==================================BSR49================================== 49. Initiation factor; RNA polymerase II; transcription. Messenger RNA; stability; protein factors. DNA-binding proteins; transcription; eukaryotic. 1 UI - 87041494 AU - Lewis ED ; Manley JL TI - Polyadenylylation of an mRNA precursor occurs independently of transcription by RNA polymerase II in vivo. AB - Most eukaryotic messenger RNAs are transcribed as precursor molecules that must be processed by capping, splicing, 3' cleavage, and polyadenylylation to yield mature mRNAs. An important, unresolved issue is whether any of these reactions are linked either to transcription by RNA polymerase II or to each other. To address one aspect of this question, we constructed a chimeric gene containing an RNA polymerase III promoter (the adenovirus VAI promoter) fused to the body and 3'-flanking sequences of a protein-coding gene (the herpesvirus tk gene). Here we show that this hybrid gene was transcribed from the RNA polymerase III promoter following transfection of human 293 cells and that the transcripts produced were stable and efficiently transported to the cytoplasm. Although a significant proportion of the transcripts were prematurely terminated at specific sites within the gene, a high percentage of the full-length RNA was accurately cleaved and polyadenylylated. These results demonstrate that cleavage and polyadenylylation of mRNA precursors are not obligatorily coupled to transcription by RNA polymerase II in vivo. MH - Endonucleases/PHARMACOLOGY ; Human ; Nucleic Acid Precursors/*METABOLISM ; Nucleotide Mapping ; Poly A/*METABOLISM ; Promoter Regions (Genetics) ; RNA Polymerase II/*PHARMACOLOGY ; RNA Polymerase III/PHARMACOLOGY ; RNA, Messenger/*METABOLISM ; Support, U.S. Gov't, P.H.S. ; Thymidine Kinase/ GENETICS ; *Transcription, Genetic SO - Proc Natl Acad Sci USA 1986 Nov;83(22):8555-9 2 UI - 87002456 AU - Hope IA ; Struhl K TI - Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast. AB - Yeast GCN4 protein binds specifically to the promoters of amino acid biosynthetic genes and coordinately induces their transcription. Serially deleted GCN4 and hybrid LexA-GCN4 proteins were assayed for specific DNA binding activity in vitro, and for stimulation of transcription in vivo. The specific DNA binding activity resides in the 60 C-terminal amino acids, a basic region of GCN4. However, certain deletions containing the entire DNA binding region are unable to activate transcription and instead act as repressors in vivo. The activation function appears to critically involve just 19 amino acids that are centrally located in an acidic region of GCN4. In addition to their functional separation, the DNA binding and transcriptional activation regions of the protein can be separated physically by elastase cleavage. The implications of these results for the mechanisms of DNA sequence recognition and transcription activation are discussed. MH - Binding Sites ; DNA-Binding Proteins/GENETICS/METABOLISM ; Gene Expression Regulation ; Histidine/BIOSYNTHESIS ; Mutation ; Pancreatopeptidase/DIAGNOSTIC USE ; Promoter Regions (Genetics) ; Saccharomyces Cerevisiae/*GENETICS ; Structure-Activity Relationship ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Transcription Factors/*GENETICS ; Transcription, Genetic SO - Cell 1986 Sep 12;46(6):885-94 3 UI - 87106813 AU - Duvoisin RM ; Belin D ; Krisch HM TI - A plasmid expression vector that permits stabilization of both mRNAs and proteins encoded by the cloned genes. AB - Two new expression vectors have been constructed to take advantage of several useful properties of bacteriophage T4-infected Escherichia coli. These plasmids, pRDB8 and pRDB9, contain the promoter region and start codon of T4 gene 32, a contiguous multiple cloning site (MCS), and translation and transcription termination signals. DNA fragments inserted into the MCS are transcribed and translated at a high level in both uninfected and phage T4-infected cells. Furthermore, the extreme stability of the hybrid mRNA after infection permits the specific biosynthetic labeling of the protein encoded by the cloned gene. In addition, the cloned gene product is stabilized, since the host-mediated degradation of foreign proteins is inhibited by phage infection. The properties of this expression system were demonstrated with the constant region of a rabbit immunoglobulin lambda light chain (C lambda) gene. Although proteolytic degradation of the C lambda fusion protein was rapid in uninfected cells, degradation was blocked in phage-infected cells and the protein accumulated in greater amounts. MH - Base Sequence ; *Cloning, Molecular ; DNA Replication ; DNA Restriction Enzymes ; Escherichia Coli/*GENETICS ; *Genes, Structural ; *Genes, Viral ; *Genetic Vectors ; Mutation ; *Plasmids ; Promoter Regions (Genetics) ; RNA, Messenger/*GENETICS ; Support, Non-U.S. Gov't ; T-Phages/*GENETICS ; Transcription, Genetic ; Translation, Genetic ; Viral Proteins/*GENETICS SO - Gene 1986;45(2):193-201 4 UI - 87092300 AU - Ballard DW ; Bothwell A TI - Mutational analysis of the immunoglobulin heavy chain promoter region. AB - Complete immunoglobulin heavy chain (IgH) genes (gamma and mu) containing the intronic IgH enhancer and mutations in the upstream promoter region were constructed in vitro and introduced into murine J558L myeloma cells by protoplast fusion. S1-nuclease mapping experiments demonstrated that IgH gene expression was extremely sensitive to mutation in an upstream region containing the octanucleotide sequence ATGCAAAT. Significant IgH mRNA levels were detected in RNA from cells transfected with IgH gene constructs in which all upstream sequences on the 5' proximal side of this element were deleted. Similar results were obtained using the precise inverse of the IgH octamer, which is found in the upstream promoter region of immunoglobulin light chain genes. Deletion of the IgH octamer, or point mutation of adenine to guanine at position 6, resulted in the loss of correctly initiated IgH mRNA. A DNA binding factor from J558L nuclear extracts was identified that appeared to recognize the octamer on the basis of differential binding to homologous restriction fragments containing the various mutations and that bound preferentially with octamer DNA fragments derived from functional relative to nonfunctional IgH constructs. Collectively, these data suggest that the octamer element contains residues that are critical to accurate immunoglobulin gene transcription and that may serve as part of a recognition locus for nuclear factors important to B-cell-specific immunoglobulin expression. MH - Animal ; Base Sequence ; Binding, Competitive ; Cell Line ; DNA-Binding Proteins/METABOLISM ; Gene Expression Regulation ; Immunoglobulin Variable Region/*GENETICS ; Immunoglobulins, Gamma Chain/GENETICS ; Immunoglobulins, Heavy Chain/*GENETICS ; Immunoglobulins, Mu Chain/ GENETICS ; Mice ; Mutation ; *Promoter Regions (Genetics) ; Support, Non-U.S. Gov't ; Transcription Factors/*GENETICS ; Transcription, Genetic SO - Proc Natl Acad Sci USA 1986 Dec;83(24):9626-30 5 UI - 87089788 AU - Gilman MZ ; Wilson RN ; Weinberg RA TI - Multiple protein-binding sites in the 5'-flanking region regulate c-fos expression. AB - We tested