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1.
Mol Microbiol ; 42(4): 1133-45, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11737652

RESUMO

The type III secretion (TTS) system of Gram-negative pathogenic bacteria is composed of proteins that assemble into the TTS machinery, proteins that are secreted by this machinery and specific chaperones that are required for storage and sometimes secretion of these proteins. Many sequential protein interactions are involved in the TTS pathway to deliver effector proteins to host cells. We used the yeast two-hybrid system to investigate the interaction partners of the Shigella flexneri effectors and chaperones. Libraries of preys containing random fusions with fragments of the TTS proteins were screened using effectors and chaperones as baits. Interactions between the effectors IpaB and IpaC and their chaperone IpgC were detected by this method, and interaction domains were identified. Using a His-tagged IpgC protein to co-purify truncated IpaB and IpaC proteins, we showed that the chaperone-binding domain was unique and located in the N-terminus of these proteins. This domain was not required for the secretion of recombinant proteins but was involved in the stability of IpaC and instability of IpaB. Homotypic interactions were identified with the baits IpaA, IpaB and IpaC. Interactions between effectors and components of the TTS machinery were also selected that might give insights into regulation of the TTS process.


Assuntos
Chaperonas Moleculares/metabolismo , Shigella flexneri/metabolismo , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biblioteca Gênica , Plasmídeos/genética , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Shigella flexneri/química , Shigella flexneri/patogenicidade , Técnicas do Sistema de Duplo-Híbrido
2.
Curr Biol ; 11(23): 1885-90, 2001 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-11728313

RESUMO

Cells have a recurrent need for the correct assembly of protein-nucleic acid complexes. We have studied a yeast homolog of the smallest subunit of chromatin assembly factor 1 (CAF1), encoded by YMR131c and termed "RRB1". Unlike other yeast homologs, Msi1p, and Hat2p, Rrb1p is essential for cell viability. Impairment of Rrb1p function results in decreased levels of free 60S ribosomal subunits and the appearance of half-mer polysomes, suggesting its involvement in ribosome biogenesis. Using tandem affinity purification (TAP ) combined with mass spectrometry, we show that Rrb1p is associated with ribosomal protein L3. A fraction of Rrb1p is also found in a protein-precursor rRNA complex containing at least ten other early-assembling ribosomal proteins. We propose that Rrb1p is required for proper assembly of preribosomal particles during early ribosome biogenesis, presumably by targeting L3 onto the 35S precursor rRNA. This action may resemble the mechanism by which CAF1 assembles histones H3/H4 onto newly replicated DNA.


Assuntos
Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA/fisiologia , Ribossomos/metabolismo , Saccharomyces cerevisiae/fisiologia , Sequência de Bases , Fator 1 de Modelagem da Cromatina , Primers do DNA , Espectrometria de Massas , Hibridização de Ácido Nucleico , Precursores de RNA/metabolismo , RNA Ribossômico/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ribossômicas/isolamento & purificação , Proteínas Ribossômicas/metabolismo
3.
EMBO J ; 20(22): 6475-84, 2001 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-11707418

RESUMO

Eukaryotic ribosome maturation depends on a set of well ordered processing steps. Here we describe the functional characterization of yeast Nog2p (Ynr053cp), a highly conserved nuclear protein. Nog2p contains a putative GTP-binding site, which is essential in vivo. Kinetic and steady-state measurements of the levels of pre-rRNAs in Nog2p-depleted cells showed a defect in 5.8S and 25S maturation and a concomitant increase in the levels of both 27SB(S) and 7S(S) precursors. We found Nog2p physically associated with large pre-60S complexes highly enriched in the 27SB and 7S rRNA precursors. These complexes contained, besides a subset of ribosomal proteins, at least two additional factors, Nog1p, another putative GTP-binding protein, and Rlp24p (Ylr009wp), which belongs to the Rpl24e family of archaeal and eukaryotic ribosomal proteins. In the absence of Nog2p, the pre-60S ribosomal complexes left the nucleolus, but were retained in the nucleoplasm. These results suggest that transient, possibly GTP-dependent association of Nog2p with the pre-ribosomes might trigger late rRNA maturation steps in ribosomal large subunit biogenesis.


