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1.
Curr Opin Genet Dev ; 5(1): 38-43, 1995 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-7749323

RESUMO

The let-60 ras gene of Caenorhabditis elegans is required for multiple aspects of development. The vulvar differentiation pathway is the most intensively studied of these, but the ras pathway has now been shown to also be essential for male spicule development. Using vulval differentiation, molecular genetic techniques are now being used to study structure/function relationships of particular signaling components and to identify new positively and negatively acting proteins of Ras-mediated signaling pathways. Mutations affecting LET-23, a receptor tyrosine kinase homolog, which cause tissue-specific defects have been localized to the carboxyl terminus. SH2 domain specificity has been analyzed through Src/SEM-5 chimeric proteins in transgenic nematodes. A mitogen-activated protein kinase that acts downstream of LET-60 Ras in vulval differentiation has been identified. Negative regulatory genes have been cloned and found to encode novel proteins and a clathrin adaptor protein.


Assuntos
Caenorhabditis elegans/genética , Genes ras , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Sequência Conservada , Análise Mutacional de DNA , Feminino , Dados de Sequência Molecular , Vulva/crescimento & desenvolvimento
4.
Cell ; 65(6): 1023-31, 1991 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-2044150

RESUMO

To search for histone domains that may regulate transcription in vivo, we made deletions and amino acid substitutions in the histone N-termini of S. cerevisiae. Histone H4 N-terminal residues 4-23, which include the extremely conserved, reversibly acetylated lysines (at positions 5, 8, 12, and 16), were found to encompass a region required for the activation of the GAL1 promoter. Deletions in the H4 N-terminus reduce GAL1 activation 20-fold. This effect is specific to histone H4 in that large deletions in the N-termini of H2A, H2B, and H3 do not similarly decrease induction. Activation of the PHO5 promoter is reduced approximately 4- to 5-fold by these H4 deletions. Mutations in histone H4 acetylation sites and surrounding residues can cause comparable and, in some cases, even greater effects on induction of these two promoters. We postulate that the H4 N-terminus may interact with a component of the transcription initiation complex, allowing nucleosome unfolding and subsequent initiation.


Assuntos
Regulação Fúngica da Expressão Gênica , Histonas/fisiologia , Nucleossomos/fisiologia , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Transcrição Gênica , Acetilação , Sequência de Aminoácidos , Proteínas Fúngicas/genética , Histonas/química , Dados de Sequência Molecular , Nucleossomos/ultraestrutura , RNA Fúngico/biossíntese , Relação Estrutura-Atividade
5.
Proc Natl Acad Sci U S A ; 87(16): 6286-90, 1990 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-2201024

RESUMO

Repression of transcription from the silent mating loci (HML alpha and HMRa) is essential for mating ability in Saccharomyces cerevisiae. This silencing is known to require at least five proteins (SIR1, SIR2, SIR3, SIR4, and histone H4) and is accompanied by a change in chromatin structure. We show here that four positions of histone H4 (N-terminal residues 16, 17, 18, and 19) are crucial to silencing. HML alpha and HMRa are efficiently repressed when these positions are occupied by basic amino acids but are derepressed when substituted with glycine. These results suggest that acetylation of Lys-16 would lead to derepression of the silent mating loci. Three strong extragenic suppressors of the latter H4 mutations were isolated and determined to be located in SIR3. These suppressors allow high mating efficiencies in cells expressing either wild-type H4 or H4 containing single amino acid substitutions. They did not allow efficient mating in a strain that contained an H4 N-terminal deletion. These results indicate that the SIR3 mutations do not bypass the requirement for the H4 N terminus but, rather, allow repression in the presence of a less than optimal H4 N terminus. This provides a link between one of the SIR proteins and a component of chromatin.


