to the p28sis protein. ESS1 was shown to be single copy in the yeast genome and transcriptionally active during logarithmic growth. It is located on the right arm of chromosome X, 6 centimorgans distal to ilv3. The genetic map location indicates it is not allelic to any previously characterized mutation in this organism. Both inactivation of ESS1 by gene disruption and overexpression by fusion to a heterologous promoter were detrimental to growth in both haploid and diploid cell types. Under non-permissive conditions, the terminal phenotype of strains containing a suppressible amber mutation within ESS1 was one of aberrant multibudded structures. Examination of this morphology indicates that loss of ESS1 function may lead to a defect in cytokinesis or cell separation.