Assuntos
GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Transporte Ativo do Núcleo Celular , Processamento Alternativo , Sequência de Aminoácidos , Sítios de Ligação , Northern Blotting , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , DNA Complementar/metabolismo , GTP Fosfo-Hidrolases/química , Proteínas de Ligação ao GTP/metabolismo , Genótipo , Glucose/metabolismo , Proteínas de Fluorescência Verde , Humanos , Hibridização In Situ , Cinética , Proteínas Luminescentes/metabolismo , Espectrometria de Massas , Microscopia Eletrônica , Microscopia de Fluorescência , Modelos Genéticos , Dados de Sequência Molecular , Plasmídeos/metabolismo , Polirribossomos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA/metabolismo , RNA Mensageiro/metabolismo , RNA Ribossômico/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Tempo
4.
J Cell Biol ; 154(6): 1147-60, 2001 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-11564755

RESUMO

The nuclear pore complexes (NPCs) are evolutionarily conserved assemblies that allow traffic between the cytoplasm and the nucleus. In this study, we have identified and characterized a novel human nuclear pore protein, hNup133, through its homology with the Saccharomyces cerevisiae nucleoporin scNup133. Two-hybrid screens and immunoprecipitation experiments revealed a direct and evolutionarily conserved interaction between Nup133 and Nup84/Nup107 and indicated that hNup133 and hNup107 are part of a NPC subcomplex that contains two other nucleoporins (the previously characterized hNup96 and a novel nucleoporin designated as hNup120) homologous to constituents of the scNup84 subcomplex. We further demonstrate that hNup133 and hNup107 are localized on both sides of the NPC to which they are stably associated at interphase, remain associated as part of a NPC subcomplex during mitosis, and are targeted at early stages to the reforming nuclear envelope. Throughout mitosis, a fraction of hNup133 and hNup107 localizes to the kinetochores, thus revealing an unexpected connection between structural NPCs constituents and kinetochores. Photobleaching experiments further showed that the mitotic cytoplasm contains kinetochore-binding competent hNup133 molecules and that in contrast to its stable association with the NPCs the interaction of this nucleoporin with kinetochores is dynamic.


Assuntos
Evolução Molecular , Cinetocoros/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Células HeLa , Humanos , Cinetocoros/química , Cinetocoros/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Microscopia de Fluorescência , Mitose , Membrana Nuclear/metabolismo , Poro Nuclear/química , Poro Nuclear/genética , Proteínas Nucleares/química , Proteínas Nucleares/fisiologia , Testes de Precipitina , Ligação Proteica , Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-Híbrido
5.
J Biol Chem ; 276(19): 15861-7, 2001 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-11278769

RESUMO

Mitochondrial ribosomal proteins were studied best in yeast, where the small subunit was shown to contain about 35 proteins. Yet, genetic and biochemical studies identified only 14 proteins, half of which were predictable by sequence homology with prokaryotic ribosomal components of the small subunit. Using a recently described affinity purification technique and tagged versions of yeast Ykl155c and Mrp1, we isolated this mitochondrial ribosomal subunit and identified a total of 20 proteins, of which 12 are new. For a subset of the newly described ribosomal proteins, we showed that they are localized in mitochondria and are required for the respiratory competency of the yeast cells. This brings to 26 the total number of proteins described as components of the mitochondrial small ribosomal subunit. Remarkably, almost half of the previously and newly identified mitochondrial ribosomal components showed no similarity to any known ribosomal protein. Homologues could be found, however, in predicted protein sequences from Schizosaccharomyces pombe. In more distant species, putative homologues were detected for Ykl155c, which shares conserved motifs with uncharacterized proteins of higher eukaryotes including humans. Another newly identified ribosomal protein, Ygl129c, was previously shown to be a member of the DAP-3 family of mitochondrial apoptosis mediators.