Assuntos
Proteínas Fúngicas/genética , Histonas/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Sequência de Aminoácidos , Deleção Cromossômica , Cruzamentos Genéticos , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Histonas/metabolismo , Dados de Sequência Molecular , Mutação , Saccharomyces cerevisiae/fisiologia , Supressão Genética
6.
Psychiatr Clin North Am ; 13(1): 171-85, 1990 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-2315202

RESUMO

Today's consumers, faced with limited resources, want documented proof of the quality of a product or service. This shift to consumerism has caused health care providers and institutions to carefully examine their practice and move toward the establishment of standards of care in all areas. The authors are recommending a collaborative approach between standards and are advocating the use of the Marker Model as a system to define, organize, integrate, document, and monitor standards in the fields of psychiatry and psychiatric nursing.


Assuntos
Relações Interprofissionais , Enfermagem Psiquiátrica/normas , Psiquiatria/normas , Garantia da Qualidade dos Cuidados de Saúde , Competência Clínica , Defesa do Consumidor , Hospitais/normas , Humanos
7.
EMBO J ; 8(7): 2067-75, 1989 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-2551674

RESUMO

A gene from Saccharomyces cerevisiae has been mapped, cloned, sequenced and shown to encode a catalytic subunit of an N-terminal acetyltransferase. Regions of this gene, NAT1, and the chloramphenicol acetyltransferase genes of bacteria have limited but significant homology. A nat1 null mutant is viable but exhibits a variety of phenotypes, including reduced acetyltransferase activity, derepression of a silent mating type locus (HML) and failure to enter G0. All these phenotypes are identical to those of a previously characterized mutant, ard1. NAT1 and ARD1 are distinct genes that encode proteins with no obvious similarity. Concomitant overexpression of both NAT1 and ARD1 in yeast causes a 20-fold increase in acetyltransferase activity in vitro, whereas overexpression of either NAT1 or ARD1 alone does not raise activity over basal levels. A functional iso-1-cytochrome c protein, which is N-terminally acetylated in a NAT1 strain, is not acetylated in an isogenic nat1 mutant. At least 20 other yeast proteins, including histone H2B, are not N-terminally acetylated in either nat1 or ard1 mutants. These results suggest that NAT1 and ARD1 proteins function together to catalyze the N-terminal acetylation of a subset of yeast proteins.


Assuntos
Acetiltransferases/genética , Citocromos c , Genes Fúngicos , Genes Fúngicos Tipo Acasalamento , Genes , Mutação , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Grupo dos Citocromos c/genética , Histonas/genética , Dados de Sequência Molecular , Acetiltransferase N-Terminal A , Fenótipo , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência do Ácido Nucleico
8.
Cell ; 55(1): 27-39, 1988 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-3048701

RESUMO

Yeast histone H4 function was probed in vivo by deleting segments of this extremely conserved 102 amino acid protein. Deletions in the hydrophobic core of H4 are lethal and block chromosomal segregation. In contrast, deletions at the hydrophilic N terminus (residues 4-28) and C terminus (residues 100-102) are viable. However, N-terminal deletion alters normal chromatin structure and lengthens the cell cycle, especially G2. Surprisingly, removal of the H4 N terminus also derepresses the silent mating type loci, HML alpha and HMRa, disrupting mating. This activation is specific since other regulated genes (GAL10, PHO5, CUP1) are repressed and induced normally in these cells. Deletions of the hydrophilic N termini of H2A or H2B do not show this effect on mating. These experiments allow us to define a unique H4 function that is not shared by other histones (H2A and H2B).


