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T Maniatis

Publications and source records attributed to T Maniatis.

At least 145 records · Page 8Linked to original sources
PubMed1986

The human beta-interferon gene enhancer is under negative control.

The human beta-interferon gene is regulated by an inducible enhancer element. Analysis of the effect of deletions within this element on beta-interferon transcription indicates that this enhancer is under negative control. Deletion of sequences from the 3' end of the enhancer leads to a dramatic increase in the basal level of beta-interferon mRNA and a decrease in the induction ratio. The remaining 5' region of the enhancer can act as a strong constitutive transcription element, and it shares considerable homology with sequences known to be required for the activity of constitutive viral enhancers. We conclude that the beta-interferon enhancer consists of a constitutive transcription element and a negative regulatory sequence that prevents enhancer activity prior to induction. Thus, derepression of a constitutive transcription element appears to play a key role in the control of human beta-interferon gene expression.

Animals↗
PubMed1986

Detection of factors that interact with the human beta-interferon regulatory region in vivo by DNAase I footprinting.

We have used a DNAase I genomic footprinting procedure to detect interactions between cellular factors and the regulatory sequences of the human beta-interferon gene. Prior to induction with poly(I)-poly(C), factors that bind to DNA are detected in one region located between -94 and -167 from the mRNA cap site, and in another region located between -68 and -38. After induction these factors dissociate and another factor binds to a region located between -77 and -64. Correlation of these footprints with the effects of deletions in the regulatory region of the beta-interferon gene (accompanying paper) suggest that the factors that bind prior to induction are repressor molecules, while the component that binds after induction is a transcription factor. Dissociation of the repressor molecules from the DNA after induction may allow the transcription factor to bind to and activate the beta-interferon promoter. Thus, the beta-interferon gene may be controlled by a negative regulatory mechanism.

Base Sequence↗
PubMed1986

Fine structure genetic analysis of a beta-globin promoter.

A novel procedure for saturation mutagenesis of cloned DNA was used to obtain more than 100 single base substitutions within the promoter of the mouse beta-major globin gene. The effects of these promoter substitutions on transcription were determined by transfecting the cloned mutant genes into HeLa cells on plasmids containing an SV40 transcription enhancer, and measuring the levels of correctly initiated beta-globin transcripts after 2 days. Mutations in three regions of the promoter resulted in a significant decrease in the level of transcription: (i) the CACCC box, located between -87 and -95, (ii) the CCAAT box, located between -72 and -77, and (iii) the TATA box, located between -26 and -30 relative to the start site of transcription. In contrast, two different mutations in nucleotides immediately upstream from the CCAAT box resulted in a 3- to 3.5-fold increase in transcription. With two minor exceptions, single base substitutions in all other regions of the promoter had no effect on transcription. These results precisely delineate the cis-acting sequences required for accurate and efficient initiation of beta-globin transcription, and they establish a general approach for the fine structure genetic analysis of eukaryotic regulatory sequences.

Animals↗
PubMed1986

Regulatory elements involved in Drosophila Adh gene expression are conserved in divergent species and separate elements mediate expression in different tissues.

Alcohol dehydrogenase (ADH) is expressed in a complex temporal and spatial pattern from tandem promoters (proximal and distal) in Drosophila melanogaster, and from two closely linked genes (Adh-1 and Adh-2) in D. mulleri. The expression patterns of Adh-1 and the proximal promoter, and Adh-2 and the distal promoter are similar, but not identical. We show that the mulleri Adh genes are appropriately expressed when introduced into the melanogaster genome, indicating that the cis- and trans-acting elements which regulate the corresponding promoters are functionally equivalent in the two species. By analyzing the expression of in vitro generated mutants of the mulleri Adh locus, we define at least three regulatory regions of the mulleri Adh genes and show that different control elements mediate the expression of Adh in different tissues.

