Search PubMed⌕ Search

Biomedical subjects

T Maniatis

Publications and source records attributed to T Maniatis.

At least 91 records · Page 5Linked to original sources
PubMed1992

Isolation of a complementary DNA that encodes the mammalian splicing factor SC35.

The mammalian splicing factor SC35 is required for the first step in the splicing reaction and for spliceosome assembly. The cloning and characterization of a complementary DNA encoding this protein revealed that it is a member of a family of splicing factors that includes mammalian SF2/ASF. This family of proteins is characterized by the presence of a ribonucleoprotein (RNP)-type RNA binding motif and a carboxyl-terminal serine-arginine-rich (SR) domain. A search of the DNA sequence database revealed that the thymus-specific exon (ET) of the c-myb proto-oncogene is encoded on the antisense strand of the SC35 gene.

Amino Acid Sequence↗
PubMed1992

Positive control of pre-mRNA splicing in vitro.

Positive control of the sex-specific alternative splicing of doublesex (dsx) precursor messenger RNA (pre-mRNA) in Drosophila melanogaster involves the activation of a female-specific 3' splice site by the products of the transformer (tra) and transformer-2 (tra-2) genes. The mechanisms of this process were investigated in an in vitro system in which the female-specific 3' splice site could be activated by recombinant Tra or Tra-2 (or both). An exon sequence essential for regulation in vivo was shown to be both necessary and sufficient for activation in vitro. Nuclear proteins in addition to Tra and Tra-2 were found to bind specifically to this exon sequence. Therefore, Tra and Tra-2 may act by promoting the assembly of a multiprotein complex on the exon sequence. This complex may facilitate recognition of the adjacent 3' splice site by the splicing machinery.

Animals↗
PubMed1992

An ATF/CREB binding site is required for virus induction of the human interferon beta gene [corrected].

We report the characterization of a distinct regulatory element of the human interferon beta (HuIFN-beta) gene promoter, which we designate PRDIV (positive regulatory domain IV). In previous studies, sequences between -104 and -91 base pairs upstream from the start site of transcription were shown to be required for maximal levels of virus induction in mouse L929 cells. We have localized the essential sequence in this region extending from -99 to at least -91, and we show that this sequence is a binding site for a protein of the activating transcription factor/cAMP response element binding protein (ATF/CREB) family of transcription factors. Mutations in PRDIV that decrease the affinity of one member of this family (ATF-2/CRE-BP1) decrease the level of virus induction in vivo. Moreover, multiple copies of PRDIV can confer both virus and cAMP inducibility upon a minimal promoter in L929 cells, while it is constitutively active in HeLa cells. We conclude that PRDIV is a distinct regulatory element of the HuIFN-beta promoter and that the signal transduction pathways involved in virus and cAMP induction may partially overlap.

Animals↗
PubMed1992

The 35-kDa mammalian splicing factor SC35 mediates specific interactions between U1 and U2 small nuclear ribonucleoprotein particles at the 3' splice site.

The splicing factor SC35 is required for the first step of the splicing reaction and for the assembly of the earliest ATP-dependent complex detected by native gel electrophoresis (A complex). Here we investigate the role of SC35 in mediating specific interactions between U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) and the 5' and 3' splice sites of pre-mRNA. We show that U1 snRNP interacts specifically with both the 5' and 3' splice sites in the presence of ATP and that SC35 is required for these ATP-dependent interactions. Significantly, the SC35-dependent interaction between U1 snRNP and the 3' splice site requires U2 snRNP but not the 5' splice site. We also show that SC35 is required for the ATP-dependent interaction between U2 snRNP and the branch-point sequence. We conclude that SC35 may play an important role in mediating specific interactions between splicing components bound to the 5' and 3' splice sites.

Adenosine Triphosphate↗
PubMed1992

Rearrangement of upstream regulatory elements leads to ectopic expression of the Drosophila mulleri Adh-2 gene.

The Adh-2 gene of Drosophila mulleri is expressed in the larval fat body and the adult fat body and hindgut, and a 1500-bp element located 2-3 kb upstream of the Adh-2 promoter is necessary for maximal levels of transcription. Previous work demonstrated that deletion of sequences between this upstream element and the Adh-2 promoter results in Adh-2 gene expression in a novel larval tissue, the middle midgut. In this study we show that the upstream element possesses all of the characteristics of a transcriptional enhancer: its activity is independent of orientation, it acts on a heterologous promoter, and it functions at various positions both 5' and 3' to the Adh-2 gene. Full enhancer function can be localized to a 750-bp element, although other regions possess some redundant activity. The ectopic expression pattern is dependent on the proximity of at least two sequence elements. Thus, tissue-specific transcription can involve complex proximity-dependent interactions among combinations of regulatory elements.

Animals↗
PubMed1992

A conserved regulatory unit implicated in tissue-specific gene expression in Drosophila and man.