Assuntos
Genes Fúngicos , Genes Fúngicos Tipo Acasalamento , Histonas/análise , Saccharomyces cerevisiae/análise , Cromatina/análise , Deleção Cromossômica , Mapeamento Cromossômico , Nuclease do Micrococo/metabolismo , Transcrição Gênica
9.
Mol Cell Biol ; 8(9): 3717-25, 1988 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2975753

RESUMO

The adenovirus early region 3 (E3) promoter is an early viral promoter which is strongly induced by the adenovirus transactivator protein E1A. DNase I footprinting with HeLa cell extracts has identified four factor-binding domains which appear to be involved in basal and E1A-induced transcriptional regulation. These binding domains may bind TATA region-binding factors (site I), the CREB/ATF protein (site II), the AP-1 protein (site III), and nuclear factor I/CTF (site IV). Recently, it has been shown that the DNA-binding domain of transcription factor AP-1 has homology with the yeast transcription factor GCN4 and that the yeast transactivator protein GAL4 is able to stimulate transcription in HeLa cells from promoters containing GAL4-binding sites. These results suggest an evolutionary conservation of both transcription factors and the mechanisms responsible for transcriptional activation in Saccharomyces cerevisiae and higher eucaryotic organisms. To determine whether similar patterns of transcriptional regulation were seen with the E3 promoter in HeLa and yeast cells, the E3 promoter fused to the chloramphenicol acetyltransferase (cat) gene was cloned into a high-copy-number plasmid and stably introduced into yeast cells. S1 analysis revealed that similar E3 promoter mRNA start sites were found in yeast and HeLa cells. DNase I footprinting with partially purified yeast extracts revealed that four regions of the E3 promoter were protected. Several of these regions were similar to binding sites determined by using HeLa cell extracts. Oligonucleotide mutagenesis of these binding domains indicated their importance in the transcriptional regulation of the E3 promoter in yeast cells. These results suggest that similar cellular transcription factor-binding sites may be involved in the regulation of promoters in both yeast and mammalian cells.


Assuntos
Adenoviridae/genética , Genes Reguladores , Genes Virais , Genes , Proteínas Oncogênicas Virais/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Proteínas Precoces de Adenovirus , Sequência de Bases , Desoxirribonuclease I , Células HeLa/metabolismo , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas
10.
EMBO J ; 7(7): 2211-9, 1988 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-3046933

RESUMO

We have constructed a yeast strain (UKY403) in which the sole histone H4 gene is under control of the GAL1 promoter. This allows the activation of H4 mRNA synthesis on galactose and its repression on glucose. UKY403 cells, pre-synchronized in G1 with alpha-mating factor, have been used to show that glucose treatment results in the loss of approximately half the chromosomal nucleosomes. This depletion is only partially reversible when the H4 gene is reactivated on galactose. It was found that the resultant lethality manifests itself first in S phase, the period of nucleosome assembly, but leads to highly synchronous arrest in G2 and a virtually complete block in chromosomal segregation. Histone H4-depleted chromatin was analyzed for its efficiency as a template for all three RNA polymerases. Using pulse-labeling, we find no evidence for altered transcription by RNA polymerase I (25S, 18S and 5.8S rRNAs) or RNA polymerase III (5S rRNA, tRNAs). Northern blot analysis was used to measure levels of RNA polymerase II transcripts. There was little effect on the activation or repression of the CUP1 chelatin gene. While there may be some decrease in the level of certain mRNAs (e.g. HIS4, ARG4) other message levels (HIS3, TRP1) show little change upon glucose repression. Therefore, nucleosome loss certainly does not have a general effect on transcription.


Assuntos
Genes Fúngicos , Genes , Histonas/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Ciclo Celular , Genótipo , Histonas/fisiologia , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Especificidade da Espécie
11.
EMBO J ; 7(7): 2221-8, 1988 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-3046934