Alcohol Dehydrogenase↗
PubMed1986

Activation of the human beta-interferon gene requires an interferon-inducible factor.

beta-Interferon (beta-IFN) gene expression can be induced by poly(I)-poly(C) or virus, but there is considerable variation in the extent of induction between different cell lines. We characterized two poorly inducible human cell lines, HeLa and 143 thymidine kinase negative (143 tk-), to define cellular factors involved in the activation of the beta-IFN gene. We show that the deficiency in beta-IFN induction in these cells can be complemented by fusion to highly inducible mouse cells. We conclude that the human cells are deficient in a trans-acting factor required for B-IFN gene activation. The level of induction of the beta-IFN gene in HeLa and 143 tk- cells can also be increased by priming with IFN before induction. If IFN priming is carried out in the presence of cycloheximide, a approximately 200-fold increase in induction is observed. We conclude that activation of the beta-IFN gene requires an IFN-inducible factor that is only expressed at low levels in unprimed HeLa and 143 tk- cells.

Cell Fusion↗
PubMed1986

Bioavailability of a once daily-administered theophylline preparation. A comparison study.

To evaluate the bioavailability of a new theophylline preparation suitable for once-a-day (od) oral administration, we performed a nonrandomized crossover study in which the absorption of the OD and a standard twice-a-day (bid) preparation were compared. Eight stable asthmatic patients, after having achieved steady-state, received an average of 975 mg of OD preparation at 8 PM. The protocol was later repeated with the same subjects receiving 487.5 mg of the bid preparation at 8 PM and again at 8 AM using the same total dose. The maximal mean serum concentrations were 15.5 +/- 1.6 (SEM) micrograms/ml for the od preparation on the 8th hour and 12.7 +/- 2.2 for the bid regimen. The trough level was 7.4 +/- 1.2 micrograms/ml for the od regimen and 10.6 +/- 1.6 for the bid regimen. With either regimen, therapeutic theophylline levels could be observed throughout the 24-hour study period. Anhydrous theophylline may be administered as a single daily dose agent.

Administration, Oral↗
PubMed1986

Beta-thalassemia: analysis of mRNA precursors of a mutant human globin gene with defective splicing using peripheral blood nucleated red blood cells.

Studies on the effects of thalassemic mutations on gene function in vivo have clinical as well as scientific implications. Usually these studies have been performed on nucleated red blood cell (RBC) precursors normally present in bone marrow. Many patients with beta-thalassemia are splenectomized and may have high levels of nucleated RBC, orthochromatic normoblasts, in their peripheral blood (1-5% of total RBC). The possibility of exploiting these cells instead of bone marrow as a source for nuclear and cytoplasmic RNA for expression studies was investigated. A simple procedure was developed for enrichment for normoblasts in blood samples withdrawn from patients prior to transfusion. Globin transcripts were analyzed in RNA purified from 12 patients. Unspliced precursor beta-mRNA molecules were observed in a patient with beta o-thalassemia, homozygous for a mutation at the 5' IVS2 splice site of the beta-globin gene. Detailed analyses showed that his mature beta-mRNA was larger than normal, and that a cryptic 5' splice site, approximately 50 nucleotides downstream from the normal one, was utilized. We conclude that peripheral blood can be used as a reliable source of RNA for the analysis of the effects of beta-thalassemia mutations on gene expression and the relationship to the clinical condition. Moreover, this procedure facilitates the comparison of in vivo gene expression with the results obtained from DNA transfection experiments with cloned beta-thalassemia genes.

Adolescent↗
PubMed1985

Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes.

Single base substitutions can be detected and localized by a simple and rapid method that involves ribonuclease cleavage of single base mismatches in RNA:DNA heteroduplexes. A 32P-labeled RNA probe complementary to wild-type DNA is synthesized in vitro and annealed to a test DNA containing a single base substitution. The resulting single base mismatch is cleaved by ribonuclease A, and the location of the mismatch is then determined by analyzing the sizes of the cleavage products by gel electrophoresis. Analysis of every type of mismatch in many different sequence contexts indicates that more than 50 percent of all single base substitutions can be detected. The feasibility of this method for localizing base substitutions directly in genomic DNA samples is demonstrated by the detection of single base mutations in DNA obtained from individuals with beta-thalassemia, a genetic disorder in beta-globin gene expression.

Animals↗
PubMed1985

Structure and transcription of the Drosophila mulleri alcohol dehydrogenase genes.