sequences flanking the mouse c-fos gene for the ability to form specific DNA-protein complexes with factors present in crude nuclear extracts prepared from mammalian cells. Three such complexes were detected. One complex formed in a region necessary for the induction of c-fos expression by serum growth factors. Two additional complexes formed at sequences that contribute to basal c-fos promoter activity in vivo. These complexes represent three novel sequence-specific DNA-binding activities which appear to participate in the regulation of c-fos transcription. MH - Animal ; Base Sequence ; Cell Line ; Cells, Cultured ; Chromosome Deletion ; DNA Restriction Enzymes ; DNA-Binding Proteins/*METABOLISM ; *Genes, Regulator ; Lymphoma ; Methylation ; Mice ; Mice, Inbred BALB C ; *Oncogenes ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Transcription, Genetic SO - Mol Cell Biol 1986 Dec;6(12):4305-16 6 UI - 87066715 AU - Santiago TC ; Purvis IJ ; Bettany AJ ; Brown AJ TI - The relationship between mRNA stability and length in Saccharomyces cerevisiae. AB - A rapid and convenient procedure has been developed for the measurement of mRNA half-life in S.cerevisiae using the transcriptional inhibitor, 1,10-phenanthroline. A range of half-lives from 6.6 +/- 0.67 minutes to over 100 minutes, relative to the stability of the 18S rRNA control, has been obtained for fifteen mRNAs. They include the pyruvate kinase and actin mRNAs, as well as 13 randomly picked mRNAs of unknown function. The mRNAs clearly fall into two populations when their lengths and half-lives are analysed; one population is considerably more stable than the other when mRNAs of similar length are compared. Also, within each population, there is an inverse relationship between mRNA length and half-life. These results suggest that mRNA length and at least one additional factor strongly influence mRNA stability in yeast. MH - Cloning, Molecular ; DNA/METABOLISM ; Half-Life ; Kinetics ; Nucleic Acid Hybridization ; Phenanthrolines/PHARMACOLOGY ; RNA, Messenger/*GENETICS/ METABOLISM ; Saccharomyces Cerevisiae/*GENETICS/METABOLISM ; Support, Non-U.S. Gov't ; Transcription, Genetic/DRUG EFFECTS ; Translation, Genetic SO - Nucleic Acids Res 1986 Nov 11;14(21):8347-60 7 UI - 87064427 AU - Kimura S ; Gonzalez FJ ; Nebert DW TI - Tissue-specific expression of the mouse dioxin-inducible P(1)450 and P(3)450 genes: differential transcriptional activation and mRNA stability in liver and extrahepatic tissues. AB - Expression of the P(1)450 and P(3)450 genes was examined in liver and five extrahepatic tissues of mice after they were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3-methylcholanthrene. All six tissues were shown to have increased P(1)450 and P(3)450 mRNA concentrations after treatment with these inducers. P(3)450 mRNA induction was more sensitive than P(1)450 mRNA induction to small doses of TCDD in liver, kidney, and lung. When transcription run-on assays were compared with mRNA prevalence, control P(3)450 mRNA in liver, kidney, and lung was shown to be 20 to 30 times more stable than control P(1)450 mRNA. After TCDD treatment the increases in mRNA concentrations did not necessarily parallel the increases in transcriptional rate. Thus, the inducer appeared to enhance mRNA stability in some instances. This was evident for liver P(1)450 mRNA, in which an 8-fold rise in transcription was associated with a 27-fold increase in mRNA content, and for kidney P(3)450 mRNA, in which a 2-fold rise in transcription was accompanied by a 12-fold increase in mRNA content. In the kidney and lung of control and TCDD-treated mice, transcriptional rates of the P(3)450 gene were at least 10-fold less than those of the P(1)450 gene. These data indicate that even though both genes are controlled by the same receptor, striking tissue-specific differences in transcription and mRNA stabilization affect the final mRNA concentrations. MH - Animal ; Cytochrome P-450/BIOSYNTHESIS/*GENETICS ; Dioxins/*PHARMACOLOGY ; Enzyme Induction ; Genes, Structural/*DRUG EFFECTS ; Isoenzymes/ BIOSYNTHESIS/*GENETICS ; Liver/DRUG EFFECTS/*METABOLISM ; Male ; Methylcholanthrene/PHARMACOLOGY ; Mice ; Mice, Inbred C57BL ; Organ Specificity ; RNA, Messenger/*GENETICS/METABOLISM ; Tetrachlorodibenzodioxin/*PHARMACOLOGY ; Transcription, Genetic/*DRUG EFFECTS SO - Mol Cell Biol 1986 May;6(5):1471-7 8 UI - 87064420 AU - Endo T ; Nadal-Ginard B TI - Transcriptional and posttranscriptional control of c-myc during myogenesis: its mRNA remains inducible in differentiated cells and does not suppress the differentiated phenotype. AB - It is widely accepted that the cellular oncogene c-myc plays an important role in the control of cell proliferation and that its expression diminishes in differentiated cells. We examined whether there is a correlation between c-myc expression and cell proliferation or differentiation by using a subclone of a rat skeletal muscle cell line L6E9. Myoblasts irreversibly withdraw from the cell cycle, fuse to form multinucleated myotubes, and express muscle-specific genes (terminal differentiation). Muscle-specific genes can also be expressed in the absence of fusion (biochemical differentiation). Such mononucleated but biochemically differentiated cells can be stimulated to reenter the cell cycle. c-myc was induced by insulin, insulin-like growth factor, or serum factors in G0-arrested cells, whereas induction by protein synthesis inhibitors or superinduction by protein synthesis inhibitors in combination with serum factors occurred in all physiological states tested. We found that c-myc expression was reduced in biochemically and terminally differentiated cells as well as in quiescent undifferentiated cells but that it remained inducible by growth factors in all three physiological states. Results of nuclear runoff transcription assays suggested that the induction of c-myc mRNA by growth factors and its deinduction in these physiological states were regulated mainly at the transcriptional level. In contrast, induction and superinduction of c-myc mRNA by protein synthesis inhibitors alone and in combination with growth factors, respectively, were regulated posttranscriptionally mainly by stabilization of c-myc mRNA. Moreover, c-myc and muscle-specific genes could be simultaneously transcribed in both biochemically and terminally differentiated cells. These results indicate that irreversible repression of c-myc is not required for terminal myogenic differentiation and that its expression is insufficient by itself to suppress the differentiated phenotype. MH - Animal ; *Cell Differentiation/DRUG EFFECTS ; Cell Division/DRUG EFFECTS ; Cell Line ; DNA Replication ; Growth Substances/PHARMACOLOGY ; Muscles/ *CYTOLOGY ; *Oncogenes/DRUG EFFECTS ; Phenotype ; Rats ; RNA/ISOLATION & PURIFICATION ; *RNA Processing, Post-Transcriptional ; RNA, Messenger/ *GENETICS ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; *Transcription, Genetic SO - Mol Cell Biol 1986 May;6(5):1412-21 9 UI - 87064374 AU - Widelitz RB ; Magun BE ; Gerner EW TI - Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts. AB - A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis. MH - Animal ; Cell Line ; Cycloheximide/*PHARMACOLOGY ; Fibroblasts/DRUG