scholarly journals In vitro transcription of cloned 5S RNA genes of the newt Notophthalmus.

1981 ◽  
Vol 90 (2) ◽  
pp. 323-331 ◽  
Author(s):  
B K Kay ◽  
O Schmidt ◽  
J G Gall
Keyword(s):  
Dna Sequences ◽  
Gene Segment ◽  
In Vitro Transcription ◽  
Xenopus Laevis Oocyte ◽  
Coding Region ◽  
5S Rna ◽  
Rna Sequences ◽  
Repeat Units ◽  
5S Gene

Recombinant plasmids that carried genes coding for 5S ribosomal RNA of the newt, Notophthalmus viridescens, were transcribed in vitro with extracts of Xenopus laevis oocyte nuclei. Plasmids containing multiple repeats of the 5S gene and spacer directed accurate transcription of 5S RNA (120 bases). Individual repeat units were recloned by inserting Sau 3A restriction fragments into the Bam HI site of plasmid pBR322. Because each repeat was cut by the enzyme within the coding region, the inserts had incomplete coding regions at their ends and spacer sequences in the middle. The DNA of these subclones directed synthesis of a 5S-size RNA that contained both plasmid and 5S RNA sequences. Transcription initiated in the vector, proceeded through the gene segment coding for nucleotides 41-120, and terminated at the end of the gene. The initiation of in vitro transcription required neither the original 5' flanking sequences of the spacer nor the first third of the gene. We conclude that intragenic DNA sequences control the initiation of transcription. Other subclones that include pseudogenes gave rise to some transcripts 156 nucleotides long. These long transcripts represented continuation of transcription through the 36-base-pair pseudogene that is located immediately downstream from the 5S gene. However, most transcripts of these subclones terminated at the end of the normal gene before the beginning of the pseudogene. It is probable that a run of four or more Ts serves as part of the termination signal.

Simian virus 40 major late promoter: an upstream DNA sequence required for efficient in vitro transcription

1984 ◽  
Vol 4 (1) ◽  
pp. 133-141
Author(s):  
J Brady ◽  
M Radonovich ◽  
M Thoren ◽  
G Das ◽  
N P Salzman
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Simian Virus 40 ◽  
Promoter Sequence ◽  
In Vitro Transcription ◽  
Simian Virus ◽  
Upstream Promoter ◽  
Upstream Promoter Sequence

We have previously identified an 11-base DNA sequence, 5'-G-G-T-A-C-C-T-A-A-C-C-3' (simian virus 40 [SV40] map position 294 to 304), which is important in the control of SV40 late RNA expression in vitro and in vivo (Brady et al., Cell 31:625-633, 1982). We report here the identification of another domain of the SV40 late promoter. A series of mutants with deletions extending from SV40 map position 0 to 300 was prepared by nuclease BAL 31 treatment. The cloned templates were then analyzed for efficiency and accuracy of late SV40 RNA expression in the Manley in vitro transcription system. Our studies showed that, in addition to the promoter domain near map position 300, there are essential DNA sequences between nucleotide positions 74 and 95 that are required for efficient expression of late SV40 RNA. Included in this SV40 DNA sequence were two of the six GGGCGG SV40 repeat sequences and an 11-nucleotide segment which showed strong homology with the upstream sequences required for the efficient in vitro and in vivo expression of the histone H2A gene. This upstream promoter sequence supported transcription with the same efficiency even when it was moved 72 nucleotides closer to the major late cap site. In vitro promoter competition analysis demonstrated that the upstream promoter sequence, independent of the 294 to 304 promoter element, is capable of binding polymerase-transcription factors required for SV40 late gene transcription. Finally, we show that DNA sequences which control the specificity of RNA initiation at nucleotide 325 lie downstream of map position 294.

scholarly journals In vitro transcription of immunoglobulin genes in a B-cell extract: effects of enhancer and promoter sequences.

1987 ◽  
Vol 7 (5) ◽  
pp. 1989-1994 ◽  
Author(s):  
R Sen ◽  
D Baltimore
Keyword(s):  
B Cell ◽  
Dna Sequences ◽  
In Vitro Transcription ◽  
Cell Extract ◽  
Upstream Sequence ◽  
Immunoglobulin Genes ◽  
Specific Expression ◽  
Sequence Element ◽  
Transcription System

Transfection experiments have led to the identification of three DNA sequences that are responsible for the tissue-specific expression of immunoglobulin genes. As a first step toward characterizing these regulatory phenomena at the biochemical level, we report the development of an in vitro transcription system from cells of the B lymphoid lineage. In these extracts, transcription of the MOPC41 kappa promoter is correctly initiated and dependent on the presence of an upstream sequence element located between -44 and -79 base pairs from the cap site. Second, although standard in vitro transcriptions are not affected by the presence or absence of enhancer sequences, we observed that the addition of polyethylene glycol led to a B-cell extract-specific suppression of transcription from a template that carries an immunoglobulin enhancer.

scholarly journals RNA polymerase II transcription factors H4TF-1 and H4TF-2 require metal to bind specific DNA sequences.