The Drosophila melanogaster alcohol dehydrogenase (Adh) gene is expressed in a specific set of tissues during larval development and in adults. Expression in the adult fat body is controlled by the Adh adult enhancer (AAE). Previous studies identified a negative regulatory element in the AAE and a protein that binds specifically to this sequence [adult enhancer factor-1 (AEF-1)]. Here, we show that the AEF-1-binding site in the AAE and in two other Drosophila fat body enhancers overlaps a sequence recognized by the mammalian transcription factor CCAAT/enhancer-binding protein (C/EBP). Remarkably, these two proteins also bind specifically to overlapping sites in a liver-specific regulatory element of the human Adh gene. Cotransfection experiments in mammalian cells reveal that C/EBP stimulates the activity of the AAE by 50-fold, and this activity can be suppressed by AEF-1. In addition, AEF-1 prevents C/EBP binding in vitro, and displaces prebound C/EBP. Thus, a tissue-specific regulatory unit consisting of one positive and one negative regulatory element has been conserved between Drosophila and man.

Alcohol Dehydrogenase↗
PubMed1992

A Drosophila CREB/ATF transcriptional activator binds to both fat body- and liver-specific regulatory elements.

We have identified a Drosophila transcription factor that binds to fat body-specific enhancers of alcohol dehydrogenase (Adh) and yolk protein genes. DNA sequence analysis of cDNA clones encoding this protein, box B-binding factor-2 (BBF-2), indicates that it is a member of the CREB/ATF family of transcriptional regulatory proteins. A number of observations suggest that BBF-2 is involved in fat body-specific expression: Mutations that disrupt BBF-2 binding to two different Adh fat body enhancers in vitro decrease the activity of these enhancers in transgenic flies. BBF-2 mRNA is present in all cell types examined, and the protein is present in cells that express ADH. Finally, BBF-2 is a transcriptional activator in Drosophila tissue culture cells. Remarkably, BBF-2 also binds specifically to regulatory elements required for liver-specific expression of the human Adh and rat tyrosine aminotransferase genes. Thus, BBF-2 and the DNA sequence to which it binds may be important components of a tissue-specific regulatory mechanism conserved between Drosophila and man.

Activating Transcription Factors↗
PubMed1992

Only two of the five zinc fingers of the eukaryotic transcriptional repressor PRDI-BF1 are required for sequence-specific DNA binding.

The eukaryotic transcriptional repressor PRDI-BF1 contains five zinc fingers of the C2H2 type, and the protein binds specifically to PRDI, a 14-bp regulatory element of the beta interferon gene promoter. We have investigated the amino acid sequence requirements for specific binding to PRDI and found that the five zinc fingers and a short stretch of amino acids N terminal to the first finger are necessary and sufficient for PRDI-specific binding. The contribution of individual zinc fingers to DNA binding was investigated by inserting them in various combinations into another zinc finger-containing DNA-binding protein whose own fingers had been removed. We found that insertion of PRDI-BF1 zinc fingers 1 and 2 confer PRDI-binding activity on the recipient protein. In contrast, the insertion of PRDI-BF1 zinc fingers 2 through 5, the insertion of zinc finger 1 or 2 alone, and the insertion of zinc fingers 1 and 2 in reverse order did not confer PRDI-binding activity. We conclude that the first two PRDI-BF1 zinc fingers together are sufficient for the sequence-specific recognition of PRDI.

Amino Acid Sequence↗
PubMed1992

Inducible processing of interferon regulatory factor-2.

PRDI-BFc and PRDI-BFi are proteins that bind specifically to a regulatory element required for virus induction of the human beta interferon (IFN-beta). PRDI-BFc is a constitutive binding activity, while the PRDI-BFi binding activity is observed only after cells are treated with inducers such as virus or poly(I).poly(C) plus cycloheximide or in some cells by cycloheximide alone. In this paper we report that PRDI-BFc is interferon regulatory factor-2 (IRF-2), a known transcriptional repressor. In addition, we find that PRDI-BFi is a truncated form of IRF-2, lacking approximately 185 C-terminal amino acids. Thus, PRDI-BFi appears to be generated by inducible proteolysis. Although the affinity of PRDI-BFc/IRF-2 for the IFN-beta promoter does not appear to be affected by the removal of C-terminal amino acids, the ability of PRDI-BFi to function as a repressor in cotransfection experiments is significantly less than that of intact IRF-2. Studies have shown that IRF-2 can block the activity of the transcriptional activator IRF-1, which also binds specifically to the IFN-beta gene promoter. Thus, the inducible proteolysis of IRF-2 may be involved in the regulation of the IFN-beta gene or of other genes in which the ratio of IRF-1 to IRF-2 can affect the level of transcription.

Animals↗
PubMed1992

Drosophila transcriptional repressor protein that binds specifically to negative control elements in fat body enhancers.

Expression of the Drosophila melanogaster Adh gene in adults requires a fat body-specific enhancer called the Adh adult enhancer (AAE). We have identified a protein in Drosophila nuclear extracts that binds specifically to a site within the AAE (adult enhancer factor 1 [AEF-1]). In addition, we have shown that AEF-1 binds specifically to two other Drosophila fat body enhancers. Base substitutions in the AEF-1 binding site that disrupt AEF-1 binding in vitro result in a significant increase in the level of Adh expression in vivo. Thus, the AEF-1 binding site is a negative regulatory element within the AAE. A cDNA encoding the AEF-1 protein was isolated and shown to act as a repressor of the AAE in cotransfection studies. The AEF-1 protein contains four zinc fingers and an alanine-rich sequence. The latter motif is found in other eukaryotic proteins known to be transcriptional repressors.

