scholarly journals ATTAAA as well as downstream sequences are required for RNA 3'-end formation in the E3 complex transcription unit of adenovirus.

1985 ◽  
Vol 5 (11) ◽  
pp. 3183-3193 ◽  
Author(s):  
B M Bhat ◽  
W S Wold
Keyword(s):  
Base Pair ◽  
Transcription Unit ◽  
Rna Probes ◽  
Labeled Rna

We mapped the location of the E3A RNA 3' end site in the E3 transcription unit of adenovirus 2. The procedure used was nuclease-gel analysis with 32P-labeled RNA probes. The poly(A) addition sites were microheterogeneous and were located approximately 17 to 29 nucleotides downstream from an ATTAAA sequence. To identify the sequences that make up the E3A RNA 3' end signal, we constructed five viable virus mutants with deletions in or near the E3A RNA 3' end site. The mutants were analyzed for E3A RNA 3' end formation in vivo. No effect was observed from a 47-base-pair (bp) deletion (dl716) or a 72-bp deletion (dl714) located 22 and 19 nucleotides, respectively, upstream of the ATTAAA. In contrast, E3A RNA 3' end formation was abolished by a 554-bp deletion (dl708) that removes both the ATTAAA and the poly(A) addition sites, a 124-bp deletion (dl713) that removes the ATTAAA but leaves the poly(A) addition sites, and a 65-bp deletion (dl719) that leaves the ATTAAA but removes the poly(A) addition sites. These results indicate that the ATTAAA, as well as downstream sequences, including the poly(A) addition sites, are required for E3A RNA 3' end formation.

ATTAAA as well as downstream sequences are required for RNA 3'-end formation in the E3 complex transcription unit of adenovirus

1985 ◽  
Vol 5 (11) ◽  
pp. 3183-3193
Author(s):  
B M Bhat ◽  
W S Wold
Keyword(s):  
Base Pair ◽  
Transcription Unit ◽  
Rna Probes ◽  
Labeled Rna

We mapped the location of the E3A RNA 3' end site in the E3 transcription unit of adenovirus 2. The procedure used was nuclease-gel analysis with 32P-labeled RNA probes. The poly(A) addition sites were microheterogeneous and were located approximately 17 to 29 nucleotides downstream from an ATTAAA sequence. To identify the sequences that make up the E3A RNA 3' end signal, we constructed five viable virus mutants with deletions in or near the E3A RNA 3' end site. The mutants were analyzed for E3A RNA 3' end formation in vivo. No effect was observed from a 47-base-pair (bp) deletion (dl716) or a 72-bp deletion (dl714) located 22 and 19 nucleotides, respectively, upstream of the ATTAAA. In contrast, E3A RNA 3' end formation was abolished by a 554-bp deletion (dl708) that removes both the ATTAAA and the poly(A) addition sites, a 124-bp deletion (dl713) that removes the ATTAAA but leaves the poly(A) addition sites, and a 65-bp deletion (dl719) that leaves the ATTAAA but removes the poly(A) addition sites. These results indicate that the ATTAAA, as well as downstream sequences, including the poly(A) addition sites, are required for E3A RNA 3' end formation.

scholarly journals Base-Pair Substitutions in Avian Sarcoma Virus U5 and U3 Long Terminal Repeat Sequences Alter the Process of DNA Integration In Vitro

2001 ◽  
Vol 75 (3) ◽  
pp. 1132-1141 ◽  
Author(s):  
Patrick Hindmarsh ◽  
Michael Johnson ◽  
Ray Reeves ◽  
Jonathan Leis
Keyword(s):  
Base Pair ◽  
High Mobility ◽  
Transcription Unit ◽  
Avian Sarcoma Virus ◽  
High Mobility Group Protein ◽  
Dna Integration ◽  
Sarcoma Virus ◽  
Avian Sarcoma

ABSTRACT We have described a reconstituted avian sarcoma virus (ASV) concerted DNA integration system with specially designed mini-donor DNA containing a supF transcription unit, a supercoiled plasmid acceptor, purified bacterially expressed ASV integrase (IN), and human high-mobility-group protein I(Y). Integration in this system is dependent upon the mini-donor DNA having IN recognition sequences at both ends and upon both ends of the same donor integrating into the acceptor DNA. The integrated DNA product exhibits all of the features associated with integration of viral DNA in vivo (P. Hindmarsh et al., J. Virol., 73:2994–3003, 1999). Individual integrants are isolated from bacteria containing drug-resistant markers with amber mutations. This system was used to evaluate the importance of sequences in the terminal U5 and U3 long terminal repeats at positions 5 and/or 6, adjacent to the conserved CA dinucleotide. Base-pair substitutions introduced at these positions in U5 result in significant reductions in recovered integrants from bacteria, due to increases in one-ended insertion events. Among the recovered integrants from reactions with mutated U5 but not U3 IN recognition sequences were products that contain large deletions in the acceptor DNA. Base-pair substitutions at positions 5 and 6 in U3 mostly reduce the efficiency of integration of the modified donor. Together, these results indicate that sequences directly 5′ to the conserved CA dinucleotide are very important for the process of concerted DNA integration. Furthermore, IN interacts with U3 and U5 termini differently, and aberrant end-processing events leading to nonconcerted DNA integration are more common in U5 than in U3.

scholarly journals Lysozyme is an inducible marker of macrophage activation in murine tissues as demonstrated by in situ hybridization.