EFFECTS/METABOLISM ; Heat ; Heat-Shock Proteins/BIOSYNTHESIS/*GENETICS ; Kinetics ; Rats ; RNA, Messenger/*GENETICS ; Support, U.S. Gov't, P.H.S. ; Transcription, Genetic/*DRUG EFFECTS SO - Mol Cell Biol 1986 Apr;6(4):1088-94 10 UI - 87064329 AU - Pilder S ; Moore M ; Logan J ; Shenk T TI - The adenovirus E1B-55K transforming polypeptide modulates transport or cytoplasmic stabilization of viral and host cell mRNAs. AB - The adenovirus type 5 mutant H5dl338 lacks 524 base pairs within early region 1B. The mutation removed a portion of the region encoding the related E1B-55K and -17K polypeptides but did not disturb the E1B-21K coding region. The virus can be propagated in 293 cells which contain and express the adenovirus type 5 E1A and E1B regions, but it is defective for growth in HeLa cells, in which its final yield is reduced about 100-fold compared with the wild-type virus. The mutant also fails to transform rat cells at normal efficiency. The site of the dl338 defect was studied in HeLa cells. Early gene expression and DNA replication appeared normal. Late after infection, mRNAs coded by the major late transcription unit accumulated to reduced levels. At a time when transcription rates and steady-state nuclear RNA species were normal, the rate at which late mRNA accumulated in the cytoplasm was markedly reduced. Furthermore, in contrast to the case with the wild type, transport and accumulation of cellular mRNAs continued late after infection with dl338. Thus, the E1B product appears to facilitate transport and accumulation of viral mRNAs late after infection while blocking the same processes for cellular mRNAs. MH - Adenoviruses, Human/*GENETICS ; Biological Transport ; Cell Transformation, Viral ; Cytoplasm/METABOLISM ; Drug Stability ; Hela Cells/METABOLISM ; Human ; Kinetics ; Oncogene Proteins, Viral/ *METABOLISM ; RNA, Messenger/GENETICS/*METABOLISM ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Transcription, Genetic SO - Mol Cell Biol 1986 Feb;6(2):470-6 11 UI - 87064310 AU - Baker EJ ; Keller LR ; Schloss JA ; Rosenbaum JL TI - Protein synthesis is required for rapid degradation of tubulin mRNA and other deflagellation-induced RNAs in Chlamydomonas reinhardi. AB - After flagellar detachment in Chlamydomonas reinhardi, there is a rapid synthesis and accumulation of mRNAs for tubulin and other flagellar proteins. Maximum levels of these mRNAs (flagellar RNAs) are reached within 1 h after deflagellation, after which they are rapidly degraded to their predeflagellation levels. The degradation of alpha- and beta-tubulin RNAs was shown to be due to the shortening of their half-lives after accumulation (Baker et al., J. Cell Biol. 99:2074-2081, 1984). Deflagellation in the presence of protein synthesis inhibitors results in the accumulation of tubulin and other flagellar mRNAs by kinetics similar to those of controls. However, unlike controls, in which the accumulated mRNAs are rapidly degraded, these mRNAs are stabilized in cycloheximide. The stabilization by cycloheximide is specific for the flagellar mRNAs accumulated after deflagellation, since there is no change in the levels of flagellar mRNAs in nondeflagellated (uninduced) cells in the presence of cycloheximide. The kinetics of flagellar mRNA synthesis after deflagellation are shown to be the same in cycloheximide-treated and control cells by in vivo labeling and in vitro nuclear runoff experiments. These results show that protein synthesis is not required for the induced synthesis of flagellar mRNAs, and that all necessary transcriptional control factors are present in the cell before deflagellation, but that protein synthesis is required for the accelerated degradation of the accumulated flagellar mRNAs. Since cycloheximide prevents the induced synthesis and accumulation of flagellar proteins, it is possible that the product(s) of protein synthesis required for the accelerated decay of these mRNAs is a flagellar protein(s). The possibility that one or more flagellar proteins autoregulate the stability of the flagellar mRNAs is discussed. MH - Bacterial Proteins/GENETICS ; Chlamydomonas/*GENETICS/METABOLISM/ PHYSIOLOGY ; Cycloheximide/PHARMACOLOGY ; Flagella/*PHYSIOLOGY ; Nucleic Acid Hybridization ; RNA, Messenger/*GENETICS/METABOLISM ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. ; Transcription, Genetic/ DRUG EFFECTS ; *Translation, Genetic/DRUG EFFECTS ; Tubulin/*GENETICS SO - Mol Cell Biol 1986 Jan;6(1):54-61 12 UI - 87054023 AU - Williams JL ; Bownes M TI - Reduced stability of RNA coding for yolk polypeptide 3 in Drosophila melanogaster ovary. AB - In Drosophila three yolk polypeptides (YP1, YP2 and YP3) are synthesized at two sites in the adult female: in the fat body tissue, from which they are transported via the haemolymph to the ovary, and in the ovarian follicle cells which surround the developing oocytes. All three yolk polypeptides are synthesized at equal levels in the fat body. In this paper we show that the steady-state level of YP3 RNA is significantly reduced in the ovary in comparison with the fat body, and that none of the yolk protein genes is amplified either in the fat body or the follicle cells. In order to determine the basis of the reduced level of YP3 RNA in the ovary, which could result from a lower rate of transcription or through a decreased stability of the RNA, we have devised an in vivo method of determining relative rates of gene transcription. In both the fat body and the ovary all three yolk proteins are transcribed at similar rates. Thus we infer that YP3 RNA is destabilised in the ovary, accounting for the reduction in its steady-state level. MH - Drosophila Melanogaster/GENETICS/*METABOLISM ; Egg Proteins/*GENETICS ; Female ; Gene Expression Regulation ; Male ; Nucleotides/METABOLISM ; Ovary/METABOLISM ; Peptides/GENETICS ; RNA, Messenger/*METABOLISM ; Support, Non-U.S. Gov't ; Transcription, Genetic SO - Eur J Biochem 1986 Nov 17;161(1):95-101 13 UI - 87051757 AU - Schuh R ; Aicher W ; Gaul U ; C:ot:e S ; Preiss A ; Maier D ; Seifert E ; Nauber U ; Schr:oder C ; Kemler R ; et al TI - A conserved family of nuclear proteins containing structural elements of the finger protein encoded by Kr:uppel, a Drosophila segmentation gene. AB - Kr:uppel (Kr), a segmentation gene of Drosophila, encodes a protein sharing structural features of the DNA-binding "finger motif: of TFIIIA, a Xenopus transcription factor. Low-stringency hybridization of the Kr finger coding sequence revealed multiple copies of homologous DNA sequences in the genomes of Drosophila and other eukaryotes. Molecular analysis of one Kr-homologous DNA clone identified a developmentally regulated gene. Its product, a finger protein, relates to Kr by the invariant positioning of crucial amino acid residues within the finger repeats and by a stretch of seven amino acids connecting the finger loops, the "H/C link.: This H/C link is conserved in several nuclear and chromosome-associated proteins of Drosophila and other eukaryotic organisms including mammals. Our results demonstrate a new subfamily of evolutionarily conserved nuclear and possibly DNA-binding proteins that again relate to a Drosophila segmentation gene as in the case of the homeo domain. MH - Animal ; Drosophila/EMBRYOLOGY/*GENETICS ; DNA-Binding Proteins/*GENETICS ; *Genes ; Nucleic Acid Hybridization ; Sequence Homology, Nucleic Acid ; Support, Non-U.S. Gov't ; Transcription, Genetic ; Vertebrates/GENETICS SO - Cell 1986 Dec 26;47(6):1025-32 14 UI - 87016343 AU - Whitelaw E ; Coates A ; Proudfoot NJ TI - Globin gene transcripts can utilize histone gene 3' end processing signals. AB - Deletion of the poly(A) site from the human alpha globin gene results in a defective gene that produces very little stable mRNA as compared to the intact gene, presumably due to the instability of the mRNA. However, if the Alpha poly(A) site is replaced by mouse histone H4 3' end processing signals, significant levels of hybrid alpha/H4 mRNA are obtained and the transcripts formed are cytoplasmic and poly(A)-. When both mouse histone 3' end processing signals and the alpha globin poly(A) site signals are placed in tandem after the alpha globin gene promoter and coding sequence, the alpha poly(A) site signals are utilized exclusively. These results show that the histone 3' end processing signals can function independently of the histone promoter and the transcripts which are normally polyadenylated (alpha globin) can be stabilized by a poly(A)- histone mRNA 3' terminus. Furthermore, these results show that the histone 3' end processing signals are less efficient than the alpha globin poly(A) site signals, if the two are placed in direct competition. MH - Animal ; Gene Expression Regulation ; Genetic Intervention ; Globin/ *GENETICS ; Hela Cells ; Histones/*GENETICS ; Human ; Mice ; Poly A/ GENETICS ; *RNA Processing, Post-Transcriptional ; RNA, Messenger/ *GENETICS ; Support, Non-U.S. Gov't SO - Nucleic Acids Res 1986 Sep 11;14(17):7059-70 15 UI - 87008376 AU - Collins JJ ; Roberts GP ; Brill WJ TI - Posttranscriptional control of Klebsiella pneumoniae nif mRNA stability by the nifL product. AB - Posttranscriptional control of nif mRNA stability was demonstrated by functional and chemical analyses, using specific probes for four nif transcripts. In the wild type, nif transcripts (except nifLA) were stable during derepression, with half-lives of approximately 30 min. They were dramatically destabilized by O2 or elevated temperature (41 degrees C) and to a lesser extent by NH4+. In contrast, the nifLA message was not particularly stable, and posttranscriptional control was not evident. In NifL- strains, both forms of analysis indicated that the nifL product was involved in nif mRNA destabilization in the presence of O2 and NH4+. MH - Acetates/PHARMACOLOGY ; Bacterial Proteins/*PHYSIOLOGY/SECRETION ; *Gene Expression Regulation ; Genes, Bacterial ; Klebsiella Pneumoniae/ *GENETICS/METABOLISM ; Models, Genetic ; *Nitrogen Fixation ; Oxygen/ PHARMACOLOGY ; RNA, Bacterial/GENETICS/METABOLISM ; RNA, Messenger/ GENETICS/*METABOLISM ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Temperature ; Transcription, Genetic SO - J Bacteriol 1986 Oct;168(1):173-8 16 UI - 86223996 AU - Theofan G ; Norman AW TI - Effects of alpha-amanitin and cycloheximide on 1,25-dihydroxyvitamin D3-dependent calbindin-D28K and its mRNA in vitamin D3-replete chick intestine. AB - We have examined the effects of the transcriptional inhibitor alpha-amanitin and the translational inhibitor cycloheximide on levels of calbindin-D28K (28-kDa calcium binding protein, CaBP) and CaBP-mRNA in the vitamin D-replete chick intestine. Chicks were raised on one of four diets: "normal: (1% calcium, 0.6% phosphorus); high calcium (3.3% calcium, 0.5% phosphorus); low calcium (0.3% calcium, 0.6% phosphorus); or low phosphorus (1% calcium, 0.09% phosphorus). Chicks were then treated either with alpha-amanitin (20 micrograms/chick) or cycloheximide (600 micrograms/chick) 2 h prior to a dose of 6.5 nmol of 1,25-dihydroxyvitamin D3. Duodenal mucosa was collected from 0 to 120 min afterward and assayed for CaBP-mRNA by dot blot hybridization and for CaBP using an enzyme-linked immunosorbent assay. In the absence of inhibitor, CaBP levels were depressed by high calcium and elevated by low calcium or low phosphorus, as expected. These changes occurred, however, without a change in CaBP-mRNA levels. alpha-Amanitin had no effect on CaBP or on CaBP-mRNA levels in chicks raised on any of the diets. Cycloheximide inhibited CaBP levels, and surprisingly also inhibited CaBP-mRNA levels in all four dietary groups. These results indicate that continual protein synthesis is necessary for the expression of CaBP-mRNA, suggesting the existence of a rapidly turned over protein that may be required for stabilization or for processing of the chick intestinal CaBP messenger RNA. MH - Amanitins/*PHARMACOLOGY ; Animal ; Calcitriol/*PHARMACOLOGY ; Calcium-Binding Protein, Vitamin D-Dependent/*ANALYSIS/GENETICS ; Calcium-Binding Proteins/*ANALYSIS ; Chickens ; Cycloheximide/ *PHARMACOLOGY ; Intestines/*ANALYSIS ; Male ; Proteins/BIOSYNTHESIS ; RNA, Messenger/*ANALYSIS ; Support, U.S. Gov't, P.H.S. ; Transcription, Genetic/DRUG EFFECTS ; Uridine/METABOLISM SO - J Biol Chem 1986 Jun 5;261(16):7311-5 17 UI - 86315855 AU - Briggs MR ; Kadonaga JT ; Bell SP ; Tjian R TI - Purification and biochemical characterization of the promoter-specific transcription factor, Sp1. AB - The biochemical analysis of cellular trans-activators involved in promoter recognition provides an important step toward understanding the mechanisms of gene expression in animal cells. The promoter selective transcription factor, Sp1, has been purified from human cells to more than 95 percent homogeneity by sequence-specific DNA affinity chromatography. Isolation and renaturation of proteins purified from sodium dodecyl sulfate polyacrylamide gels allowed the identification of two polypeptides (105 and 95 kilodaltons) as those responsible for recognizing and interacting specifically with the GC-box promoter elements characteristic of Sp1 binding sites. MH - Animal ; Base Sequence ; Chromatography, Affinity ; Chromatography, High Pressure Liquid ; Cricetulus ; DNA/METABOLISM ; DNA-Binding Proteins/ *ISOLATION & PURIFICATION/METABOLISM ; Electrophoresis, Polyacrylamide Gel ; Gene Expression Regulation ; Hamsters ; Hela Cells/METABOLISM ; Human ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Transcription Factors/*ISOLATION & PURIFICATION/METABOLISM ; Transcription, Genetic SO - Science 1986 Oct 3;234(4772):47-52 18 UI - 86313641 AU - Lopata MA ; Cleveland DW ; Sollner-Webb B TI - RNA polymerase specificity of mRNA production and enhancer action. AB - To examine the RNA polymerase (EC 2.7.7.6) specificity of RNA maturation/utilization and transcriptional enhancement, we constructed a chimeric plasmid (pPolI-CAT) in which a promoter for mouse rRNA gene transcription was placed adjacent the coding sequences for chloramphenicol acetyltransferase (CAT; EC 2.3.1.28). A number of other constructs, including plasmids also containing a murine sarcoma virus enhancer or lacking any natural eukaryotic promoter sequences, were also prepared. In apparent agreement with earlier conclusions that an RNA polymerase I transcript can act as a messenger RNA, transient transfection of mouse L cells with pPolI-CAT yielded both high levels of transcription from the RNA polymerase I promoter and enzymatically active CAT protein. However, further