The D. melanogaster Adh gene is transcribed from two different promoters; a proximal (larval) promoter is active during late embryonic and larval stages, and a distal (adult) promoter is active primarily in third instar larvae and in adult flies (1). Genetic analyses suggest that several species of the mulleri subgroup (distant relatives of D. melanogaster) have two closely-linked Adh genes, Adh-1 and Adh-2, each of which expresses a different ADH protein (2). The temporal pattern of expression of Adh-1 and Adh-2 is similar to the expression of D. melanogaster Adh from the proximal and distal promoters (2,3,4). We are interested in the molecular basis for the pattern of Adh expression in the mulleri subgroup species and in the mechanism of the switch in Adh promoter utilization. For these reasons, we have studied the structure and transcription of the Adh locus of D. mulleri, a species of the mulleri subgroup. We show that the ADH-1 and ADH-2 proteins are expressed from two distinct genes separated by 2 kilobase pairs, and that Adh-1 and Adh-2 are transcribed in the expected temporal pattern. In addition, we find a pseudogene 1.2 kb upstream from Adh-2, which is transcribed in a temporal pattern similar to Adh-2.

Age Factors↗
PubMed1985

A general method for saturation mutagenesis of cloned DNA fragments.

A new procedure for generating and isolating random single-base substitutions in cloned DNA fragments is presented. The mutations are generated by treatment of single-stranded DNA with various chemicals, followed by the synthesis of the complementary strand with reverse transcriptase. Misincorporation frequently occurs when the enzyme encounters a damaged base in the mutagenized template DNA. The resulting duplex DNA fragments containing random single-base substitutions are cloned, amplified as a population, and isolated from wild-type DNA by preparative denaturing gradient gel electrophoresis. The physical separation of mutant DNA fragments makes it possible to isolate and characterize large numbers of site-directed single-base substitutions in the absence of a phenotypic selection. This procedure should be generally applicable to the fine-structure genetic analysis of regulatory and protein-coding sequences.

Animals↗
PubMed1985

Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis.

The melting behavior of a DNA fragment carrying the mouse beta maj-globin promoter was investigated as a means of establishing procedures for separating DNA fragments differing by any single base substitution using the denaturing gradient gel electrophoresis procedure of Fischer and Lerman (1,2). We find that attachment of a 300 base pair GC-rich DNA sequence, termed a GC-clamp, to a 135 bp DNA fragment carrying the mouse beta-globin promoter significantly alters the pattern of DNA melting within the promoter. When the promoter is attached to the clamp, the promoter sequences melt without undergoing strand dissociation. The calculated distribution of melting domains within the promoter differs markedly according to the relative orientation of the clamp and promoter sequences. We find that the behavior of DNA fragments containing the promoter and clamp sequences on denaturing gradient polyacrylamide gels is in close agreement with the theoretical melting calculations. These studies provide the basis for critical evaluation of the parameters for DNA melting calculations, and they establish conditions for determining whether all single base substitutions within the promoter can be separated on denaturing gradient gels.

Animals↗
PubMed1985

Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis.

Duplex DNA fragments differing by single base substitutions can be separated by electrophoresis in denaturing gradient polyacrylamide gels, but only substitutions in a restricted part of the molecule lead to a separation (1). In an effort to circumvent this problem, we demonstrated that the melting properties and electrophoretic behavior of a 135 base pair DNA fragment containing a beta-globin promoter are changed by attaching a GC-rich sequence, called a 'GC-clamp' (2). We predicted that these changes should make it possible to resolve most, if not all, single base substitutions within fragments attached to the clamp. To test this possibility we examined the effect of several different single base substitutions on the electrophoretic behavior of the beta-globin promoter fragment in denaturing gradient gels. We find that the GC-clamp allows the separation of fragments containing substitutions throughout the promoter fragment. Many of these substitutions do not lead to a separation when the fragment is not attached to the clamp. Theoretical calculations and analysis of a large number of different mutations indicate that approximately 95% of all possible single base substitutions should be separable when attached to a GC-clamp.

Animals↗
PubMed1985

Intron sequences involved in lariat formation during pre-mRNA splicing.

