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T Maniatis

Publications and source records attributed to T Maniatis.

At least 127 records · Page 7Linked to original sources
PubMed1989

The role of specific enhancer-promoter interactions in the Drosophila Adh promoter switch.

The Drosophila melanogaster alcohol dehydrogenase (Adh) gene is transcribed from two promoters active at different developmental stages. In this paper we show that the promoters are differentially stimulated by two enhancers, the Adh larval enhancer and the Adh adult enhancer. In early larval stages, the larval enhancer stimulates transcription from the proximal promoter; in late larval stages, the two enhancers act synergistically to stimulate transcription from the distal promoter; and in adults, the adult enhancer stimulates transcription from the distal promoter. To determine the basis for these enhancer-promoter interactions, we examined the effect of each enhancer on three different promoters. We found that the adult enhancer is stage specific and stimulates transcription from all three promoters. In contrast, the larval enhancer is potentially active in all stages and stimulates transcription from only two of the three promoters. These observations suggest that normal temporal expression of Adh depends on the stage-specific activity of the adult enhancer and the differential response of the proximal and distal promoters to the larval enhancer.

Alcohol Dehydrogenase↗
PubMed1988

Purification and visualization of native spliceosomes.

Mammalian spliceosomes were purified in preparative amounts by gel filtration chromatography and shown to be functional by in vitro complementation experiments. The column fractions containing spliceosomes are enriched in the snRNAs U1, U2, U4, U5, and U6 and a subset of proteins present in the nuclear extract. Splicing intermediates, the entire set of snRNAs, and the enriched proteins can be immunoprecipitated with three different monoclonal antibodies that recognize snRNP determinants. At least one U1 snRNP is present in each spliceosome since the particles are quantitatively immunoprecipitated by an anti-U1 snRNP monoclonal antibody. Examination of the spliceosome fractions by EM revealed a relatively homogeneous population of 40-60 nm particles with a striking morphology. Evidence that these particles are spliceosomes is their sensitivity to micrococcal nuclease, their ATP-dependent assembly, and their immunoprecipitation with a trimethyl cap monoclonal antibody. In addition, pre-mRNA was visualized in the particles by EM.

Adenosine Triphosphate↗
PubMed1988

Drosophila Adh: a promoter element expands the tissue specificity of an enhancer.

Drosophila alcohol dehydrogenase (Adh) genes are expressed in the fat body and in species-specific sets of other tissues during larval and adult development. The Drosophila mulleri Adh-1 gene is expressed in the larval fat body and in three other larval tissues. In this paper, we show that Adh-1 expression in multiple cell types is the result of synergistic interactions between fat body-specific enhancers and a specific Adh-1 promoter element.

Alcohol Dehydrogenase↗
PubMed1988

GAL4 activates transcription in Drosophila.

GAL4 is a yeast regulatory protein that binds to specific sites within a DNA sequence called UASG (galactose upstream activating sequence) and activates transcription of linked genes. This activation requires two functions of the protein: a DNA binding domain located near the amino terminus, and one or more 'activating regions'. The 'activating regions' are highly acidic (see also ref. 12) and can be replaced, for example, by a short peptide designed to form a negatively charged, amphipathic alpha-helix. GAL4, as well as deletion derivatives bearing one or more 'activating regions' attached to the DNA binding domain, activates transcription in cultured mammalian cells from mammalian promoters linked to a UASG (refs 14, 15). Here we show that GAL4, when expressed in particular tissues of Drosophila larvae, stimulates tissue-specific transcription of a Drosophila promoter linked to GAL4 binding sites.

Animals↗
PubMed1988

2-Aminopurine selectively inhibits the induction of beta-interferon, c-fos, and c-myc gene expression.

The protein kinase inhibitor 2-aminopurine (2AP) blocks the induction of the human beta-interferon gene by virus or poly(I)-poly(C) at the level of transcription. This inhibition is specific, since 2AP does not inhibit induction of either the hsp70 heat-shock gene by high temperature or the metallothionein gene by cadmium or dexamethasone. However, 2AP does block the induction of the c-fos and c-myc proto-oncogenes by serum growth factors or virus, suggesting that a protein kinase may be involved in the regulation of these genes, as well as of the beta-interferon gene. However, different factors must be required for the induction of these three genes, since they are not coordinately regulated by the same inducers in most of the cell lines examined.

2-Aminopurine↗
PubMed1988

Identification of an inducible factor that binds to a positive regulatory element of the human beta-interferon gene.

