Alternative splicing of the Vupur3 transcript in cowpea produces multiple mRNA species with a single protein product that is present in both plastids and mitochondria

2005 ◽  
Vol 32 (8) ◽  
pp. 683
Author(s):  
John D. Bussell ◽  
Doug J. Hall ◽  
Anthea J. Mann ◽  
Danica E. Goggin ◽  
Craig A. Atkins ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Alternative Splicing ◽  
In Vitro Transcription ◽  
Protein Product ◽  
Heterogeneous Population ◽  
Primary Transcript ◽  
Single Protein ◽  
Genes Encoding ◽  
Molecular Masses

A heterogeneous population of cDNAs (designated Vupur3) encoding phosphoribosylglycinamide formyltransferase (GART; EC 2.1.2.2) was isolated from a cowpea (Vigna unguiculata L. Walp.) nodule library. Three classes of cDNA with the same ORF, but differing in their 3′-UTRs, were identified. Southern analysis and sequencing of genomic DNA confirmed that these differences result from alternative splicing of the primary transcript of a single Vupur3 gene. Alternative splicing does not appear to play a role in the production of soybean (Glycine max Merrill.) pur3 transcripts. The presence of the protein product of the Vupur3 gene, GART, in plastids and mitochondria was confirmed by immunoblotting with antibodies raised against the recombinant protein. The antibodies recognised two proteins with apparent molecular masses of 27 and 27.5 kDa in both mitochondria and plastids. All Vupur3 transcripts have two in-frame start codons that are active in wheatgerm in vitro transcription / translation experiments suggesting a mechanism by which the gene product could be targeted to two organelles. Like other genes encoding enzymes for purine synthesis, Vupur3 is expressed in nodules before nitrogen fixation begins but in contrast to these genes its expression does not increase markedly after nitrogen fixation begins.

scholarly journals Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites.

1989 ◽  
Vol 9 (9) ◽  
pp. 3603-3613 ◽  
Author(s):  
J C Kennell ◽  
A M Lambowitz
Keyword(s):  
Mitochondrial Dna ◽  
Neurospora Crassa ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
In Vitro Transcription ◽  
Transcription System ◽  
Group I ◽  
Genes Encoding

We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-TTAGARA(T/G)G(T/G)ARTRR-3', all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5'-TTAGARR(T/G)R(T/G)A-3', was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5' end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.

scholarly journals The Staphylococcus aureus rsbW(orf159) Gene Encodes an Anti-Sigma Factor of SigB

1999 ◽  
Vol 181 (9) ◽  
pp. 2846-2851 ◽  
Author(s):  
Eishi Miyazaki ◽  
Jong-Min Chen ◽  
Chiew Ko ◽  
William R. Bishai
Keyword(s):  
Staphylococcus Aureus ◽  
Stress Responses ◽  
Sigma Factor ◽  
Specific Binding ◽  
In Vitro Transcription ◽  
Alternative Sigma Factor ◽  
Consensus Sequences ◽  
Its Gene ◽  
Genes Encoding

ABSTRACT SigB, a newly discovered alternative sigma factor ofStaphylococcus aureus, has been shown to play an important role in stress responses and the regulation of virulence factors. ThersbW (orf159) gene is immediately upstream ofsigB. Its gene product is homologous to Bacillus subtilis RsbW which under appropriate conditions binds toB. subtilis SigB and functions as an anti-sigma factor or negative posttranslational regulator. To define the function ofS. aureus RsbW, both the S. aureus SigB and RsbW proteins were expressed in Escherichia coli and purified. Cross-linking experiments with these purified proteins revealed that RsbW was capable of specific binding to SigB. In an in vitro transcription runoff assay, RsbW prevented SigB-directed transcription from the sar P3 promoter, a known SigB-dependent promoter, and the inhibitory activity of RsbW was found to be concentration dependent. We also identified SigB promoter consensus sequences upstream of the genes encoding alkaline shock protein 23 and coagulase and have demonstrated SigB and RsbW dependence for the promoters in vitro. These results show that RsbW is a protein sequestering anti-sigma factor of S. aureus SigB and suggest that SigB activity in S. aureus is regulated posttranslationally.

Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites

1989 ◽  
Vol 9 (9) ◽  
pp. 3603-3613
Author(s):  
J C Kennell ◽  
A M Lambowitz
Keyword(s):  
Mitochondrial Dna ◽  
Neurospora Crassa ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
In Vitro Transcription ◽  
Transcription System ◽  
Group I ◽  
Genes Encoding

We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-TTAGARA(T/G)G(T/G)ARTRR-3', all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5'-TTAGARR(T/G)R(T/G)A-3', was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5' end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.

scholarly journals Characterization of human and rat brain myristoyl-CoA:protein N-myristoyltransferase: evidence for an alternative splice variant of the enzyme

1998 ◽  
Vol 333 (3) ◽  
pp. 491-495 ◽  
Author(s):  
R. A. Jeffrey McILHINNEY ◽  
Kate YOUNG ◽  
Mark EGERTON ◽  
Roger CAMBLE ◽  
Ann WHITE ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Splice Variant ◽  
In Vitro Translation ◽  
Rat Tissues ◽  
Human Cell Lines ◽  
Alternative Splice Variant ◽  
Molecular Masses ◽  
Rapid Amplification

Using 5´-rapid amplification of cDNA ends, we have identified an extended 5´-end of mRNA coding for human myristoyl-CoA:protein N-myristoyltransferase (NMT). PCR using primers based on this new 5´-sequence and reverse primers within the currently accepted coding sequence of the enzyme resulted in the identification of a novel splice variant of NMT. In vitro translation of these cDNAs resulted in the production of proteins with apparent molecular masses of 63 kDa and 48 kDa. Immunoprecipitation of NMT from human cell lines and immunoblotting of a range of rat tissues has identified proteins with molecular masses corresponding to those derived from these cDNAs, and provided evidence that their relative abundance differs among tissues. Our results provide evidence that this enzyme exists in different forms resulting from alternative splicing of the mRNA.

Identification of a transcription factor, an 80-kDa protein that interacts with the HLH recognition motif of the rat p53 promoter

2001 ◽  
Vol 79 (2) ◽  
pp. 153-158 ◽  
Author(s):  
Haisun Song ◽  
Minhyung Lee ◽  
Sunhee Yu ◽  
Jong-sang Park
Keyword(s):  
Transcription Factor ◽  
In Vitro Transcription ◽  
Mobility Shift ◽  
P53 Expression ◽  
Dnase I Footprinting ◽  
E Box ◽  
Molecular Masses ◽  
Mobility Shift Assay ◽  
Stimulating Factor

The p53 promoter has been shown to contain a number of potential regulatory motifs. It was previously reported that the upstream stimulating factor (USF) played a central role in regulating the p53 expression. The USF binding site, E-box, is located around 40 bp upstream of the major transcription start site. In this study, it was confirmed that the E-box binds to proteins by DNase I footprinting assay. In the electrophoretic mobility shift assay (EMSA), two retarded bands were detected. One band was abolished by the competition of USF consensus oligonucleotide, but the other band was not. This result indicated that a factor, other than USF, was bound to the E-box. The molecular masses of the binding proteins were determined by a Southwestern-blotting assay. As a result, 46- and 80-kDa proteins were detected. The 46-kDa protein was eliminated by the competition of USF consensus oligonucleotide. Also, the Southwestern-blotting assay with 32P-labeled USF consensus oligonucleotide showed only a 46-kDa protein. Therefore, the 46-kDa protein was USF. These results showed that USF and the 80-kDa protein were bound to the E-box. In addition, it was proved by in vitro transcription assay that this 80-kDa protein had a basal transcriptional activity.Key words: E-box, HLH, rat p53 promoter, transcription factor, upstream stimulating factor (USF).

scholarly journals Molecular Characterization and Sequence of a Methionine Biosynthetic Locus from Pseudomonas syringae

1998 ◽  
Vol 180 (17) ◽  
pp. 4497-4507 ◽  
Author(s):  
Gary L. Andersen ◽  
Gwyn A. Beattie ◽  
Steven E. Lindow
Keyword(s):  
Pseudomonas Syringae ◽  
In Vitro Transcription ◽  
Open Reading Frames ◽  
Predicted Amino Acid Sequence ◽  
Wild Type ◽  
Methionine Biosynthesis ◽  
Leaf Surfaces ◽  
Bacterial Fitness ◽  
Molecular Masses

ABSTRACT Two methionine biosynthetic genes in Pseudomonas syringae pv. syringae, metX andmetW, were isolated, sequenced, and evaluated for their roles in methionine biosynthesis and bacterial fitness on leaf surfaces. The metXW locus was isolated on a 1.8-kb DNA fragment that was required for both methionine prototrophy and wild-type epiphytic fitness. Sequence analysis identified two consecutive open reading frames (ORFs), and in vitro transcription-translation experiments provided strong evidence that the ORFs encode proteins with the predicted molecular masses of 39 and 22.5 kDa. The predicted amino acid sequence of MetX (39 kDa) showed homology to several known and putative homoserineO-acetyltransferases. This enzyme is the first enzyme in the methionine biosynthetic pathway of fungi, gram-negative bacteria of the genus Leptospira, and several gram-positive bacterial genera. Both metX andmetW were required for methionine biosynthesis, and transcription from both genes was not repressed by methionine. MetW (22.5 kDa) did not show significant homology to any known protein, including prokaryotic and eukaryotic methionine biosynthetic enzymes. Several classes of methionine auxotrophs, includingmetX and metW mutants, exhibit reduced fitness on leaf surfaces, indicating a requirement for methionine prototrophy in wild-type epiphytic fitness. This requirement is enhanced under environmentally stressful conditions, suggesting a role for methionine prototrophy in bacterial stress tolerance.

