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T Maniatis

Publications and source records attributed to T Maniatis.

At least 163 records · Page 9Linked to original sources
PubMed1984

Human alpha- and beta-globin gene transcription in mouse erythroleukaemia cells.

Human beta-globin genes introduced into mouse erythroleukaemia (MEL) cells by DNA co-transformation are correctly regulated when erythroid cell differentiation is induced by dimethylsulphoxide (DMSO). In contrast, cloned human alpha-globin genes are efficiently transcribed in MEL cells before induction, and no increase in the level alpha-globin mRNA is observed when the cells differentiate. These observations suggest that the mechanisms by which alpha- and beta-globin genes are activated during erythroid cell differentiation are fundamentally different. Analysis of the transcription of hybrid human alpha-beta-globin genes in MEL cells revealed that the sequences responsible for differences in transcription of the intact alpha- and beta-globin genes are located on the 3' side of the mRNA capping site of the two genes, suggesting that cis-acting regulatory sequences are located within the structural genes.

Animals↗
PubMed1984

Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter.

A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).

Base Sequence↗
PubMed1984

Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro.

Human beta-globin mRNA precursors (pre-mRNAs) synthesized in vitro from a bacteriophage SP6 promoter/beta-globin gene fusion are accurately and efficiently spliced when added to a HeLa cell nuclear extract. Under optimal conditions, the first intervening sequence (IVS 1) is removed by splicing in up to 90% of the input pre-mRNA. Splicing requires ATP and in its absence the pre-mRNA is neither spliced nor cleaved at splice junctions. Splicing does not require that the pre-mRNA contain a correct 5' or 3' end, a 3' poly A tail, or a 5'-terminal cap structure. However, capping of the pre-mRNA significantly affects the specificity of in vitro processing. In the absence of a cap approximately 30%-40% of the pre-mRNA is accurately spliced, and a number of aberrantly cleaved RNAs are also detected. In contrast, capped pre-mRNAs are spliced more efficiently and produce fewer aberrant RNA species. The specificity of splice-site selection in vitro was tested by analyzing pre-mRNAs that contain beta-thalassemia splicing mutations in IVS 1. Remarkably, these mutations cause the same abnormal splicing events in vitro and in vivo. The ability to synthesize mutant pre-mRNAs and study their splicing in a faithful in vitro system provides a powerful approach to determine the mechanisms of RNA splice-site selection.

Adenosine Triphosphate↗
PubMed1984

Differences in human alpha- and beta-globin gene expression in mouse erythroleukemia cells: the role of intragenic sequences.

Human beta-globin genes introduced into mouse erythroleukemia (MEL) cells by DNA cotransformation are correctly regulated when erythroid cell differentiation is induced by dimethylsulfoxide (DMSO). In contrast, cloned human alpha-globin genes are efficiently transcribed in MEL cells prior to induction, and no increase in the level of alpha-globin mRNA is observed when the cells differentiate. These observations suggest that the mechanisms by which alpha- and beta-globin genes are activated during erythroid cell differentiation are fundamentally different. Analysis of the transcription of hybrid human alpha/beta-globin genes in MEL cells revealed that the sequences responsible for differences in transcription of the intact alpha- and beta-globin genes are located on the 3' side of the mRNA capping site of the two genes, suggesting that cis-acting regulatory sequences are located within the structural genes.

Animals↗
PubMed1984

Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro.

To study the mechanisms of RNA splicing we have analyzed the products generated by in vitro processing of a truncated 32P-labeled human beta-globin RNA precursor that contains the first two exons and the first intervening sequence (IVS1). Six major RNA products were detected and characterized. The first detectable RNA processing event is cleavage at the 5' GT of IVS1. Subsequently, accurately spliced RNA and the excised, intact IVS1 are simultaneously observed. The IVS1-containing RNA processing products have several unusual properties, which include: anomalous electrophoretic mobilities on polyacrylamide gels; a block to reverse transcription near the 3' end of IVS1; the presence of a nuclease-resistant component within IVS1. The block to reverse transcription and the nuclease-resistant component map to the same site near the 3' end of IVS1. The nuclease-resistant component appears to be a modified adenosine residue that contains an RNA branch. Based upon these and other structural studies we propose that the 5' end of IVS1 is joined by a 2'-5' phosphodiester linkage to the A residue in the RNAase T1 oligonucleotide ACTCTCTCTG located 28-37 nucleotides upstream from the IVS1 3' end. The IVS1 is therefore in the form of a lariat. These results imply that sequences within IVS1 actively participate in splicing.

Base Sequence↗
PubMed1984

High-level expression of a cloned HLA heavy chain gene introduced into mouse cells on a bovine papillomavirus vector.