Human beta-interferon gene expression is induced by virus or poly(I).poly(C). This induction is due at least in part to an increase in the rate of transcription and does not require protein synthesis. A 40-base-pair DNA sequence within the beta-interferon promoter, termed the interferon gene regulatory element (IRE), is an inducible enhancer in mouse fibroblasts, and both positive and negative regulatory DNA sequences have been identified within this element. In this paper we identify three factors that bind specifically to two positive regulatory domains within the IRE. Two of these factors are present in nuclear extracts prepared from uninduced and induced cells; one is present only in extracts from induced cells. The functional significance of these binding activities was demonstrated by showing that point mutations within the IRE that decrease human beta-interferon gene transcription in vivo prevent binding in vitro. We propose that induction of the beta-interferon gene involves the modification of a protein to a form that binds specifically to a positive regulatory sequence within the IRE.

Base Sequence↗
PubMed1988

Identification of a functional mammalian spliceosome containing unspliced pre-mRNA.

Functional 60S spliceosomes were assembled under conditions that block the first step of the mRNA splicing reaction. This block was imposed by carrying out the splicing reaction in nuclear extracts preincubated in 2.5 mM EDTA. Preparative amounts of the spliceosomes were isolated by gel filtration chromatography and shown to be functional by in vitro complementation assays. The unspliced pre-mRNA in the complex was converted to spliced products when incubated in cytoplasmic S100 extracts or in heat-treated or micrococcal nuclease-treated nuclear extracts. The latter result, in conjunction with the size of the complex, suggests that the spliceosome contains all of the small nuclear ribonucleoproteins (snRNPs) required for both steps of the splicing reaction. Biochemical characterization of the 5' cleavage reaction revealed that ATP and MgCl2 are required for this step in the splicing pathway. The presence of U1 snRNP in the blocked complex was demonstrated by quantitative immunoprecipitation of this complex by an anti-U1 snRNP monoclonal antibody.

Animals↗
PubMed1988

Overlapping positive and negative regulatory domains of the human beta-interferon gene.

Virus or poly(I).poly(C) induction of human beta-interferon gene expression requires a 40-base-pair DNA sequence designated the interferon gene regulatory element (IRE). Previous studies have shown that the IRE contains both positive and negative regulatory DNA sequences. To localize these sequences and study their interactions, we have examined the effects of a large number of single-base mutations within the IRE on beta-interferon gene regulation. We find that the IRE consists of two genetically separable positive regulatory domains and an overlapping negative control sequence. We propose that the beta-interferon gene is switched off in uninduced cells by a repressor that blocks the interaction between one of the two positive regulatory sequences and a specific transcription factor. Induction would then lead to inactivation or displacement of the repressor and binding of transcription factors to both positive regulatory domains.

DNA Mutational Analysis↗
PubMed1988

The role of the mammalian branchpoint sequence in pre-mRNA splicing.

We show that base substitutions in the mammalian branchpoint sequence (BPS) YNCUGAC dramatically reduce the efficiency of pre-mRNA splicing in vitro and alter 3' splice-site selection in vivo. Contrary to current dogma that an adenine residue at the appropriate distance from the 3' splice site is the primary determinant of lariat formation, we find that many mutations in the BPS virtually abolish splicing even though the position of this adenine is unchanged. Comparison of the analogous single-base changes in the mammalian and yeast BPSs revealed similar relative effects on splicing efficiency. However, in contrast to yeast, mammalian branchpoint mutations that decrease splicing efficiency severely do not prevent spliceosome assembly. Thus, mutations in the mammalian BPS appear to uncouple spliceosome assembly from cleavage at the 5' splice site and lariat formation.

Adenine↗
PubMed1988

Inspiratory muscle strength in asthma.

Augmentation of inspiratory muscle strength (Pimax) represents an adaptive response to airway obstruction. We explore the possibility that respiratory muscle weakness may herald hospital admission during acute bronchospasm. The Pimax measured 81 +/- 25 percent of a predicted value in 20 patients with acute bronchospasm (forced expiratory volume in one second, 36 +/- 17 percent predicted). Pimax was related to both hyperinflation (functional residual capacity, as percent predicted) and body weight (subjects were 122 +/- 29 percent ideal body weight), but not to the degree of airway obstruction per se. Furthermore, measurements of axial (craniocaudal) motion of the rib cage and asynchrony of rib cage and abdominal motions during tidal breathing did not correlate with either the degree of air flow obstruction or Pimax. We conclude that little if any respiratory muscle weakness occurs with bronchospasm. Furthermore, Pimax does not correlate with the degree of airway obstruction and does not explain abnormalities of rib cage and abdominal motion associated with asthma.

Abdominal Muscles↗
PubMed1987

Regulation of inducible and tissue-specific gene expression.

