scholarly journals Minor transcription initiation events indicate that both human mitochondrial promoters function bidirectionally.

1986 ◽  
Vol 6 (1) ◽  
pp. 294-301 ◽  
Author(s):  
D D Chang ◽  
J E Hixson ◽  
D A Clayton
Keyword(s):  
Mitochondrial Dna ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Site Directed Mutagenesis ◽  
Deletion Mapping ◽  
Rich Region ◽  
Human Mitochondrial Dna ◽  
Loop Region

Human mitochondrial DNA is transcribed from two distinct, strand-specific promoters located in the displacement loop region of the genome. The transcriptional control sequences identified by deletion mapping and site-directed mutagenesis studies span short regions surrounding the initiation sites and bear no obvious sequence homology to any nuclear or procaryotic promoters. In vitro transcription analyses also revealed several minor initiation sites that are characterized by a pyrimidine-rich region followed by a purine-rich region, a feature that is shared by the two major promoters. In this paper, we report a new class of minor promoters in human mitochondrial DNA. These minor promoters were localized to the same duplex DNA sequences that direct major transcriptional events, but they had transcriptional polarity opposite to that of the major promoters. Furthermore, nucleotide changes that affected the major form of transcription similarly affected transcription in the opposite direction. For one of these minor promoters, a corresponding in vivo RNA species initiating from the same site was identified. These observations indicate that the major transcriptional promoters in human mitochondria can function bidirectionally both in vivo and in vitro.

Minor transcription initiation events indicate that both human mitochondrial promoters function bidirectionally

1986 ◽  
Vol 6 (1) ◽  
pp. 294-301
Author(s):  
D D Chang ◽  
J E Hixson ◽  
D A Clayton
Keyword(s):  
Mitochondrial Dna ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Site Directed Mutagenesis ◽  
Deletion Mapping ◽  
Rich Region ◽  
Human Mitochondrial Dna ◽  
Loop Region

Human mitochondrial DNA is transcribed from two distinct, strand-specific promoters located in the displacement loop region of the genome. The transcriptional control sequences identified by deletion mapping and site-directed mutagenesis studies span short regions surrounding the initiation sites and bear no obvious sequence homology to any nuclear or procaryotic promoters. In vitro transcription analyses also revealed several minor initiation sites that are characterized by a pyrimidine-rich region followed by a purine-rich region, a feature that is shared by the two major promoters. In this paper, we report a new class of minor promoters in human mitochondrial DNA. These minor promoters were localized to the same duplex DNA sequences that direct major transcriptional events, but they had transcriptional polarity opposite to that of the major promoters. Furthermore, nucleotide changes that affected the major form of transcription similarly affected transcription in the opposite direction. For one of these minor promoters, a corresponding in vivo RNA species initiating from the same site was identified. These observations indicate that the major transcriptional promoters in human mitochondria can function bidirectionally both in vivo and in vitro.

Precise assignment of the heavy-strand promoter of mouse mitochondrial DNA: cognate start sites are not required for transcriptional initiation

1986 ◽  
Vol 6 (9) ◽  
pp. 3262-3267
Author(s):  
D D Chang ◽  
D A Clayton
Keyword(s):  
Mitochondrial Dna ◽  
Dna Sequences ◽  
High Rate ◽  
In Vitro Assays ◽  
Regulatory Sequences ◽  
Sufficient Information ◽  
Mitochondrial Rna ◽  
Loop Region ◽  
Heavy Strand

Transcription of the heavy strand of mouse mitochondrial DNA starts from two closely spaced, distinct sites located in the displacement loop region of the genome. We report here an analysis of regulatory sequences required for faithful transcription from these two sites. Data obtained from in vitro assays demonstrated that a 51-base-pair region, encompassing nucleotides -40 to +11 of the downstream start site, contains sufficient information for accurate transcription from both start sites. Deletion of the 3' flanking sequences, including one or both start sites to -17, resulted in the initiation of transcription by the mitochondrial RNA polymerase from alternative sites within vector DNA sequences. This feature places the mouse heavy-strand promoter uniquely among other known mitochondrial promoters, all of which absolutely require cognate start sites for transcription. Comparison of the heavy-strand promoter with those of other vertebrate mitochondrial DNAs revealed a remarkably high rate of sequence divergence among species.

scholarly journals Preferential amplification of a human mitochondrial DNA deletion in vitro and in vivo

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Oliver M. Russell ◽  
Isabelle Fruh ◽  
Pavandeep K. Rai ◽  
David Marcellin ◽  
Thierry Doll ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Mitochondrial Dna Deletion ◽  
Human Mitochondrial Dna ◽  
Dna Deletion ◽  
Preferential Amplification

Identification of a new promoter upstream of the murine dihydrofolate reductase gene

1989 ◽  
Vol 9 (10) ◽  
pp. 4568-4570
Author(s):  
L J Schilling ◽  
P J Farnham
Keyword(s):  
Dihydrofolate Reductase ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Deletion Mapping ◽  
Reductase Gene ◽  
Bona Fide ◽  
Translational Start

In vitro reactions identified a transcription initiation site located 740 nucleotides upstream of the dihydrofolate reductase translational start. Transcription from this site proceeded in the direction opposite to that of dihydrofolate reductase mRNA. Deletion mapping indicated that this new promoter can be separated from the dihydrofolate reductase promoter and that separation increased transcription at -740. Transcripts that initiate at -740 were also detected in cellular RNA, indicating that this is a bona fide transcription initiation site in vivo.

scholarly journals Interdependent transcription control elements regulate the expression of the SPRR2A gene during keratinocyte terminal differentiation.