scholarly journals Preparation of Labeled DNA, RNA, and Oligonucleotide Probes

2022 ◽  
Vol 2022 (1) ◽  
pp. pdb.top100578
Author(s):  
Michael R. Green ◽  
Joseph Sambrook
Keyword(s):  
Nucleic Acids ◽  
In Vitro Transcription ◽  
Heterogeneous Population ◽  
Oligonucleotide Probes ◽  
Nick Translation ◽  
Chain Reaction ◽  
Random Priming ◽  
Polymerase Chain ◽  
Enzymatic Methods

Labeled nucleic acids and oligonucleotides are typically generated by enzymatic methods such as end-labeling, random priming, nick translation, in vitro transcription, and variations of the polymerase chain reaction (PCR). Some of these methods place the label in specific locations within the nucleic acid (e.g., at the 5′ or 3′ terminus); others generate molecules that are labeled internally at multiple sites. Some methods yield labeled single-stranded products, whereas others generate double-stranded nucleic acids. Finally, some generate probes of defined length, whereas others yield a heterogeneous population of labeled molecules. Options available for generating and detecting labeled nucleic acids, as well as advice on designing oligonucleotides for use as probes, is included here.

scholarly journals p53 inhibits RNA polymerase III-directed transcription in a promoter-dependent manner.

1996 ◽  
Vol 16 (12) ◽  
pp. 7084-7088 ◽  
Author(s):  
I Chesnokov ◽  
W M Chu ◽  
M R Botchan ◽  
C W Schmid
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
Dependent Manner ◽  
Template Activity ◽  
7Sl Rna ◽  
Polymerase Iii ◽  
Genes Encoding

Wild-type p53 represses Alu template activity in vitro and in vivo. However, upstream activating sequence elements from both the 7SL RNA gene and an Alu source gene relieve p53-mediated repression. p53 also represses the template activity of the U6 RNA gene both in vitro and in vivo but has no effect on in vitro transcription of genes encoding 5S RNA, 7SL RNA, adenovirus VAI RNA, and tRNA. The N-terminal activation domain of p53, which binds TATA-binding protein (TBP), is sufficient for repressing Alu transcription in vitro, and mutation of positions 22 and 23 in this region impairs p53-mediated repression of an Alu template both in vitro and in vivo. p53's N-terminal domain binds TFIIIB, presumably through its known interaction with TBP, and mutation of positions 22 and 23 interferes with TFIIIB binding. These results extend p53's transcriptional role to RNA polymerase III-directed templates and identify an additional level of Alu transcriptional regulation.

scholarly journals Syntrophin binds to an alternatively spliced exon of dystrophin.

1995 ◽  
Vol 128 (3) ◽  
pp. 363-371 ◽  
Author(s):  
A H Ahn ◽  
L M Kunkel
Keyword(s):  
Recombinant Expression ◽  
Expression System ◽  
Electric Organ ◽  
In Vitro Transcription ◽  
Protein Product ◽  
Translation System ◽  
Postsynaptic Protein ◽  
Alternatively Spliced ◽  
Human Dystrophin

Dystrophin, the protein product of the Duchenne muscular dystrophy locus, is a protein of the membrane cytoskeleton that associates with a complex of integral and membrane-associated proteins. Of these, the 58-kD intracellular membrane-associated protein, syntrophin, was recently shown to consist of a family of three related but distinct genes. We expressed the cDNA of human beta 1-syntrophin and the COOH terminus of human dystrophin in reticulocyte lysates using an in vitro transcription/translation system. Using antibodies to dystrophin we immunoprecipitated these two interacting proteins in a variety of salt and detergent conditions. We demonstrate that the 53 amino acids encoded on exon 74 of dystrophin, an alternatively spliced exon, are necessary and sufficient for interaction with translated beta 1-syntrophin in our assay. On the basis of its alternative splicing, dystrophin may thus be present in two functionally distinct populations. In this recombinant expression system, the dystrophin relatives, human dystrophin related protein (DRP or utrophin) and the 87K postsynaptic protein from Torpedo electric organ, also bind to translated beta 1-syntrophin. We have found a COOH-terminal 37-kD fragment of beta 1-syntrophin sufficient to interact with translated dystrophin and its homologues, suggesting that the dystrophin binding site on beta 1-syntrophin occurs on a region that is conserved among the three syntrophin homologues.

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