1991 ◽  
Vol 174 (5) ◽  
pp. 1049-1058 ◽  
Author(s):  
S Keshav ◽  
P Chung ◽  
G Milon ◽  
S Gordon
Keyword(s):  
In Situ Hybridization ◽  
White Pulp ◽  
Mrna Levels ◽  
Necrosis Factor Alpha ◽  
Constitutive Function ◽  
Rna Probes ◽  
Factor Alpha ◽  
Labeled Rna

This study demonstrates the induction of lysozyme mRNA expression in situ in tissue macrophages (M phi) of mice following in vivo stimulation. The resting resident tissue M phi of most tissues do not contain enough lysozyme mRNA to be detected by in situ hybridization using 35S-labeled RNA probes. Following Bacille Calmette Guerin or Plasmodium yoelli infection, however, M phi recruited to liver and spleen hybridize strongly to the lysozyme probe. Within 24 h of infection, cells found in the marginal zone of the spleen begin to produce lysozyme mRNA. This response is also evoked by a noninfectious agent (intravenously injected sheep erythrocytes), and is possibly the result of an early phagocytic interaction. Later in the infection, other cells in the red and white pulp of the spleen, and cells in granulomas in the liver, become lysozyme-positive. Kupffer cells are rarely lysozyme-positive. Lysozyme mRNA levels in liver granulomas remain relatively constant during the infection, and lysozyme is produced by most granuloma cells. This contrasts with tumor necrosis factor alpha (TNF alpha) mRNA, which is produced by fewer cells in the granuloma, and which can be massively induced by lipopolysaccharide administration. The production of lysozyme, previously considered a constitutive function of M phi, is therefore an indicator of M phi activation in vivo, where immunologically specific and nonspecific stimuli both stimulate lysozyme production at high levels in subpopulations of cells occupying discrete anatomical locations.

scholarly journals Drosophila Hormone Receptor 38 Functions in Metamorphosis: A Role in Adult Cuticle Formation

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1465-1475 ◽  
Author(s):  
T Kozlova ◽  
G V Pokholkova ◽  
G Tzertzinis ◽  
J D Sutherland ◽  
I F Zhimulev ◽  
...  
Keyword(s):  
Pupal Stage ◽  
Transcription Unit ◽  
P Element ◽  
Specific Response ◽  
Orphan Receptors ◽  
Mrna Isoforms ◽  
A Cell ◽  
In Vivo Analysis

Abstract DHR38 is a member of the steroid receptor superfamily in Drosophila homologous to the vertebrate NGFI-B-type orphan receptors. In addition to binding to specific response elements as a monomer, DHR38 interacts with the USP component of the ecdysone receptor complex in vitro, in yeast and in a cell line, suggesting that DHR38 might modulate ecdysone-triggered signals in the fly. We characterized the molecular structure and expression of the Dhr38 gene and initiated an in vivo analysis of its function(s) in development. The Dhr38 transcription unit spans more than 40 kb in length, includes four introns, and produces at least four mRNA isoforms differentially expressed in development; two of these are greatly enriched in the pupal stage and encode nested polypeptides. We characterized four alleles of Dhr38: a P-element enchancer trap line, l(2)02306, which shows exclusively epidermal staining in the late larval, pre-pupal and pupal stages, and three EMS-induced alleles. Dhr38 alleles cause localized fragility and rupturing of the adult cuticle, demonstrating that Dhr38 plays an important role in late stages of epidermal metamorphosis.