1996 ◽  
Vol 16 (10) ◽  
pp. 5365-5374 ◽  
Author(s):  
D F Fischer ◽  
S Gibbs ◽  
P van De Putte ◽  
C Backendorf
Keyword(s):  
Transcription Factors ◽  
Molecular Mechanisms ◽  
Terminal Differentiation ◽  
Site Directed Mutagenesis ◽  
Deletion Mapping ◽  
Transcription Control ◽  
Mobility Shift ◽  
Response Element

Expression of the SPRR2A gene, a member of the small proline-rich family of cornified cell envelope precursor proteins, is strictly linked to keratinocyte terminal differentiation both in vivo and in vitro. In this study, we explored the molecular mechanisms underlying this regulation in transiently transfected primary keratinocytes induced to differentiate in vitro. Deletion mapping and site-directed mutagenesis of SPRR2A promoter-chloramphenicol acetyltransferase constructs indicate that four transcription control elements are essential and sufficient for promoter activity. These elements were further characterized by electrophoretic mobility shift and identified as (i) an inverted octamer doublet, bound by the POU domain factor Oct-11 (Skn-1a/i, Epoc-1), (ii) an interferon-stimulated response element recognized by interferon regulatory factors 1 and 2, (iii) an Ets binding site partially overlapping the interferon-stimulated response element, and (iv) a TG box recognized by the Sp1 family of zinc finger transcription factors. Destruction of a single terminal differentiation element is sufficient to completely abolish transcription from the SPRR2A promoter, indicating that these transcription control elements function in concert in an interdependent manner. Apparently, integration of signals transmitted by the above-mentioned transcription factors is necessary and sufficient to promote gene expression during keratinocyte terminal differentiation.

scholarly journals Precise assignment of the heavy-strand promoter of mouse mitochondrial DNA: cognate start sites are not required for transcriptional initiation.

1986 ◽  
Vol 6 (9) ◽  
pp. 3262-3267 ◽  
Author(s):  
D D Chang ◽  
D A Clayton
Keyword(s):  
Mitochondrial Dna ◽  
Dna Sequences ◽  
High Rate ◽  
In Vitro Assays ◽  
Regulatory Sequences ◽  
Sufficient Information ◽  
Mitochondrial Rna ◽  
Loop Region ◽  
Heavy Strand

Transcription of the heavy strand of mouse mitochondrial DNA starts from two closely spaced, distinct sites located in the displacement loop region of the genome. We report here an analysis of regulatory sequences required for faithful transcription from these two sites. Data obtained from in vitro assays demonstrated that a 51-base-pair region, encompassing nucleotides -40 to +11 of the downstream start site, contains sufficient information for accurate transcription from both start sites. Deletion of the 3' flanking sequences, including one or both start sites to -17, resulted in the initiation of transcription by the mitochondrial RNA polymerase from alternative sites within vector DNA sequences. This feature places the mouse heavy-strand promoter uniquely among other known mitochondrial promoters, all of which absolutely require cognate start sites for transcription. Comparison of the heavy-strand promoter with those of other vertebrate mitochondrial DNAs revealed a remarkably high rate of sequence divergence among species.

scholarly journals Archaeal Intrinsic Transcription Termination In Vivo

2009 ◽  
Vol 191 (22) ◽  
pp. 7102-7108 ◽  
Author(s):  
Thomas J. Santangelo ◽  
L'ubomíra Cubonová ◽  
Katherine M. Skinner ◽  
John N. Reeve
Keyword(s):  
Reporter Gene ◽  
Dna Sequences ◽  
Transcription Termination ◽  
Intergenic Sequence ◽  
Loop Region ◽  
Intergenic Sequences ◽  
Archaeal Transcription ◽  
Rna Hairpin

ABSTRACT Thermococcus kodakarensis (formerly Thermococcus kodakaraensis) strains have been constructed with synthetic and natural DNA sequences, predicted to function as archaeal transcription terminators, identically positioned between a constitutive promoter and a β-glycosidase-encoding reporter gene (TK1761). Expression of the reporter gene was almost fully inhibited by the upstream presence of 5′-TTTTTTTT (T8) and was reduced >70% by archaeal intergenic sequences that contained oligo(T) sequences. An archaeal intergenic sequence (t mcrA ) that conforms to the bacterial intrinsic terminator motif reduced TK1761 expression ∼90%, but this required only the oligo(T) trail sequence and not the inverted-repeat and loop region. Template DNAs were amplified from each T. kodakarensis strain, and transcription in vitro by T. kodakarensis RNA polymerase was terminated by sequences that reduced TK1761 expression in vivo. Termination occurred at additional sites on these linear templates, including at a 5′-AAAAAAAA (A8) sequence that did not reduce TK1761 expression in vivo. When these sequences were transcribed on supercoiled plasmid templates, termination occurred almost exclusively at oligo(T) sequences. The results provide the first in vivo experimental evidence for intrinsic termination of archaeal transcription and confirm that archaeal transcription termination is stimulated by oligo(T) sequences and is different from the RNA hairpin-dependent mechanism established for intrinsic bacterial termination.

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.

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.

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