Assuntos
Proteínas Fúngicas/genética , Mitocôndrias/genética , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Sequência de Aminoácidos , Animais , Proteínas Fúngicas/química , Deleção de Genes , Genes Fúngicos , Genótipo , Humanos , Mamíferos , Dados de Sequência Molecular , Células Procarióticas , Proteínas Ribossômicas/química , Ribossomos/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
6.
Yeast ; 17(2): 95-110, 2000 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-10900456

RESUMO

A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (Like Sm) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein-protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale.


Assuntos
Proteínas Fúngicas/metabolismo , Genoma Fúngico , RNA Mensageiro/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Fúngicas/genética , Proteoma/análise , Splicing de RNA , RNA Fúngico/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-Híbrido
7.
Proc Natl Acad Sci U S A ; 95(7): 3752-7, 1998 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-9520439

RESUMO

Thousands of genes have recently been sequenced in organisms ranging from Escherichia coli to human. For the majority of these genes, however, available sequence does not define a biological role. Efficient functional characterization of these genes requires strategies for scaling genetic analyses to the whole genome level. Plasmid-based library selections are an established approach to the functional analysis of uncharacterized genes and can help elucidate biological function by identifying, for example, physical interactors for a gene and genetic enhancers and suppressors of mutant phenotypes. The application of these selections to every gene in a eukaryotic genome, however, is generally limited by the need to manipulate and sequence hundreds of DNA plasmids. We present an alternative approach in which identification of nucleic acids is accomplished by direct hybridization to high-density oligonucleotide arrays. Based on the complete sequence of Saccharomyces cerevisiae, high-density arrays containing oligonucleotides complementary to every gene in the yeast genome have been designed and synthesized. Two-hybrid protein-protein interaction screens were carried out for S. cerevisiae genes implicated in mRNA splicing and microtubule assembly. Hybridization of labeled DNA derived from positive clones is sufficient to characterize the results of a screen in a single experiment, allowing rapid determination of both established and previously unknown biological interactions. These results demonstrate the use of oligonucleotide arrays for the analysis of two-hybrid screens. This approach should be generally applicable to the analysis of a range of genetic selections.


Assuntos
Escherichia coli/genética , Genoma , Oligonucleotídeos/genética , Seleção Genética , Animais , Genoma Bacteriano , Genoma Humano , Humanos
8.
Nat Genet ; 16(3): 277-82, 1997 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9207794

RESUMO

The genome of the yeast Saccharomyces cerevisiae is now completely sequenced. Despite successful genetic work in recent years, 60% of yeast genes have no assigned function and half of those encode putative proteins without any homology with known proteins. Genetic analyses, such as suppressor or synthetic lethal screens, have suggested many functional links between gene products, some of which have been confirmed by biochemical means. Altogether, these approaches have led to a fairly extensive knowledge of defined biochemical pathways. However, the integration of these pathways against the background of complexity in a living cell remains to be accomplished. The two-hybrid method applied to the yeast genome might allow the characterization to the network of interactions between yeast proteins, leading to a better understanding of cellular functions. Such an analysis has been performed for the bacteriophage T7 genome that encodes 55 proteins and for Drosophila cell cycle regulators. However, the currently available two-hybrid methodology is not suitable for a large-scale project without specific methodological improvements In particular, the exhaustivity and selectivity of the screens must first be greatly improved. We constructed a new yeast genomic library and developed a highly selective two-hybrid procedure adapted for exhaustive screens of the yeast genome. For each bait we selected a limited set of interacting preys that we classified in categories of distinct heuristic values. Taking into account this classification, new baits were chosen among preys and, in turn, used for second-round screens. Repeating this procedure several times led to the characterization of the network of interactions. Using known pre-mRNA splicing factors as initial baits, we were able to characterize new interactions between known splicing factors, identify new yeast splicing factors, including homologues of human SF1 and SAP49, and reveal novel potential functional links between cellular pathways. Using different cellular pathways as anchor points, this novel strategy allows us to envision the building of an interaction map of the yeast proteome. In addition, this two-hybrid strategy could be applied to other genomes and might help to resolve the human protein linkage map.