RESUMO

We have previously constructed a yeast strain (UKY403) whose sole histone H4 gene is under control of the GAL1 promoter. This yeast arrests in G2 upon glucose treatment as a result of histone H4 depletion. The yeast PHO5 gene contains phase nucleosomes covering promoter (UAS) sequences in the PHO5 repressed state and it has been suggested that nucleosomes prevent the binding of positively acting factors to these UAS sequences. Using UKY403 we examined the length of polynucleosomes and nucleosome phasing in the PHO5 upstream region by the use of micrococcal nuclease and indirect end-labeling. It was found that glucose arrest led to a severe disruption in PHO5 chromatin structure and that most nucleosomes had their position altered or were lost from the PHO5 promoter region. Cell undergoing nucleosome depletion synthesized large quantities of accurate PHO5 transcripts even under repressive, high inorganic phosphate conditions. Histone H4 depletion did not appear to affect the repression or activation of another inducible yeast gene, CUP1. Arrest with landmarks in early G1 (in the cell division cycle mutant cdc28) or in various stages of G2 (in cdc15, cdc17 and cdc20) does not activate PHO5; nor does arrest due to chromosome topology changes (in top2 or the top1top2 topoisomerase mutants). cdc14, which has its arrest landmark at a similar point in the cell cycle as cdc15, does derepress PHO5. However, since it also leads to derepression of CUP1 it is probably functioning through an independent mechanism. Therefore, our data suggest that nucleosomes regulate PHO5 transcription.


Assuntos
Regulação da Expressão Gênica , Genes Fúngicos , Genes , Histonas/genética , Nucleossomos/fisiologia , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Histonas/fisiologia , Mutação , Especificidade da Espécie , Transcrição Gênica
12.
Gene ; 46(2-3): 297-300, 1986.
Artigo em Inglês | MEDLINE | ID: mdl-2879772

RESUMO

To aid in the interpretation of the 3.5 A resolution electron density map of glutamine synthetase (GS) from Salmonella typhimurium, the nucleotide sequence of the gene coding for this enzyme has been determined. The predicted sequence of 468 amino acids (Mr = 51,628) has been compared to the sequence and sequence fragments reported by others for GS of Anabaena and Escherichia coli. The homology between the pairs of sequences is sufficiently strong to suggest that the overall three-dimensional structures of the three GS are similar. The predicted positions of alpha helices are in moderately good agreement with the electron-density map.


Assuntos
Genes Bacterianos , Glutamato-Amônia Ligase/genética , Salmonella typhimurium/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Glutamato-Amônia Ligase/metabolismo , Conformação Proteica , Salmonella typhimurium/enzimologia
13.
Mol Biochem Parasitol ; 13(3): 263-75, 1984 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-6396515

RESUMO

Four fragments from the maxicircle DNA of Leishmania tarentolae cloned into the selectable Saccharomyces cerevisiae shuttle vector, YIp5, exhibited autonomous replicating sequence (ars) activity. Two of the fragments (pSK120, pSK152) produced large yeast transformant colonies and two (pSK30, pSK150) produced small colonies. All yeast transformants contained extrachromosomal self replicating YIp5 hybrid plasmids as shown by mitotic instability in non selective medium and by the transformation of Escherichia coli with yeast minilysates and recovery of the plasmid from the transformed bacteria. The copy numbers of pSK30, pSK150 and pSK152 in the transformed yeast were approximately the same as that of the YRp12 control, which contains the yeast arsl element; the copy number of pSK120, however, was at least 10 fold lower. A 1.87 kb subfragment of the pSK120 fragment also showed strong ars activity. The entire DNA sequences of the pSK120, pSK152 and pSK150 fragments are known, and several yeast 11 mer consensus ars sequences are present within each fragment. In addition there is a sequence (Lt ars 189) within the pSK152 subclone that has 78% similarity with a 189 nt sequence of an ars element from the Crithidia fasciculata maxicircle (Cf ars 189), implying an evolutionary conservation of this putative origin of replication in at least two different kinetoplastid species. The relative positions of the Lt ars 189 sequence in the L. tarentolae maxicircle map and the Cf ars 189 sequence in the C. fasciculata map with respect to the 9 and 12 S ribosomal genes are similar, implying an overall conservation of gene order in this portion of the transcribed regions of these two species and perhaps in all kinetoplastid species.


Assuntos
Replicação do DNA , Leishmania/genética , Animais , Sequência de Bases , Crithidia/genética , Plasmídeos , Saccharomyces cerevisiae/genética , Transcrição Gênica , Transformação Genética
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