A gene encoding the heavy chain of an HLA human histocompatibility antigen was isolated from a library of human DNA by recombination and selection in vivo. After insertion into a bovine papillomavirus (BPV) DNA expression vector, the gene was introduced into cultured mouse cells. Cells transformed with the HLA-BPV plasmids did not appear to contain extrachromosomal viral DNA, whereas BPV recombinants usually replicated as plasmids in transformed cell lines. Large amounts of HLA RNA were produced by the transformed cells, and the rate of synthesis of human heavy chain was several-fold higher than in the JY cell line, a well-characterized human lymphoblastoid cell line which expresses high levels of surface HLA antigen. Substantial amounts of human heavy chain accumulated in the transformed cells, and HLA antigen was present at the cell surface. These observations establish the feasibility of using BPV vectors to study the structure and function of HLA antigens and the expression of cloned HLA genes.

Animals↗
PubMed1983

Transcriptional regulation of globin gene expression in the human erythroid cell line K562.

The effect of hemin on the rate of synthesis and the level of globin messenger RNA's in the human erythroid cell line K562 was examined by means of cloned hybridization probes specific for each of the human embryonic, fetal, and adult globin genes. Hemin increases both the rate of transcription and the level of accumulation of zeta-, epsilon-, gamma-, and alpha-globin messenger RNA's by a factor of 3 to 5. Thus, hemin induction of globin gene expression in K562 cells is at the level of transcription.

Cell Line↗
PubMed1983

Specific transcription and RNA splicing defects in five cloned beta-thalassaemia genes.

Transcriptional analysis of five different cloned beta-thalassaemia genes introduced into cultured mammalian cells revealed specific defects in transcription and RNA splicing. A single base change 87 base pairs to the 5' side of the mRNA cap site significantly lowers the level of transcription and therefore appears to represent a promoter mutation. Three genes contain different single base changes in the first intervening sequence (IVS) 5' splice site. One mutation, at IVS1 position 1, inactivates the splice site completely; the other two, at IVS1 positions 5 and 6, reduce its activity. Each mutation activates the same three cryptic splice sites. The fifth gene contains a single base change within IVS2 at position 745, which results in the formation of abnormal beta-globin RNA that contains an extra exon.

Alleles↗
PubMed1983

Human beta-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei.

To study the mechanisms of RNA splicing we have synthesized beta-globin mRNA precursors by in vitro transcription using a plasmid in which a human beta-globin gene is fused to an efficient bacteriophage promoter. The structural requirements for accurate splicing of the in vitro synthesized pre-mRNAs were investigated by injection into Xenopus oocyte nuclei. We detect splicing only if the pre-mRNA is capped in vitro prior to injection; uncapped RNA is rapidly degraded. In addition, we find that in vitro synthesized pre-mRNAs that are not polyadenylated or lack a normal 3' end are spliced following injection into oocytes. The observation that a purified pre-mRNA can be spliced in oocytes indicates that transcription and splicing are not obligatorily coupled. When an in vitro synthesized beta-globin pre-mRNA containing a splice junction mutation is microinjected, the affected junction is not spliced, indicating that sequences necessary for accurate splicing in human cells are also necessary for splicing in oocytes.

Animals↗
PubMed1983

Correct developmental expression of a cloned alcohol dehydrogenase gene transduced into the Drosophila germ line.

We have used P-element-mediated transformation to introduce a cloned Drosophila alcohol dehydrogenase (Adh) gene into the germ line of ADH null flies. Six independent transformants expressing ADH were identified by their acquired resistance to ethanol. Each transformant carries a single copy of the cloned Adh gene in a different chromosomal location. Four of the six transformant lines exhibit normal Adh expression by the following criteria: quantitative levels of ADH enzyme activity in larvae and adults; qualitative tissue specificity; the size of stable Adh mRNA; and the characteristic developmental switch in utilization of two different Adh promoters. The remaining two transformants express ADH enzyme activity with the correct tissue specificity, but at a lower level than wild type. These results demonstrate that an 11.8 kb chromosomal fragment containing the Adh gene includes the cis-acting sequences necessary for its correct developmental expression, and that a variety of chromosomal sites permit proper Adh gene function.

Alcohol Dehydrogenase↗
PubMed1983

Transcriptional activation of cloned human beta-globin genes by viral immediate-early gene products.

When the human beta-globin gene is transfected into Hela cells, no beta-globin RNA is detected unless the gene is linked to a viral transcription enhancer. In this paper we show that trans-acting adenovirus and herpesvirus (pseudorabies) transcriptional regulatory proteins can circumvent this enhancer requirement for detectable beta-globin transcription in transient expression assays. The viral gene products can be provided by constitutively expressed, integrated viral genes in established cell lines, by viral infection of permissive cells, or by transfection of cells with bacterial plasmids carrying the viral immediate-early genes. These results demonstrate the utility of transient expression assays for studying regulatory mechanisms involving trans-acting factors. Analysis of beta-globin promoter mutants indicates that between 75 and 128 bp of sequence 5' to the mRNA cap site is required for enhancer-dependent transcription in Hela cells. In contrast, beta-globin transcription in the presence of viral immediate-early gene products requires only 36 bp of 5'-flanking sequence, which includes the TATA box. Thus both cis and trans-acting viral factors activate beta-globin gene transcription in transient expression experiments, but the mechanisms by which they act appear to be fundamentally different.