Assuntos
Proteínas Fúngicas/metabolismo , Técnicas Genéticas , Genoma Fúngico , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Cruzamentos Genéticos , Proteínas Fúngicas/genética , Biblioteca Genômica , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Splicing de RNA/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Alinhamento de Sequência
9.
Proc Natl Acad Sci U S A ; 94(11): 5628-33, 1997 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-9159123

RESUMO

In yeast, the major mRNA degradation pathway is initiated by poly(A) tail shortening that triggers mRNA decapping. The mRNA is then degraded by 5'-to-3' exonucleolysis. In mammalian cells, even though poly(A) tail shortening also precedes mRNA degradation, the degradation pathway has not been elucidated. We have used a reverse transcription-PCR approach that relies on mRNA circularization to measure the poly(A) tail length of four mammalian mRNAs. This approach allows for the simultaneous analysis of the 5' and 3' ends of the same mRNA molecule. For all four mRNAs analyzed, this strategy permitted us to demonstrate the existence of small amounts of decapped mRNA species which have a shorter poly(A) tail than their capped counterparts. Kinetic analysis of one of these mRNAs indicates that the decapped species with a short poly(A) tail are mRNA degradation products. Therefore, our results indicate that decapping is preceded by a shortening of the poly(A) tail in mammalian cells, as it is in yeast, suggesting that this mRNA degradation pathway is conserved throughout eukaryotic evolution.


Assuntos
Apolipoproteínas/biossíntese , Fígado/metabolismo , Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , Proteína Amiloide A Sérica/biossíntese , Animais , Sequência de Bases , Evolução Biológica , Caseínas , Primers do DNA , Feminino , Inflamação , Cinética , Mamíferos , Camundongos , Camundongos Endogâmicos CBA , Fosfoenolpiruvato Carboxiquinase (GTP)/biossíntese , Plantas Tóxicas , Poli A/metabolismo , Reação em Cadeia da Polimerase , Pirofosfatases/biossíntese , RNA Mensageiro/química , RNA Mensageiro/isolamento & purificação , Albumina Sérica/biossíntese , Nicotiana/enzimologia
10.
Methods ; 11(2): 151-63, 1997 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8993027

RESUMO

Analysis of the interaction of proteins with either DNA or RNA sequences by in vivo footprinting involves two steps: (i) the in situ modification of nucleic acids by the footprinting reagent and (ii) the visualization of the footprints. Ligation-mediated PCR (LM-PCR) procedures provide a level of sensitivity and specificity that is suitable for visualization of footprints of single-copy genes or low-abundance mRNAs in higher eukaryotes. In this article, we discuss several of the technical aspects of these multistep procedures that contribute to the quality of the results, particularly the parameters that affect the specificity and fidelity of the reactions: (i) the design of the primers, which is important to achieve optimal specificity; (ii) the choice of polymerases so that the amplified material represents faithfully the initial nucleic acid population; and (iii) the impact of the plateau effect within the PCR on the interpretation of the data. We then discuss aspects of in vivo nucleic acid manipulation that may affect the quality of the footprinting image, in particular the choice of the footprinting reagent and its condition of use (e.g., on intact or permeabilized cells or prepared nuclei) and the extent of nucleic acid modification. Finally, we provide detailed experimental procedures corresponding to the techniques we have developed or modified: LM-PCR, reverse ligation-mediated PCR, and nuclease treatment of RNAs in vivo.