Alcohol Dehydrogenase↗
PubMed1991

Generation of p50 subunit of NF-kappa B by processing of p105 through an ATP-dependent pathway.

The transcription factor NF-kappa B is a heterodimer consisting of two proteins encoded by different members of the rel gene family (p50 and p65). The p50 subunit is unusual among DNA-binding proteins in that its functional form is encoded in an open reading frame of relative molecular mass 105,000 (p105; ref. 4). The N-terminal region of this open reading frame encodes p50, whereas the remaining C terminus contains ankyrin repeats. Although p50 binds to DNA, full-length p105 translated in vitro does not. The mechanism by which p50 is generated in vivo, and the fate of the C-terminal region of p105 have not been established. Here we show that functional p50 is produced by ATP-dependent proteolysis of p105. Moreover, we find that the C-terminal half of p105 is not required for processing in vivo, and is rapidly degraded on processing. We propose that the C-terminal region of p105 is involved in the cytoplasmic assembly of the complex between the p50/p65 heterodimer and the inhibitor I kappa B.

Adenosine Triphosphate↗
PubMed1991

A role for the Drosophila neurogenic genes in mesoderm differentiation.

The neurogenic genes of Drosophila have long been known to regulate cell fate decisions in the developing ectoderm. In this paper we show that these genes also control mesoderm development. Embryonic cells that express the muscle-specific gene nautilus are overproduced in each of seven neurogenic mutants (Notch, Delta, Enhancer of split, big brain, mastermind, neuralized, and almondex), at the apparent expense of neighboring, nonexpressing mesodermal cells. The mesodermal defect does not appear to be a simple consequence of associated neural hypertrophy, suggesting that the neurogenic genes may function similarly and independently in establishing cell fates in both ectoderm and mesoderm. Altered patterns of beta 3-tubulin and myosin heavy chain gene expression in the mutants indicate a role for the neurogenic genes in development of most visceral and somatic muscles. We propose that the signal produced by the neurogenic genes is a general one, effective in both ectoderm and mesoderm.

Animals↗
PubMed1991

Selection of sequences recognized by a DNA binding protein using a preparative southwestern blot.

We have tested the feasibility of using a preparative scale Southwestern blot to select DNA sequences specifically recognized by a DNA binding protein. We constructed a library of random 15-mer oligonucleotides and screened it using a preparative Southwestern blot with the transcriptional repressor, PRDI-BF1. Several sequences specifically recognized by PRDI-BF1 were isolated. All of these sequences are similar to PRDI, a regulatory element previously demonstrated to bind PRDI-BF1.

Base Sequence↗
PubMed1991

Sex-specific splicing and polyadenylation of dsx pre-mRNA requires a sequence that binds specifically to tra-2 protein in vitro.

Somatic sex determination in Drosophila involves a hierarchy of regulated alternative pre-mRNA processing. Female-specific splicing and/or polyadenylation of doublesex (dsx) pre-mRNA, the final gene in this pathway, requires transformer (tra) and transformer-2 (tra-2) proteins. The mechanisms by which these proteins regulate RNA processing has not been characterized. In this paper we show that tra-2 produced in Escherichia coli binds specifically to a site within the female-specific exon of dsx pre-mRNA. This site, which contains six copies of a 13 nucleotide repeat, is required not only for female-specific splicing, but also for female-specific polyadenylation. These observations suggest that tra-2 is a positive regulator of dsx pre-mRNA processing.

Animals↗
PubMed1991

Associations between distinct pre-mRNA splicing components and the cell nucleus.

SC-35 is a non-snRNP spliceosome component that is specifically recognized by the anti-spliceosome monoclonal antibody alpha SC-35. In this paper we provide direct evidence that SC-35 is an essential splicing factor and we examine the immunolocalization of SC-35 by confocal laser scanning microscopy and by electron microscopy. We have found that the speckled staining pattern observed by fluorescence microscopy corresponds to structures previously designated as interchromatin granules and perichromatin fibrils. Although snRNP antigens are also concentrated in these nuclear regions, we show that the two types of spliceosome components are localized through different molecular interactions: The distribution of SC-35 was not affected by treatment with DNase I or RNase A, or when the cells were heat shocked. In contrast, snRNP antigens become diffusely distributed after RNase A digestion or heat shock. Examination of cells at different stages of mitosis revealed that the SC-35 speckled staining pattern is lost during prophase and speckles containing SC-35 begin to reform in the cytoplasm of anaphase cells. In contrast, snRNP antigens do not associate with speckled regions until late in telophase. These studies reveal a dynamic pattern of assembly and disassembly of the splicing factor SC-35 into discrete nuclear structures that colocalize with interchromatin granules and perichromatin fibrils. These subnuclear regions may therefore be nuclear organelles involved in the assembly of spliceosomes, or splicing itself.

Antigens↗