1987 ◽  
Vol 7 (12) ◽  
pp. 4582-4584 ◽  
Author(s):  
L Dailey ◽  
S B Roberts ◽  
N Heintz
Keyword(s):  
Dna Binding ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Chelating Agents ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Rna Polymerase Ii Transcription

Specific DNA-binding and in vitro transcription activities of H4TF-1 and H4TF-2 are inactivated by chelating agents. Binding activity is restored by addition of Zn2+, and H4TF-2 is also reactivated by Fe2+. In contrast, preformed factor-DNA complexes are resistant to chelators. Therefore, metal ions are a required component of the H4TF-1 and H4TF-2 DNA-binding domains.

scholarly journals Formation of an active transcription complex in the Drosophila melanogaster 5S RNA gene is dependent on an upstream region.

1987 ◽  
Vol 7 (6) ◽  
pp. 2046-2051 ◽  
Author(s):  
A D Garcia ◽  
A M O'Connell ◽  
S J Sharp
Keyword(s):  
Drosophila Melanogaster ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
Upstream Region ◽  
Nucleotide Position ◽  
Wild Type ◽  
5S Rna ◽  
Cell Extracts

We constructed deletion-substitution and linker-scanning mutations in the 5'-flanking region of the Drosophila melanogaster 5S RNA gene. In vitro transcription of these templates in Drosophila and HeLa cell extracts revealed the presence of an essential control region (-30 region) located between nucleotides -39 and -26 upstream of the transcription initiation site: deletion of sequences upstream of nucleotide position -39 had no detectable effect on the wild-type level of in vitro transcription, whereas mutations extending between positions -39 and 1 resulted in templates with decreased transcriptional levels; specifically, deletion and linker-scanning mutations in the -34 to -26 region (-30 region) resulted in loss of transcription. The -30 region is essential for transcription and therefore forms part of the Drosophila 5S RNA gene transcription promoter. Compared with the activity of the wild-type gene, mutant 5S DNAs exhibited no impairment in the ability to sequester limiting transcription factors in a template exclusion competition assay. While we do not know which transcription factor(s) interacts with the -30 region, the possible involvement of RNA polymerase III at this region is discussed.

scholarly journals Prolonged exposure to methylglyoxal causes disruption of vascular KATPchannel by mRNA instability

2012 ◽  
Vol 303 (10) ◽  
pp. C1045-C1054 ◽  
Author(s):  
Yang Yang ◽  
Shanshan Li ◽  
Anuhya S. Konduru ◽  
Shuang Zhang ◽  
Timothy C. Trower ◽  
...  
Keyword(s):  
Prolonged Exposure ◽  
In Vitro Transcription ◽  
Untranslated Regions ◽  
Luciferase Reporter ◽  
Coding Region ◽  
Intermediary Metabolites ◽  
Vascular Walls ◽  
Reactive Carbonyl Species ◽  
Reporter Assays

Diabetes mellitus is characterized by hyperglycemia and excessive production of intermediary metabolites including methylglyoxal (MGO), a reactive carbonyl species that can lead to cell injuries. Interacting with proteins, lipids, and DNA, excessive MGO can cause dysfunction of various tissues, especially the vascular walls where diabetic complications often take place. However, the potential vascular targets of excessive MGO remain to be fully understood. Here we show that the vascular Kir6.1/SUR2B isoform of ATP-sensitive K+(KATP) channels is likely to be disrupted with an exposure to submillimolar MGO. Up to 90% of the Kir6.1/SUR2B currents were suppressed by 1 mM MGO with a time constant of ∼2 h. Consistently, MGO treatment caused a vast reduction of both Kir6.1 and SUR2B mRNAs endogenously expressed in the A10 vascular smooth muscle cells. In the presence of the transcriptional inhibitor actinomycin-D, MGO remained to lower the Kir6.1 and SUR2B mRNAs to the same degree as MGO alone, suggesting that the MGO effect is likely to compromise the mRNA stability. Luciferase reporter assays indicated that the 3′-untranslated regions (UTRs) of the Kir6.1 but not SUR2 mRNA were targeted by MGO. In contrast, the SUR2B mRNAs obtained with in vitro transcription were disrupted by MGO directly, while the Kir6.1 transcripts were unaffected. Consistent with these results, the constriction of mesenteric arterial rings was markedly augmented with an exposure to 1 mM MGO for 2 h, and such an MGO effect was totally eliminated in the presence of glibenclamide. These results therefore suggest that acting on the 3′-UTR of Kir6.1 and the coding region of SUR2B, MGO causes instability of Kir6.1 and SUR2B mRNAs, disruption of vascular KATPchannels, and impairment of arterial function.

scholarly journals Delineation of DNA sequences that are important for in vitro transcription from the adenovirus EIIa late promoter.