We have shown that lariat formation during in vitro splicing of several RNA precursors, from Drosophila to man, occurs at a unique and identifiable but weakly conserved site, 18 to 37 nucleotides proximal to the 3' splice site. Lariat formation within an artificial intron lacking a normal branch-point sequence occurs at a cryptic site a conserved distance (approximately 23 nucleotides) from the 3' splice site. Analysis of beta-thalassemia splicing mutations revealed that lariat formation in the first intron of the human beta-globin gene occurs at the same site in normal and mutant precursors, even though alternate 5' and 3' splice sites are utilized in the mutants. Remarkably, cleavage at the 5' splice site and lariat formation do not occur when the precursor contains a beta-thalassemia deletion removing the polypyrimidine stretch and AG dinucleotide at the 3' splice site. In contrast, a single base substitution in the AG dinucleotide blocks cleavage at the 3' splice site but not at the 5' site.

Animals↗
PubMed1985

Multiple factors including the small nuclear ribonucleoproteins U1 and U2 are necessary for pre-mRNA splicing in vitro.

We have identified six distinct factors necessary for pre-mRNA splicing in vitro by selective inactivation and complementation studies, and by fractionation procedures. Splicing factor 1 (SF1) is sensitive to micrococcal nuclease, and appears to consist of at least U1 and U2 snRNPs, since splicing is inhibited when the 5' termini of U1 and U2 snRNAs are removed by site-directed cleavage with RNAase H. SF2 is a micrococcal nuclease-resistant factor present in the nuclear extract but absent from an S100 extract. SF3 is a factor that can be preferentially inactivated by moderate heat treatment. Two additional factors (SF4A and SF4B) were identified by fractionation of the nuclear extract using spermine-agarose and CM-sepharose chromatography. SF1, SF2, and SF4B appear to be required for cleavage of the pre-mRNA at the 5' splice site and lariat formation, whereas SF3 and SF4A are only required for cleavage at the 3' splice site and exon ligation.

Base Sequence↗
PubMed1985

Human beta-interferon gene expression is regulated by an inducible enhancer element.

We have localized the regulatory sequence required for viral or poly(I)-poly(C) activation of human beta-interferon gene expression to a region located between -37 and -77 from the mRNA cap site. This sequence has the characteristics of an inducible enhancer element: it can act upstream or downstream of the beta-interferon gene regardless of its orientation, and at distances up to approximately 1 kilobase from its normal location. Moreover, this element can confer inducibility on a heterologous promoter. Further analysis has identified a minimal regulatory element of 14 base pairs within this enhancer. Sequences closely related to this element are present five times within the 5'-flanking regions of both the alpha- and beta-interferon genes. The number of these minimal regulatory elements required for maximal beta-interferon gene expression appears to differ in different cell lines.

Animals↗
PubMed1985

Linker scanning mutagenesis of the 5'-flanking region of the mouse beta-major-globin gene: sequence requirements for transcription in erythroid and nonerythroid cells.

We analyzed the sequences required for transcription of the mouse beta-major-globin gene by introducing deletion and linker scanning mutations into the 5'-flanking region and then studying the effects of these mutations on beta-globin gene transcription in a HeLa cell transient expression assay or after stable introduction into mouse erythroleukemia cells. Consistent with earlier studies, we found that three distinct regions upstream from the RNA capping site are required for efficient beta-globin gene transcription in HeLa cells: the ATA box located 30 base pairs upstream from the mRNA capping site (-30), the CCAAT box located at -75, and the distal sequence element CCACACCC located at -90. In the ATA and CAAT box regions, the sequences necessary for efficient transcription extend beyond the limits of the canonical sequences. Mutations in the sequences located between the three transcriptional control elements do not significantly affect transcription in HeLa cells. Although the promoter defined in HeLa cell transfection experiments is also required for efficient transcription in mouse erythroleukemia cells, none of the mutations tested affects the regulation of beta-globin gene transcription during mouse erythroleukemia cell differentiation. Thus, DNA sequences downstream from the mRNA cap site appear to be sufficient for the regulation of beta-globin gene expression during the differentiation of mouse erythroleukemia cells in culture.

Animals↗