In vivo analysis of sequences necessary for CBP1-dependent accumulation of cytochrome b transcripts in yeast mitochondria

1993 ◽  
Vol 13 (7) ◽  
pp. 4203-4213
Author(s):  
T M Mittelmeier ◽  
C L Dieckmann
Keyword(s):  
Cytochrome B ◽  
Mitochondrial Gene ◽  
Microprojectile Bombardment ◽  
Nuclear Gene ◽  
State Level ◽  
Transcription Unit ◽  
Endonucleolytic Cleavage ◽  
In Vivo Analysis ◽  
Nucleotide Region

In Saccharomyces cerevisiae, cytochrome b, an essential component of the respiratory chain, is encoded by the mitochondrial gene cob. The cob transcription unit includes the tRNA(Glu) gene from positions -1170 to -1099 relative to the cob ATG at +1. The initial tRNA(Glu)-cob transcript undergoes several processing events, including removal of tRNA(Glu) and production of the mature 5' end of cob mRNA at nucleotide -954. The nuclear gene product CBP1 is specifically required for the accumulation of cob mRNA. In cbp1 mutant strains, cob transcripts are not detectable by Northern (RNA) blot analysis, but the steady-state level of tRNA(Glu) is similar to that of wild type. The results of a previous study led to the conclusion that a 400-nucleotide region just downstream of tRNA(Glu) is sufficient for CBP1 function. In the present study, the microprojectile bombardment method of mitochondrial transformation was used to introduce deletions within this region of cob. The analysis of cob transcripts in strains carrying the mitochondrial deletion genomes indicates that a 63-nucleotide sequence that encompasses the cleavage site at -954 is sufficient both for CBP1 function and for correct positioning of the cleavage. Furthermore, the data indicate that CBP1 prevents the degradation of unprocessed cob transcripts produced by endonucleolytic cleavage at the 3' end of tRNA(Glu).

Identification of a protein complex that binds to a dodecamer sequence found at the 3' ends of yeast mitochondrial mRNAs

1993 ◽  
Vol 13 (7) ◽  
pp. 4167-4173
Author(s):  
J Min ◽  
H P Zassenhaus
Keyword(s):  
Binding Activity ◽  
Nucleoside Triphosphate ◽  
Yeast Mitochondria ◽  
Mobility Shift ◽  
Saccharomyces Cerevisiae Mitochondria ◽  
Molecular Masses ◽  
Labeled Rna ◽  
Gel Mobility Shift ◽  
A Site

An activity from Saccharomyces cerevisiae mitochondria was identified that specifically bound to a 12-nucleotide sequence, AAUAA(U/C)AUUCUU, that is a site for processing of pre-mRNAs so as to generate the mature 3' ends of mRNAs. Because processing occurs 3' to the end of the dodecamer site, all mRNAs in yeast mitochondria terminate with that sequence. RNase T1 digestion fragments which terminated precisely at their 3' ends with the dodecamer sequence bound the activity, indicating that mRNAs in vivo would be capable of binding. Gel mobility shift analyses using RNA oligonucleotides showed that binding was reduced by a U-to-A substitution at position 3 of the dodecamer sequence; a C-to-A substitution at position 10 eliminated binding. UV cross-linking identified three polypeptides with approximate molecular masses of 19, 60, and 70 kDa as constituents of the binding activity. These estimates included the contribution of the 32P-labeled RNA oligonucleotide used to tag these polypeptides. An oligonucleotide with a UA-->AU substitution at positions 3 and 4 of the dodecamer site formed complexes deficient in the 19-kDa species, suggesting that binding specificity was inherent to the higher-molecular-weight polypeptides. Assembly of the complex at a dodecamer site on an RNA protected sequences located 5' to the dodecamer site from digestion by a nucleoside triphosphate-dependent 3' exoribonuclease found in yeast mitochondria. Since mitochondrial mRNAs terminate with an intact dodecamer sequence, the binding activity may function in the stabilization of mRNAs in addition to 3'-end formation of mRNAs.

scholarly journals In vitro analysis of a transcription termination site for RNA polymerase II.

1990 ◽  
Vol 10 (11) ◽  
pp. 5782-5795 ◽  
Author(s):  
D K Wiest ◽  
D K Hawley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Transcription Unit ◽  
Dependent Manner ◽  
Wide Range ◽  
Vitro Analysis

Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.

scholarly journals Supercoiling DNA locates mismatches

2017 ◽  
Author(s):  
Andrew Dittmore ◽  
Sumitabha Brahmachari ◽  
Yasuhara Takagi ◽  
John F. Marko ◽  
Keir C. Neuman
Keyword(s):  
Dna Sequence ◽  
Base Pair ◽  
Dna Supercoiling ◽  
Magnetic Tweezers ◽  
Relative Probability ◽  
Base Pairs ◽  
Damage Sensing ◽  
Mismatched Base Pair ◽  
Monovalent Salt

We present a method of detecting sequence defects by supercoiling DNA with magnetic tweezers. The method is sensitive to a single mismatched base pair in a DNA sequence of several thousand base pairs. We systematically compare DNA molecules with 0 to 16 adjacent mismatches at 1 M monovalent salt and 3.5 pN force and show that, under these conditions, a single plectoneme forms and is stably pinned at the defect. We use these measurements to estimate the energy and degree of end-loop kinking at defects. From this, we calculate the relative probability of plectoneme pinning at the mismatch under physiologically relevant conditions. Based on this estimate, we propose that DNA supercoiling could contribute to mismatch and damage sensing in vivo.