Assuntos
Pegada de DNA/métodos , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Reação em Cadeia da Polimerase/métodos , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Sequência de Bases , Carcinoma Hepatocelular , Linhagem Celular , Primers do DNA , DNA Polimerase Dirigida por DNA , Desoxirribonuclease I , Humanos , Neoplasias Hepáticas , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/química , Receptores da Transferrina/biossíntese , Receptores da Transferrina/química , Sensibilidade e Especificidade , Taq Polimerase
12.
Proc Natl Acad Sci U S A ; 92(16): 7197-201, 1995 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-7638167

RESUMO

The glucocorticoid-responsive units (GRUs) of the rat tyrosine aminotransferase were associated with the regulatory sequences of a cellular gene expressed ubiquitously--that coding for the largest subunit of RNA polymerase II. In transient expression assays, glucocorticoid responsiveness of the hybrid regulatory regions depends on the spatial relationship and number of regulatory elements. Two parameters affect the ratio of induction by glucocorticoids: the basal level of the hybrid promoter that is affected by the RNA polymerase II regulatory sequences and the glucocorticoid-induced level that depends on the distance between the GRUs and the TATA box. A fully active glucocorticoid-responsive hybrid gene was used to generate transgenic mice. Results show that a composite regulatory pattern is obtained: ubiquitous basal expression characteristic of the RNA polymerase II gene and liver-specific glucocorticoid activation characteristic of the tyrosine aminotransferase GRUs. This result demonstrates that the activity of the tyrosine aminotransferase GRUs is cell-type-specific not only in cultured cells but also in the whole animal.


Assuntos
Elementos Facilitadores Genéticos , Receptores de Glucocorticoides/genética , Animais , Dexametasona/farmacologia , Elementos Facilitadores Genéticos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , TATA Box , Distribuição Tecidual , Tirosina Transaminase/genética
14.
Proc Natl Acad Sci U S A ; 90(8): 3496-500, 1993 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-7682707

RESUMO

We have adapted to RNA molecules the ligation-mediated polymerase chain reaction (LMPCR) procedure of genomic sequencing [Mueller, P. R. & Wold, B. (1989) Science 246, 780-786]. This new procedure, the reverse ligation-mediated PCR (RLPCR), is sufficiently sensitive to allow "in vivo" footprinting of minor RNA species. It is based on the ligation of an RNA linker of known sequence to every 5' end resulting from the cleavage of total cellular RNA. Target RNA molecules are specifically reverse-transcribed and the resulting products are amplified by PCR. The localization of the initial 5' ends is ultimately determined on a sequencing gel. To demonstrate the validity of this strategy, we have used RNase T1 treatment of permeabilized cells and RLPCR and have detected in vivo iron-depletion-dependent footprints on two iron-responsive elements of the transferrin receptor mRNA.


Assuntos
RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores da Transferrina/genética , Sequência de Bases , Carcinoma Hepatocelular , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Desferroxamina/farmacologia , Regulação Neoplásica da Expressão Gênica , Hemina/farmacologia , Humanos , Neoplasias Hepáticas , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/síntese química , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , DNA Polimerase Dirigida por RNA/metabolismo , Ribonuclease T1/metabolismo , Células Tumorais Cultivadas
15.
J Cell Sci ; 101 ( Pt 4): 795-9, 1992 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-1356112

RESUMO

RW cells are pancreatic endocrine RIN cells that have been stably transfected with a chimeric gene that places the expression of the dominant selection gpt gene under the control of the insulin gene regulatory sequences. These RW cells were examined for hormone content using immunocytochemistry. This analysis shows that: first, there are cells that are negative for insulin although they were cultured under selective pressure. Second, there is a higher proportion of somatostatin-producing cells than in the parental RIN cells; these somatostatin cells form two populations: one of cells containing only somatostatin and, surprisingly, one made of cells containing both insulin and somatostatin. Thus: (1) expression of the transfected and endogenous insulin regulatory sequences is not regulated in a coordinate fashion; (2) the presence of both hormones in the same cell suggests that the regulation of the expression of insulin and somatostatin genes and the differentiation pathway of the two respective cell types may be closely related.