examination revealed that CAT protein is not translated from RNA that begins at the normal rRNA transcription initiation site. Polysomal RNA is devoid of such RNA and instead consists of CAT-encoding transcripts that begin elsewhere in the mouse ribosomal DNA (rDNA) region. Since transcription of these aberrant RNAs is stimulated by the addition of a murine sarcoma virus enhancer segment, they are probably transcribed by RNA polymerase II. Transcripts that map to the authentic rRNA start site are not similarly enhanced. Moreover, unlike the RNAs deriving from the rRNA initiation site, these aberrant RNAs are more stable and the level of translatable CAT transcripts is suppressed by inclusion of larger segments of the rDNA promoter regions. Fortuitously initiated mRNAs are also formed in the absence of any natural eukaryotic promoter sequence. From these data we conclude that there is no evidence that normal RNA polymerase I transcription yields functional mRNA and that transcriptional enhancement appears to be RNA polymerase specific. MH - Acetyltransferases/ANALYSIS/GENETICS ; Base Sequence ; *Enhancer Elements (Genetics) ; *Genes, Regulator ; Plasmids ; Promoter Regions (Genetics) ; RNA Polymerases/*PHARMACOLOGY ; RNA, Messenger/*BIOSYNTHESIS ; Support, U.S. Gov't, P.H.S. ; Transcription, Genetic ; Transfection SO - Proc Natl Acad Sci USA 1986 Sep;83(18):6677-81 19 UI - 86304477 AU - Kindy MS ; Sonenshein GE TI - Regulation of oncogene expression in cultured aortic smooth muscle cells. Post-transcriptional control of c-myc mRNA. AB - Proliferation of normally quiescent vascular smooth muscle cells plays a major role in the development of an atherosclerotic lesion. Since cellular homologues of oncogenes have been implicated in the control of normal cell proliferation, we have analyzed the regulation of expression of two proto-oncogenes (c-fos and c-myc) in primary cultures of calf aortic smooth muscle cells during the transition from quiescence to proliferation. Quiescent (serum-deprived) smooth muscle cells were stimulated to proliferative by serum addition. DNA synthesis began 12 h post-serum addition and peaked between 16 and 20 h. Following addition of serum, c-fos mRNA levels increased rapidly from an undetectable amount to the maximal level at 30 min after serum addition and then rapidly returned to the levels found in quiescent cells. Changes in the rate of c-fos gene transcription, as measured by nuclear "runoff: assays, paralleled the alterations in mRNA levels, indicating that regulation of c-fos was at the level of mRNA synthesis. The mRNA for the c-myc oncogene was expressed at a detectable level in quiescent cells, peaked in abundance at approximately 2 h after stimulation, and then returned to the level found in quiescent cells. No significant change in the rate of c-myc gene transcription was detectable over the time course. The rate of decay of c-myc mRNA following the inhibition of transcription by actinomycin D was measured at the 1- and 4-h time points and in exponentially growing cells. There was a transient stabilization of the normally labile c-myc mRNA at 1 h after serum stimulation. Thus c-myc gene expression was regulated at the level of mRNA turnover. MH - Animal ; Cattle ; Cells, Cultured ; Dactinomycin/PHARMACOLOGY ; Female ; Flow Cytometry ; *Gene Expression Regulation ; Muscle, Smooth, Vascular/ *METABOLISM ; *Oncogenes ; RNA, Messenger/*METABOLISM ; Support, U.S. Gov't, P.H.S. ; *Transcription, Genetic SO - J Biol Chem 1986 Sep 25;261(27):12865-8 20 UI - 86278093 AU - Zhu YS ; Kiley PJ ; Donohue TJ ; Kaplan S TI - Origin of the mRNA stoichiometry of the puf operon in Rhodobacter sphaeroides. AB - The LH-I structural genes are located 5' of the RC-L and -M structural genes on what has been designated as the puf operon of Rhodobacter sphaeroides. Analysis of puf operon expression in R. sphaeroides by Northern hybridization with probes specific for individual structural gene has identified two transcripts encoded by this operon. The large (2.6 kilobase pairs (kb] transcript contains sequences for all four polypeptides of the puf operon, whereas the small (0.5 kb) transcript, which is more abundant (10-15-fold) than the large transcript under photosynthetic growth, is homologous only to the two LH-I structural genes. Transcription of the puf operon during photosynthetic growth under saturating light conditions is increased approximately 3-fold relative to growth in the presence of oxygen while the relative ratio of these two transcripts is independent of the incident light intensity. Analysis of the turnover of the two transcripts (t1/2 of 9 and 20 min for the large and small transcripts, respectively) indicates that 5' processing is the initial step in the degradation of the large transcript and that the molar excess of the small transcript cannot be accounted for by differences in the rates of turnover of these two mRNA species. Analysis of the 5' ends of the 2.6- and 0.5-kb transcripts, their relative abundance, and stabilities indicates that these two transcripts have different 5'-ends corresponding to 75 and 104 base pairs upstream from the start of the LH-I beta structural gene, respectively. Northern hybridization analysis with specific synthetic deoxyoligonucleotide probes confirmed that the two transcripts differ by 29 bases at their 5'-ends, suggesting that differential transcript initiation may be involved in regulating the relative levels of these two mRNA species in vivo, although we cannot rule out complex mechanisms of post-transcriptional processing. MH - Bacterial Proteins/GENETICS ; DNA Restriction Enzymes ; DNA, Bacterial/ GENETICS ; Genes, Structural ; Light ; Nucleic Acid Hybridization ; *Operon ; Oxygen/PHARMACOLOGY ; Photosynthesis ; Rhodopseudomonas Spheroides/DRUG EFFECTS/*GENETICS/RADIATION EFFECTS ; RNA, Bacterial/ *GENETICS ; RNA, Messenger/*GENETICS ; Support, U.S. Gov't, P.H.S. ; Transcription, Genetic SO - J Biol Chem 1986 Aug 5;261(22):10366-74 21 UI - 86269898 AU - Cockayne D ; Sterling KM Jr ; Shull S ; Mintz KP ; Illeyne S ; Cutroneo KR TI - Glucocorticoids decrease the synthesis of type I procollagen mRNAs. AB - Glucocorticoids selectively decrease procollagen synthesis in animal and human skin fibroblasts. beta-Actin content and beta-actin mRNA are not affected by glucocorticoid treatment of chick skin fibroblasts. The inhibitory effect of glucocorticoids on procollagen synthesis is associated with a decrease in total cellular type I procollagen mRNAs in chick skin fibroblasts. These effects of dexamethasone are receptor mediated as determined by pretreatment with the glucocorticoid antagonists progesterone and RU-486 and with the agonist beta-dihydrocortisol. Dexamethasone has a small but significant inhibitory effect on cell growth of chick skin fibroblasts. The ability of this corticosteroid to decrease the steady-state levels of type I procollagen mRNAs in nuclei, cytoplasm, and polysomes varies. The largest decrease of type I procollagen mRNAs is observed in the nuclear and cytoplasmic subcellular fractions 24 h after dexamethasone treatment. Type I procollagen hnRNAs are also decreased as determined by Northern blot analysis of total nuclear RNA. The synthesis of total cellular