Adenoviruses, Human↗
PubMed1983

The regulated expression of beta-globin genes introduced into mouse erythroleukemia cells.

We have introduced a hybrid mouse-human beta-globin gene as well as the intact human beta-globin gene into murine erythroleukemia (MEL) cells and have demonstrated that these genes are appropriately regulated during differentiation of the MEL cell in culture. The addition of chemical inducers to cotransformed cells results in a 5 to 50 fold increase in the level of mRNA transcribed from the exogenous globin gene. S1 nuclease and primer extension analyses demonstrate that these mRNAs initiate and terminate correctly. Nuclear transcription experiments indicate that induction of hybrid mRNA results at least in part from the increase in the rate of globin gene transcription. Furthermore, the induction appears to be specific for globin genes within an erythroid cell. These results permit the study of expression of the globin gene during erythroid differentiation and suggest that the specific induction of the globin gene is an inherent property of DNA sequences within or flanking the beta-globin genes. Moreover, the fact that the human and hybrid globin genes are both inducible in MEL cells suggests that these regulatory sequences are conserved between mouse and human cells.

Animals↗
PubMed1983

Identification of two distinct regulatory regions adjacent to the human beta-interferon gene.

To study the regulation of the human beta-interferon (beta-IFN) gene by poly(I)-poly(C), we analyzed the expression of deletion mutants of the cloned gene introduced into mouse cells on a new bovine papilloma virus (BPV) vector. In stable cell lines transformed by a BPV-IFN plasmid containing the beta-IFN structural gene with 210 bp of DNA to the 5' side of its mRNA cap site (denoted -210), human beta-IFN mRNA is induced approximately 400-fold by poly(I)-poly(C), and reproducible levels of expression are observed for independent cell lines. Our studies indicate that there are two distinct regulatory regions adjacent to the gene, located between -77 and -19, and between -210 and -107. The -77 to -19 region is required for constitutive and induced IFN gene expression, and both are drastically reduced by deletion to -73. When sequences between -210 and -107 are deleted, the constitutive level of IFN gene expression is increased 5- to 10-fold, while induced expression is essentially unaffected. Deletion of the -210 to -107 region also alters the kinetics of induction of the gene.

Base Sequence↗
PubMed1983

cis and trans activation of globin gene transcription in transient assays.

We examined the effects of the simian virus 40 enhancer sequence on transcription of cloned human alpha- and beta-globin genes shortly after their introduction into cultured mammalian cells. We find that (i) detectable transcription of the beta-globin gene but not the alpha-globin gene requires linkage to the enhancer; (ii) the enhancer increases the amount of beta-globin RNA at least 100-fold but results in only a 5- to 10-fold increase in the amount of alpha-globin RNA; (iii) plasmid replication does not increase the level of beta-globin RNA, regardless of linkage to the enhancer, but does result in an approximately equal to 50-fold increase in the level of alpha-globin RNA; (iv) the enhancer is not required for and does not increase transcription of either gene in 293 cells, an adenovirus 5-transformed human kidney cell line. We also show that an enhancer sequence is not required for activity of the normally enhancer-dependent simian virus 40 early promoter in 293 cells, indicating that these cells contain a trans-acting factor(s) that circumvents the requirement for the enhancer sequence.

Alpha-Globulins↗
PubMed1983

Plasmid vectors for the rapid isolation and transcriptional analysis of human beta-globin gene alleles.

We describe the construction and characterization of miniplasmid vectors that can be used to isolate and express normal and mutant alleles of the human beta-globin gene. These vectors, designated pi SV beta plasmids, contain a bacterial origin of replication and selectable marker, a 5'-flanking beta-globin DNA fragment that can be used for recombination screening (Seed, 1983), and simian virus 40 (SV40) sequences that allow accurate and efficient expression of the beta-globin gene transfected into mammalian cells. We show that pi SV beta plasmids can be used to select cloned beta-globin genes from a bacteriophage lambda library of genomic DNA, and that plasmids containing the beta-globin gene linked to the SV40 enhancer sequence can be excised from the phage, circularized and recovered by transformation of Escherichia coli. Analysis of the beta-globin transcripts produced by the recovered pi SV beta recombinant plasmids after transfection into COS cells and replication to high copy number, indicates that the beta-globin gene is accurately transcribed, but a substantial fraction of the transcripts are the result of readthrough from sites within the vector. In contrast, when these plasmids are transferred into HeLa cells beta-globin RNA is accurately initiated and little readthrough transcription is observed. These results indicate that HeLa cells are more suitable than COS cells for studying mutant beta-globin genes, even though the copy number of the pi SV beta plasmids is much higher in COS cells.

Alleles↗