1988 ◽  
Vol 8 (5) ◽  
pp. 1906-1914 ◽  
Author(s):  
D H Huang ◽  
R G Roeder
Keyword(s):  
Dna Sequences ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Initiation Site ◽  
Late Promoter ◽  
Fold Reduction ◽  
Sequence Elements ◽  
A Cell ◽  
Circular Template

Late in infection, transcription of the EIIa gene is initiated primarily at map unit 72 of the adenovirus genome. A cell-free nuclear extract system was used to determine sequence elements important for the function of this late promoter. In such a system, the transcriptional activity of a circular template was found to be much higher (5- to 10-fold) than that of a linear template. The effect of template topology appeared to be dependent on two distal upstream elements with 5' boundaries located near -265 to -223 and -147 to -133 (in relation to the initiation site), since deletions of these regions reduced transcription of the circular template, in a stepwise fashion, to a level similar to that observed with the linear template. Further deletions revealed an element in the -116 region that appeared to be more important for transcription of the circular template (10-fold reduction) than for transcription of the linear template (3-fold reduction). Lastly, deletion of the TACAAA sequence in the -29 region resulted in further reduction in transcription, indicating that this element functions as a promoter in vitro.

scholarly journals Transcriptionally inactive oocyte-type 5S RNA genes of Xenopus laevis are complexed with TFIIIA in vitro.

1987 ◽  
Vol 7 (10) ◽  
pp. 3503-3510 ◽  
Author(s):  
L J Peck ◽  
L Millstein ◽  
P Eversole-Cire ◽  
J M Gottesfeld ◽  
A Varshavsky
Keyword(s):  
Xenopus Laevis ◽  
Differential Expression ◽  
Gene Transcription ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
5S Rna ◽  
Differential Binding ◽  
5S Rna Genes ◽  
Rna Genes

An extract from whole oocytes of Xenopus laevis was shown to transcribe somatic-type 5S RNA genes approximately 100-fold more efficiently than oocyte-type 5S RNA genes. This preference was at least 10-fold greater than the preference seen upon microinjection of 5S RNA genes into oocyte nuclei or upon in vitro transcription in an oocyte nuclear extract. The approximately 100-fold transcriptional bias in favor of the somatic-type 5S RNA genes observed in vitro in the whole oocyte extract was similar to the transcriptional bias observed in developing Xenopus embryos. We also showed that in the whole oocyte extract, a promoter-binding protein required for 5S RNA gene transcription, TFIIIA, was bound both to the actively transcribed somatic-type 5S RNA gene and to the largely inactive oocyte-type 5S RNA genes. These findings suggest that the mechanism for the differential expression of 5S RNA genes during Xenopus development does not involve differential binding of TFIIIA to 5S RNA genes.

scholarly journals Identification of an RNA Hairpin in Poliovirus RNA That Serves as the Primary Template in the In Vitro Uridylylation of VPg

2000 ◽  
Vol 74 (22) ◽  
pp. 10359-10370 ◽  
Author(s):  
Aniko V. Paul ◽  
Elizabeth Rieder ◽  
Dong Wook Kim ◽  
Jacques H. van Boom ◽  
Eckard Wimmer
Keyword(s):  
Rna Synthesis ◽  
Rna Replication ◽  
Covalent Attachment ◽  
Minus Strand ◽  
Coding Region ◽  
Rna Sequences ◽  
Cellular Factors ◽  
Rna Hairpin ◽  
Made In

ABSTRACT The first step in the replication of the plus-stranded poliovirus RNA is the synthesis of a complementary minus strand. This process is initiated by the covalent attachment of UMP to the terminal protein VPg, yielding VPgpU and VPgpUpU. We have previously shown that these products can be made in vitro in a reaction that requires only synthetic VPg, UTP, poly(A), purified poliovirus RNA polymerase 3Dpol, and Mg2+ (A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280–284, 1998). Since such a poly(A)-dependent process cannot confer sufficient specificity to poliovirus RNA replication, we have developed a new assay to search for a viral RNA template in conjunction with viral or cellular factors that could provide this function. We have now discovered a small RNA hairpin in the coding region of protein 2C as the site in PV1(M) RNA that is used as the primary template for the in vitro uridylylation of VPg. This hairpin has recently been described in poliovirus RNA as being an essential structure for the initiation of minus strand RNA synthesis (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590–4600, 2000). The uridylylation reaction either with transcripts of cre(2C) RNA or with full-length PV1(M) RNA as the template is strongly stimulated by the addition of purified viral protein 3CDpro. Deletion of the cre(2C) RNA sequences from minigenomes eliminates their ability to serve as template in the reaction. A similar signal in the coding region of VP1 in HRV14 RNA (K. L. McKnight and S. M. Lemon, RNA 4:1569–1584, 1998) and the poliovirus cre(2C) can be functionally exchanged in the assay. The mechanism by which the VPgpUpU precursor, made specifically on the cre(2C) template, might be transferred to the site where it serves as primer for poliovirus RNA synthesis, remains to be determined.

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