type I procollagen mRNAs is reversibly decreased by dexamethasone treatment. In addition the synthesis of total nuclear type I procollagen mRNA sequences is decreased at 2, 4, and 24 h following the addition of radioactive nucleoside and dexamethasone to cell cultures. Although the synthesis of pro alpha 1(I) and pro alpha 2(I) mRNAs is decreased in dexamethasone-treated chick skin fibroblasts, the degradation of the total cellular procollagen mRNAs is not altered while the degradation of total cellular RNA is stabilized. These data indicate that the dexamethasone-mediated decrease of procollagen synthesis in embryonic chick skin fibroblasts results from the regulation of procollagen gene expression. MH - Actins/METABOLISM ; Animal ; Chick Embryo ; Collagen/BIOSYNTHESIS ; Dexamethasone/*PHARMACOLOGY ; Fibroblasts/METABOLISM ; Glucocorticoids/ *PHARMACOLOGY ; Kinetics ; Nucleic Acid Hybridization ; Procollagen/ *GENETICS ; Proline/METABOLISM ; RNA/ISOLATION & PURIFICATION ; RNA, Heterogeneous Nuclear/GENETICS/ISOLATION & PURIFICATION ; RNA, Messenger/ *GENETICS ; Skin/*METABOLISM ; Support, U.S. Gov't, P.H.S. ; Transcription, Genetic/*DRUG EFFECTS SO - Biochemistry 1986 Jun 3;25(11):3202-9 22 UI - 86245070 AU - Belasco JG ; Nilsson G ; von Gabain A ; Cohen SN TI - The stability of E. coli gene transcripts is dependent on determinants localized to specific mRNA segments. AB - To map the structural features responsible for the 5-fold difference in stability of the E. coli ompA and bla gene transcripts, we have constructed gene fusions that encode chimeric ompA/bla transcripts and a deletion that eliminates a large internal segment of bla mRNA. Shortening of bla transcripts by internal deletion or replacement of the 3' end with the corresponding segment of the ompA transcript had little effect on bla mRNA stability. However, fusion of a 5'-terminal 147 nucleotide segment of the ompA message 5' to full-length or truncated bla transcripts increased the half-life of the bla segments 3- to 5-fold. These and other findings indicate that E. coli transcripts contain discrete structural determinants of stability and instability that can influence the decay rate of linked mRNA segments derived from other genes. MH - Chimera ; Escherichia Coli/*GENETICS/METABOLISM ; *Genes, Structural ; Half-Life ; Nucleic Acid Hybridization ; Plasmids ; RNA, Messenger/ *GENETICS/METABOLISM ; Support, U.S. Gov't, P.H.S. ; *Transcription, Genetic ; Translation, Genetic SO - Cell 1986 Jul 18;46(2):245-51 23 UI - 86242742 AU - Bird RC ; Jacobs FA ; Sells BH TI - Stability of histone mRNAs is related to their location in polysomes. AB - Synthesis of histone mRNAs is closely coupled to DNA synthesis. Following inhibition of DNA synthesis in L6 myoblasts with cytosine arabinoside, a coordinate and exaggerated rate of degradation of histone mRNAs occurs while other mRNAs, encoding ribosomal protein L32 and actin, are unaffected. Inhibition of protein synthesis by puromycin, emetine, or cycloheximide stabilizes histone mRNAs and results in their accumulation. When inhibition of DNA synthesis was followed immediately by inhibition of protein synthesis, the exaggerated rate of decay of the existing subspecies of histone H4 mRNAs was prevented and histone mRNA accumulated. If inhibition of protein synthesis was delayed longer than 3 minutes following inhibition of DNA synthesis, the ability to accumulate H4 mRNAs was lost. Furthermore, new protein synthesis was required to activate the mechanism which specifically destabilized histone mRNA. Puromycin was able to prevent the exaggerated rate of degradation of the various subspecies of H4 mRNA when added up to 15 min after inhibition of DNA synthesis, whereas emetine was effective only when added up to 5 min following inhibition of DNA synthesis. These data suggest that histone H4 mRNAs in polysomes are better targets than those released from polysomes for the specific mechanism which destabilizes histone mRNAs upon inhibition of DNA synthesis. MH - Animal ; Cell Line ; Cycloheximide/PHARMACOLOGY ; Cytarabine/PHARMACOLOGY ; DNA Replication ; Emetine/PHARMACOLOGY ; Histones/*GENETICS ; Kinetics ; Polyribosomes/DRUG EFFECTS/*METABOLISM/ULTRASTRUCTURE ; Puromycin/ PHARMACOLOGY ; Rats ; RNA, Messenger/*GENETICS ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Transcription, Genetic ; Translation, Genetic/DRUG EFFECTS SO - Biochem Cell Biol 1986 Feb;64(2):99-105 24 UI - 86233305 AU - Mayrand SH ; Pedersen N ; Pederson T TI - Identification of proteins that bind tightly to pre-mRNA during in vitro splicing. AB - Incubation of a human beta-globin pre-mRNA in a HeLa cell nuclear extract under conditions permissive for efficient splicing resulted in the assembly of the RNA into ribonucleoprotein (RNP) complexes. This RNP formation occurred largely within the characteristic lag period that precedes splicing. Two classes of RNP were detected by the criterion of their stability in Cs2SO4 gradients. One was unstable and contained mainly aberrant RNA cleavage products. The other class of RNP complexes comprised 50-85% of the beta-globin RNA, formed only under splicing-permissive conditions, was stable in Cs2SO4 gradients, and contained both unspliced pre-mRNA molecules and the lariat intron 1-exon 2 splicing intermediate. This latter class of RNP complexes banded at approximately equal to 1.30 g/cm3, a density very similar to that of native heterogeneous nuclear RNP particles that contain pre-mRNA. RNA-protein crosslinking revealed major proteins of Mr approximately equal to 38,000 and 41,000 in the stable class of RNP. The use of antibodies specific for heterogeneous nuclear RNP core proteins and for small nuclear RNA-associated proteins, in conjunction with [32P]RNA-protein crosslinking, revealed polypeptides having the molecular weights of both sets of antigens. These results show that both heterogeneous nuclear RNP particle core proteins and small nuclear RNA-associated proteins bind tightly to pre-mRNA during splicing in vitro. MH - Autoantibodies/IMMUNOLOGY ; Female ; Globin/GENETICS ; Hela Cells ; Human ; In Vitro ; Nucleic Acid Precursors/*METABOLISM ; Ribonucleoproteins/ *METABOLISM ; *RNA Processing, Post-Transcriptional ; *RNA Splicing ; RNA, Messenger/*METABOLISM ; RNA, Small Nuclear/IMMUNOLOGY/METABOLISM ; Support, U.S. Gov't, P.H.S. SO - Proc Natl Acad Sci USA 1986 Jun;83(11):3718-22 25 UI - 86218097 AU - Grummt I ; Rosenbauer H ; Niedermeyer I ; Maier U ; Ohrlein A TI - A repeated 18 bp sequence motif in the mouse rDNA spacer mediates binding of a nuclear factor and transcription termination. AB - DNA sequences and protein factors directing termination of mouse rDNA transcription in a nuclear extract system were examined. Termination is specific and requires a sequence element AGGTCGACCAGATTANTCCG (the Sall box) that is present eight times in the spacer region downstream of the 3' end of pre-rRNA. Exonuclease III protection experiments reveal the binding of a nuclear protein to the Sall box. Deletions, insertions, and point mutations in the Sall box reduce or abolish the interaction with the nuclear factor and disrupt transcription termination. A synthetic oligonucleotide corresponding to the Sall box consensus sequence governs transcription