Assuntos
Expressão Gênica , Insulina/genética , Pâncreas/metabolismo , Somatostatina/genética , Transcrição Gênica , Animais , Linhagem Celular , Imuno-Histoquímica , Insulina/metabolismo , Microscopia Eletrônica , Pâncreas/citologia , Pâncreas/ultraestrutura , Coelhos , Somatostatina/metabolismo
16.
Mol Endocrinol ; 4(5): 669-77, 1990 May.
Artigo em Inglês | MEDLINE | ID: mdl-2274051

RESUMO

Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific transcripts in pancreas, but not in other tissues, 3) the specific immunofluorescence staining of pancreatic islets for human C-peptide, and 4) the synthesis and accumulation of human (pro)insulin in isolated islets. Deletions in the injected DNA fragment of sequences upstream from positions -353, -258, and -168 allowed correct initiation of the transcripts and cell specificity of expression, while quantitative expression gradually decreased. Deletion to -58 completely abolished the expression of the gene. The amount of human product that in mice harboring the longest fragment contributes up to 50% of the total insulin does not alter the normal proportion of mice insulins I and II. These results suggest that expression of the human insulin gene in vivo results from the cooperation of several cis-regulatory elements present in the various deleted fragments. With none of the deletions used, expression of the transgene was observed in cell types other than beta-islet cells.


Assuntos
Insulina/genética , Animais , Peptídeo C/metabolismo , Peptídeo C/urina , Deleção Cromossômica , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Insulina/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Pâncreas/anatomia & histologia , Pâncreas/metabolismo , RNA Mensageiro/genética
17.
Exp Cell Res ; 180(1): 220-33, 1989 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-2562829

RESUMO

To define a selective system for the study of rat tyrosine aminotransferase (TAT; EC 2.6.1.5) gene expression, we have introduced into cultured cells the selectable bacterial gene gpt linked to TAT gene flanking sequences. After integration in host cell DNA, the chimeric gene exhibits the same pattern of regulation as the TAT gene. In hepatoma cells, its expression is induced after glucocorticoid hormone treatment and repressed after fusion with fibroblasts. In fibroblasts, the chimeric gene is not expressed. The correct pattern of regulation is lost when the number of integrated copies is high. At copy number above 10, the transfected gene responds poorly to glucocorticoids in hepatoma cells. At copy number above 50, the gene is expressed in fibroblasts. Another gene present in the same construction and controlled by the SV40 early promoter and enhancer is positively regulated by glucocorticoids in hepatoma cells but not after fusion with fibroblasts. These data indicate that in hybrid cells, both TAT promoter and glucocorticoid-responsive elements are negatively regulated.


Assuntos
Regulação da Expressão Gênica , Transfecção , Tirosina Transaminase/genética , Animais , Linhagem Celular , Quimera , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Híbridas , Camundongos , Pentosiltransferases/genética , Plasmídeos , Regiões Promotoras Genéticas , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Células Tumorais Cultivadas
19.
Biomed Biochim Acta ; 47(4-5): 349-53, 1988.
Artigo em Inglês | MEDLINE | ID: mdl-3071363

RESUMO

Transgenic mouse lines carrying the human insulin gene flanked by 4 kilobases (kb) in 5' and 5.5 kb in 3' were obtained. The presence of the human C-peptide in serum and urine, and of specific transcripts of the human gene in pancreas RNA indicate that the human DNA fragment contains the sequences necessary for correct phenotypic expression of the gene.


Assuntos
Genes , Insulina/genética , Animais , Southern Blotting , Peptídeo C/sangue , DNA/administração & dosagem , DNA/genética , Sondas de DNA , Humanos , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Transgênicos , Microinjeções , Proteínas Recombinantes/sangue , Transcrição Gênica
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