Molecular genetics approaches have been used to identify and characterize cis-acting DNA sequences required for eukaryotic gene regulation. These sequences are modular in nature, consisting of arrays of short (10- to 12-base pair) recognition elements that interact with specific transcription factors. Some transcription factors have been extensively purified and the corresponding genes have been cloned, but the mechanisms by which they promote transcription are not yet understood. Positive and negative regulatory elements that function only in specific cell types or in response to extracellular inducers have been identified. A number of cases of inducible and tissue-specific gene expression involve the activation of preexisting transcription factors, rather than the synthesis of new proteins. This activation may involve covalent modification of the protein or an allosteric change in its structure. The modification of regulatory proteins may play a central role in the mechanisms of eukaryotic gene regulation.

Animals↗
PubMed1987

Drosophila Krüppel gene product produced in a baculovirus expression system is a nuclear phosphoprotein that binds to DNA.

The product of the Drosophila segmentation gene Krüppel was produced in cultured insect cells using the baculovirus expression system. When a cloned Krüppel cDNA sequence was inserted into the viral genome downstream from the promoter of the polyhedrin gene, a polypeptide with an apparent molecular weight of approximately equal to 72,000 was observed in the nuclei of infected cells. Antibodies were raised against this protein and used to detect Krüppel in Drosophila embryos. Characterization of the Krüppel protein extracted from infected cells showed that it is tightly bound to the nucleus, it binds to calf thymus DNA-cellulose, and it is phosphorylated. These results support the hypothesis that Krüppel is a regulatory protein that acts by binding DNA.

Animals↗
PubMed1987

A new mutation in IVS-1 of the human beta globin gene causing beta thalassemia due to abnormal splicing.

A G to T transversion at the fifth nucleotide of the first intervening sequence (IVS-1) of the beta-globin gene has been identified in cloned beta-thalassemia genes of two unrelated individuals, one of Mediterranean and the other of Anglo Saxon ancestry. In each patient the mutation was present in a different beta globin gene framework, defined by intragenic restriction site polymorphisms, thereby suggesting the occurrence of independent mutations. The study of the RNA products of one of these cloned genes, after transfer and transient expression in HeLa cells, showed partial inactivation of the normal donor splice site of IVS-1 and activation of two major and one minor cryptic splice sites. Only one of the two major cryptic sites was utilized in a cell-free splicing extract. The effects of this mutation on messenger RNA (mRNA) splicing are similar to that of another beta thalassemia gene with a G to C transition at the same position.

Base Sequence↗
PubMed1986

A role for exon sequences and splice-site proximity in splice-site selection.

Analysis of the in vitro splicing products of RNA precursors containing tandem duplications of the 5' or 3' splice sites of human beta-globin IVS 1 revealed that exon sequences play an important role in the relative use of the duplicated sites. These studies also show that the proximity of the 5' and 3' splice sites is an important determinant in the selection of splice-sites. Deletion, substitution, or even subtle changes of exon sequences can alter the pattern of splice-site selection, and in many cases the splice site adjacent to the altered exon is not used. The relative use of the duplicated splice sites can also be altered by diluting the splicing extract, suggesting that factors involved in splice-site selection are limiting.

Base Sequence↗
PubMed1986

Rapid reprogramming of globin gene expression in transient heterokaryons.

Interspecific heterokaryons were formed by fusing adult mouse erythroleukemia (MEL) cells and human embryonic/fetal erythroid (K562) cells with each other, or with a variety of mouse and human nonerythroid cell types. Analysis of total cellular RNA isolated 24 hr after fusion revealed that normally inactive globin genes can be activated in these "transient" heterokaryons, in which the nuclei do not fuse. In general, the types of globin genes expressed in the donor erythroid cell are activated in the nucleus of the recipient cell. Therefore, erythroid cells contain transacting regulatory factors that are capable of activating the expression of globin genes in a stage- and tissue-specific manner. These observations also indicate that globin genes are not irreversibly repressed in differentiated cells and that their expression can be rapidly reprogrammed in the presence of the appropriate regulatory factors.

Animals↗
PubMed1986

Tuberculosis as a manifestation of the acquired immunodeficiency syndrome (AIDS).

Tuberculosis has not been well documented as a complication of the acquired immunodeficiency syndrome (AIDS). We studied 48 cases of mycobacterial diseases among a group of 136 adult patients with AIDS over a 43-month period. Twenty-nine of them had severe and unusual manifestations of disease due to Mycobacterium tuberculosis, predominantly extrapulmonary and disseminated. Tuberculosis was more common among Haitians (4/8) and intravenous drug abusers (24/102) than among homosexuals who did not abuse drugs (0/22). Twelve of 21 patients with tuberculosis who were treated responded well, whereas three developed progressive disease indicative of treatment failure. Severe and unusual presentation of overwhelming tuberculosis in appropriate clinical circumstances may be considered an infection predictive of the presence of AIDS.

Acquired Immunodeficiency Syndrome↗