termination in vitro, although with reduced activity. Therefore, other sequences normally surrounding the Sall box appear to contribute to the accuracy and efficiency of termination. MH - Animal ; Cell Line ; DNA-Binding Proteins/METABOLISM ; DNA, Recombinant ; DNA, Ribosomal/*GENETICS ; *Genes, Regulator ; Mice ; Mutation ; Oligonucleotides/GENETICS ; Promoter Regions (Genetics) ; Repetitive Sequences, Nucleic Acid ; RNA Polymerase I/METABOLISM ; Support, Non-U.S. Gov't ; *Terminator Regions (Genetics) ; Transcription Factors/ *METABOLISM ; *Transcription, Genetic SO - Cell 1986 Jun 20;45(6):837-46 26 UI - 86205871 AU - Wong HC ; Chang S TI - Identification of a positive retroregulator that stabilizes mRNAs in bacteria. AB - A positive retroregulator that enhances the expression of an upstream gene(s) has been identified. It resides within a 381-base pair (bp) restriction fragment containing the transcriptional terminator of the crystal protein (cry) gene from Bacillus thuringiensis vs. Kurstaki HD-1. This fragment was fused to the distal ends of either the penicillinase (penP) gene of Bacillus licheniformis or the interleukin 2 cDNA from the human Jurkat cell line. In both cases, the half-lives of the mRNAs derived from the fusion genes were increased from approximately equal to 2 to 6 min in both Escherichia coli and Bacillus subtilis. Synthesis of the corresponding polypeptides in the bacteria carrying the fusion genes was also increased correspondingly. The enhancement of expression of the upstream genes was independent of the insertional orientation of the distal cry terminator fragment. Deletion analysis showed that the locus conferring the enhancing activity coincided with the terminator sequence and was located within a 89-bp fragment that includes an inverted repeat, the 19-bp upstream-, and the 27-bp downstream-flanking sequences. We propose that transcription of the retroregulator sequence leads to the incorporation of the corresponding stem-and-loop structure at the 3' end of the mRNA; the presence of this structure protects the mRNAs from exonucleolytic degradation from the 3' end and, thereby, increases the mRNA half-life and enhances protein synthesis of the target genes. MH - Bacillus thuringiensis/*GENETICS ; Bacillus Subtilis/*GENETICS ; Bacterial Proteins/*GENETICS ; Base Sequence ; DNA, Recombinant ; Escherichia Coli/*GENETICS ; *Gene Expression Regulation ; Genes, Bacterial ; *Genes, Regulator ; Genes, Structural ; Human ; Interleukin 2/ GENETICS ; Nucleic Acid Conformation ; Recombinant Proteins/GENETICS ; RNA, Messenger/*GENETICS/METABOLISM ; *Transcription, Genetic SO - Proc Natl Acad Sci USA 1986 May;83(10):3233-7 27 UI - 86187761 AU - Berger FG ; Loose D ; Meisner H ; Watson G TI - Androgen induction of messenger RNA concentrations in mouse kidney is posttranscriptional. AB - The concentrations of several mRNAs in mouse kidney increase in response to testosterone. To determine if the increases are generated at the level of gene transcription, we have assayed transcription rates for several androgen-inducible mRNAs in kidney nuclei in vitro. No significant changes were found in the synthesis of three mRNAs whose concentrations increase 10-20-fold during testosterone treatment. Kinetic analysis of changes in transcript levels after testosterone administration and withdrawal suggests that mRNA stabilization is a major factor in the inductions. Thus, the androgen-mediated induction of these kidney mRNAs is generated predominantly at the posttranscriptional level. MH - Animal ; Female ; Kidney/DRUG EFFECTS/*METABOLISM ; Kinetics ; Mice ; Mice, Inbred A ; Mice, Inbred DBA ; RNA Processing, Post-Transcriptional/ *DRUG EFFECTS ; RNA, Messenger/BIOSYNTHESIS/*GENETICS ; Support, U.S. Gov't, P.H.S. ; Testosterone/*PHARMACOLOGY ; Transcription, Genetic/DRUG EFFECTS SO - Biochemistry 1986 Mar 11;25(5):1170-5 28 UI - 86168174 AU - Sazer S ; Schimke RT TI - A re-examination of the 5' termini of mouse dihydrofolate reductase RNA. AB - Using primer extension and nuclease S1-mapping techniques we have re-examined the 5' termini of RNA transcribed from the mouse dihydrofolate reductase gene. We characterize a previously undescribed transcription initiation site at position -55 relative to the AUG codon, in addition to the previously identified start site at position -115. Differences in the 5' noncoding regions of these two transcripts with respect to their length and relative G + C content result in their differential ability to form stable hybrids with the DNA probe used in previous analyses of these transcripts and thus precluded the detection of transcripts initiated at -55. We show that changes in the temperature of the hybridization reaction result in the ability to detect the RNA having a shorter noncoding region and a lower G + C content. That position -55 represents an authentic transcription start site is confirmed by use of a DNA probe with which the two transcripts can form S1-resistant hybrids of equal stability and by primer extension analysis using an oligonucleotide primer that hybridizes near the AUG codon. These analyses also demonstrate that the transcript with a 5' end mapping near position -55 accounts for the majority of cellular dihydrofolate reductase RNA. MH - Animal ; Base Sequence ; Cell Line ; Cells, Cultured ; Clone Cells ; DNA Restriction Enzymes ; Endonucleases ; Mice ; Nucleic Acid Conformation ; Nucleic Acid Hybridization ; RNA, Messenger/*GENETICS ; Support, U.S. Gov't, P.H.S. ; Tetrahydrofolate Dehydrogenase/*GENETICS ; *Transcription, Genetic SO - J Biol Chem 1986 Apr 5;261(10):4685-90 29 UI - 86149304 AU - Belanger FC ; Brodl MR ; Ho TH TI - Heat shock causes destabilization of specific mRNAs and destruction of endoplasmic reticulum in barley aleurone cells. AB - In response to a phytohormone, gibberellic acid, the aleurone layers of barley seeds synthesize and secrete alpha-amylases, which are coded by a set of stable mRNAs. When aleurone layers are subjected to heat shock treatment, the synthesis of alpha-amylase is suppressed while heat shock proteins are induced. The suppression of alpha-amylase synthesis is not the result of translational control as reported in several other systems. Rather, the sequences of alpha-amylase mRNA are rapidly degraded during heat shock as shown by in vitro translation and dot blot hybridization with a cDNA probe. Upon recovery from heat shock, the tissue resumes the synthesis of alpha-amylase in 2-4 hr. However, in the presence of a transcription inhibitor, cordycepin, the resumption of synthesis of alpha-amylase does not take place, indicating that new transcription of alpha-amylase genes is necessary for this recovery process. The degradation of alpha-amylase mRNAs correlates with the rapid destruction of endoplasmic reticulum as observed by electron microscopy, a phenomenon that has not been reported previously as a heat shock response. Since alpha-amylase mRNA is associated with the endoplasmic reticulum via membrane-bound polyribosomes, we suggest that the destruction of the endoplasmic reticulum during heat shock causes the destabilization and the eventual degradation of alpha-amylase mRNA. MH - Alpha-Amylase/GENETICS/METABOLISM ; Barley/CYTOLOGY/METABOLISM ; Deoxyadenosine/PHARMACOLOGY ; Endoplasmic Reticulum/ULTRASTRUCTURE ; Enzyme Induction/DRUG EFFECTS ; Gibberellins/PHARMACOLOGY ; Heat ; Heat-Shock Proteins/*GENETICS ; RNA, Messenger/*METABOLISM ; Support, U.S. Gov't, Non-P.H.S. ; Time Factors ; Transcription, Genetic SO - Proc Natl Acad Sci USA 1986 Mar;83(5):1354-8 30 UI - 86149278 AU - Toulm:e JJ ; Krisch HM ; Loreau N ; Thuong NT ; H:el:ene C TI - Specific inhibition of mRNA translation by complementary oligonucleotides covalently linked to intercalating agents. AB - Synthetic oligodeoxynucleotides that are covalently linked at their 3' end to an acridine derivative and are complementary to the repeated sequence UUAAAUUAAAUUAAA adjacent to the ribosome binding site of the gene 32-encoded mRNA from phage T4 have been used to regulate the synthesis of gene 32-encoded protein in vitro. These modified, synthetic oligonucleotides specifically block the translation of gene 32-encoded mRNA with a higher efficiency than the homologous unsubstituted oligonucleotides. The inhibition produced by these short "anti-messengers: is due to the formation of specific mRNA . oligodeoxynucleotide hybrids that are stabilized by the intercalation of the acridine ring in the RNA . DNA duplex. MH - Acridines ; Base Sequence ; Escherichia Coli ; Genes, Viral ; Intercalating Agents ; Nucleic Acid Hybridization ; Oligonucleotides/ *PHARMACOLOGY ; Ribosomes/*METABOLISM ; RNA, Double-Stranded/*METABOLISM ; RNA, Messenger/*METABOLISM ; RNA, Viral/GENETICS ; Support, Non-U.S. Gov't ; T-Phages/GENETICS ; Transcription, Genetic ; *Translation, Genetic SO - Proc Natl Acad Sci USA 1986 Mar;83(5):1227-31 31 UI - 86136133 AU - Arcangioli B ; Lescure B TI - Structural features of the DNA template required for transcription in vitro by yeast RNA polymerase B (II). AB - Yeast RNA polymerase II initiates in vitro transcription at two sites located within the vector DNA and the cloned promoter, on a recombinant plasmid DNA containing the yeast iso1 cytochrome c promoter. Both initiation sites are found within a DNA fragment hypersensitive to osmium tetroxide modification. Using a series of yeast iso1 cytochrome c promoter deletions, we have characterized an upstream DNA sequence required for optimal transcription from this site and shown in this case a correlation between osmium sensitivity and the capacity of RNA polymerase to initiate. However, perturbation of the double helix is not sufficient to generate a transcription initiation site. Insertion of 28 alternating AT residues at the EcoRV site of pBR322 generates an site hypersensitive to osmium tetroxide modification, that does not serve as a transcription start site. MH - Binding Sites ; DNA, Fungal/*METABOLISM ; DNA, Recombinant ; Osmium Tetroxide ; Peptide Chain Initiation ; Plasmids ; Promoter Regions (Genetics) ; RNA Polymerase II/*METABOLISM ; Saccharomyces Cerevisiae/ ENZYMOLOGY/*GENETICS ; Support, Non-U.S. Gov't ; Templates ; *Transcription, Genetic SO - Eur J Biochem 1986 Feb 17;155(1):69-75 32 UI - 86122884 AU - Keegan L ; Gill G ; Ptashne M TI - Separation of DNA binding from the transcription-activating function of a eukaryotic regulatory protein. AB - The yeast GAL4 protein (881 amino acids) binds to specific DNA sites upstream of target genes and activates transcription. Derivatives of this protein bearing as few as 74 amino terminal residues bind to these sites but fail to activate transcription. When appropriately positioned in front of a gene these derivatives act as repressors. These and related findings support the idea that GAL4 activates transcription by touching other DNA-bound proteins. MH - Amino Acid Sequence ; Base Sequence ; Beta-Galactosidases/GENETICS ; DNA-Binding Proteins/*GENETICS/METABOLISM ; DNA, Fungal/GENETICS/ METABOLISM ; DNA, Recombinant ; Fungal Proteins/GENETICS ; Galactose ; Gene Expression Regulation ; Repressor Proteins/GENETICS ; Saccharomyces Cerevisiae/*GENETICS ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Transcription Factors/*GENETICS/METABOLISM ; *Transcription, Genetic SO - Science 1986 Feb 14;231(4739):699-704 USER: 33 UI - 86111932 AU - Rabek JP ; Papaconstantinou J TI - Identification of a stable nuclear RNA complementary to the 3'-end flanking sequences of the mouse beta-major globin gene. AB - A stable nuclear RNA of approximately 1600 nucleotides (nt) isolated from dimethyl sulfoxide-induced Friend erythroleukemia cells has been characterized. This RNA has been shown to be homologous to a region of unique sequences situated 3' to the mouse beta-major globin gene between the poly(A) addition site and a BglII site located 1400 nt farther downstream. It is transcribed from the same DNA strand as the beta-major globin mRNA, and the amount of this RNA present in the cell is directly proportional to the level of beta-globin mRNA. Therefore, the 1600-nt RNA appears to be related to the large primary transcript of the beta-major globin gene. We feel that this RNA species is an unusually stable intermediate product of the early processing event which cleaves the primary transcript at the poly(A) addition site. The observed stability and discrete length which are contrary to the expected properties of such a processing intermediate may reflect peculiarities of the transformed state of the Friend erythroleukemia cells. MH - Animal ; Base Sequence ; Cell Line ; Cell Nucleus/ANALYSIS ; Comparative Study ; Erythroleukemia/ANALYSIS ; Friend Virus ; Globin/*GENETICS ; Mice ; RNA Processing, Post-Transcriptional ; RNA, Messenger/*METABOLISM ; RNA, Neoplasm/METABOLISM ; Sequence Homology, Nucleic Acid ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Transcription, Genetic SO - J Biol Chem 1986 Feb 15;261(5):2304-8 34 UI - 86106203 AU - Cockerill PN ; Garrard WT TI - Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. AB - Introduction of torsional stress into active chromatin domains requires that linear DNA molecules be anchored in vivo to impede free rotation. While searching for these anchorage elements, we have localized a nuclear matrix association region (MAR) within the mouse immunoglobulin kappa gene that contains two topoisomerase II sites and is adjacent to the tissue-specific enhancer. The same matrix contact occurs when the kappa locus is in germ-line (inactive) or rear-ranged (transcribed) configurations. This constitutive anchorage site partitions the gene into V-J and C region chromatin domains. We demonstrate that at least 10,000 similar and evolutionarily conserved MAR binding sites exist in the nucleus. We propose that these sites, in association with topoisomerase II and possibly in conjunction with enhancers, play fundamental roles in the functional organization of chromatin loop domains. MH - Animal ; B Lymphocytes/PHYSIOLOGY/ULTRASTRUCTURE ; Cell Nucleus/ *ULTRASTRUCTURE ; Chromosome Mapping ; Chromosomes/PHYSIOLOGY/ *ULTRASTRUCTURE ; DNA Gyrase/*METABOLISM ; DNA-Binding Proteins/GENETICS ; *Enhancer Elements (Genetics) ; Gene Expression Regulation ; *Genes, Regulator ; Immunoglobulin Constant Region/GENETICS ; Immunoglobulin Variable Region/GENETICS ; Immunoglobulins, Kappa Chain/*GENETICS ; Mice ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Tissue Distribution ; Transcription, Genetic SO - Cell 1986 Jan 31;44(2):273-82