scholarly journals Regulation of Sulfur Assimilation Pathways in Burkholderia cenocepacia: Identification of Transcription Factors CysB and SsuR and Their Role in Control of Target Genes

2006 ◽  
Vol 189 (5) ◽  
pp. 1675-1688 ◽  
Author(s):  
Roksana Iwanicka-Nowicka ◽  
Agata Zielak ◽  
Anne M. Cook ◽  
Mark S. Thomas ◽  
Monika M. Hryniewicz
Keyword(s):  
Target Genes ◽  
Positive Control ◽  
Transcription Unit ◽  
Burkholderia Cenocepacia ◽  
Sulfur Assimilation ◽  
Allelic Replacement ◽  
Genes Encoding ◽  
Target Promoters

ABSTRACT Two genes encoding transcriptional regulators involved in sulfur assimilation pathways in Burkholderia cenocepacia strain 715j have been identified and characterized functionally. Knockout mutations in each of the B. cenocepacia genes were constructed and introduced into the genome of 715j by allelic replacement. Studies on the utilization of various sulfur sources by 715j and the obtained mutants demonstrated that one of the B. cenocepacia regulators, designated CysB, is preferentially involved in the control of sulfate transport and reduction, while the other, designated SsuR, is required for aliphatic sulfonate utilization. Using transcriptional promoter-lacZ fusions and DNA-binding experiments, we identified several target promoters for positive control by CysB and/or SsuR—sbpp (preceding the sbp cysT cysW cysA ssuR cluster), cysIp (preceding the cysI cysD1 cysN cysH cysG cluster), cysD2p (preceding a separate cluster, cysD2 cysNC), and ssuDp (located upstream of the ssuDCB operon)—and we demonstrated overlapping functions of CysB and SsuR at particular promoters. We also demonstrated that the cysB gene is negatively controlled by both CysB and SsuR but the ssuR gene itself is not significantly regulated as a separate transcription unit. The function of B. cenocepacia CysB (in vivo and in vitro) appeared to be independent of the presence of acetylserine, the indispensable coinducer of the CysB regulators of Escherichia coli and Salmonella. The phylogenetic relationships among members of the “CysB family” in the γ and β subphyla are presented.

Identification of an evolutionarily conserved 110 base-pair cis-acting regulatory sequence that governs Wnt-1 expression in the murine neural plate

Development ◽  
1998 ◽  
Vol 125 (14) ◽  
pp. 2735-2746 ◽  
Author(s):  
D.H. Rowitch ◽  
Y. Echelard ◽  
P.S. Danielian ◽  
K. Gellner ◽  
S. Brenner ◽  
...  
Keyword(s):  
Base Pair ◽  
Regulatory Element ◽  
Regulatory Region ◽  
Neural Plate ◽  
Homologous Region ◽  
Regulatory Sequence ◽  
Embryonic Mouse ◽  
Cis Acting ◽  
Evolutionarily Conserved

The generation of anterior-posterior polarity in the vertebrate brain requires the establishment of regional domains of gene expression at early somite stages. Wnt-1 encodes a signal that is expressed in the developing midbrain and is essential for midbrain and anterior hindbrain development. Previous work identified a 5.5 kilobase region located downstream of the Wnt-1 coding sequence which is necessary and sufficient for Wnt-1 expression in vivo. Using a transgenic mouse reporter assay, we have now identified a 110 base pair regulatory sequence within the 5.5 kilobase enhancer, which is sufficient for expression of a lacZ reporter in the approximate Wnt-1 pattern at neural plate stages. Multimers of this element driving Wnt-1 expression can partially rescue the midbrain-hindbrain phenotype of Wnt-1(−/−) embryos. The possibility that this region represents an evolutionarily conserved regulatory module is suggested by the identification of a highly homologous region located downstream of the wnt-1 gene in the pufferfish (Fugu rubripes). These sequences are capable of appropriate temporal and spatial activation of a reporter gene in the embryonic mouse midbrain; although, later aspects of the Wnt-1 expression pattern are absent. Genetic evidence has implicated Pax transcription factors in the regulation of Wnt-1. Although Pax-2 binds to the 110 base pair murine regulatory element in vitro, the location of the binding sites could not be precisely established and mutation of two putative low affinity sites did not abolish activation of a Wnt-1 reporter transgene in vivo. Thus, it is unlikely that Pax proteins regulate Wnt-1 by direct interactions with this cis-acting regulatory region. Our analysis of the 110 base pair minimal regulatory element suggests that Wnt-1 regulation is complex, involving different regulatory interactions for activation and the later maintenance of transgene expression in the dorsal midbrain and ventral diencephalon, and at the midbrain-hindbrain junction.

Sign in / Sign up

Export Citation Format

Share Document