scholarly journals Characterization of mutant mitochondrial plasmids of Neurospora spp. that have incorporated tRNAs by reverse transcription.

1989 ◽  
Vol 9 (2) ◽  
pp. 678-691 ◽  
Author(s):  
R A Akins ◽  
R L Kelley ◽  
A M Lambowitz
Keyword(s):  
Mitochondrial Dna ◽  
Reverse Transcription ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
Trna Genes ◽  
Wild Type ◽  
Mitochondrial Trna ◽  
Mitochondrial Plasmids ◽  
Circular Dnas ◽  
Mitochondrial Introns

The Mauriceville and Varkud mitochondrial plasmids of Neurospora spp. are closely related, closed-circular DNAs (3.6 and 3.7 kilobases, respectively) whose nucleotide sequences and genetic organization suggest relationships to mitochondrial introns and retroelements. We have characterized nine suppressive mutants of these plasmids that outcompete mitochondrial DNA and lead to impaired growth. All nine suppressive plasmids contain small insertions, corresponding to or including a mitochondrial tRNA (tRNATrp, tRNAGly, or tRNAVal) or a tRNA-like sequence. The insertions are located at the position corresponding to the 5' end of the major plasmid transcript or 24 nucleotides downstream near a cognate of the sequence at the major 5' RNA end. The structure of the suppressive plasmids suggests that the tRNAs were inserted via an RNA intermediate. The 3' end of the wild-type plasmid transcript can itself be folded into a secondary structure which has tRNA-like characteristics, similar to the tRNA-like structures at the 3' ends of plant viral RNAs. This structure may play a role in replication of the plasmids by reverse transcription. Major transcripts of the suppressive plasmids begin at the 5' end of the inserted mitochondrial tRNA sequence and are present in 25- to 100-fold-higher concentrations than are transcripts of wild-type plasmids. Mapping of 5' RNA ends within the inserted mtDNA sequences identifies a short consensus sequence (PuNPuAG) which is present at the 5' ends of a subset of mitochondrial tRNA genes. This sequence, together with sequences immediately upstream in the plasmids, forms a longer consensus sequence, which is similar to sequences at transcription initiation sites in Neurospora mitochondrial DNA. The suppressive behavior of the plasmids is likely to be directly related to the insertion of tRNAs leading to overproduction of plasmid transcripts.

Characterization of mutant mitochondrial plasmids of Neurospora spp. that have incorporated tRNAs by reverse transcription

1989 ◽  
Vol 9 (2) ◽  
pp. 678-691
Author(s):  
R A Akins ◽  
R L Kelley ◽  
A M Lambowitz
Keyword(s):  
Mitochondrial Dna ◽  
Reverse Transcription ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
Trna Genes ◽  
Wild Type ◽  
Mitochondrial Trna ◽  
Mitochondrial Plasmids ◽  
Circular Dnas ◽  
Mitochondrial Introns

The Mauriceville and Varkud mitochondrial plasmids of Neurospora spp. are closely related, closed-circular DNAs (3.6 and 3.7 kilobases, respectively) whose nucleotide sequences and genetic organization suggest relationships to mitochondrial introns and retroelements. We have characterized nine suppressive mutants of these plasmids that outcompete mitochondrial DNA and lead to impaired growth. All nine suppressive plasmids contain small insertions, corresponding to or including a mitochondrial tRNA (tRNATrp, tRNAGly, or tRNAVal) or a tRNA-like sequence. The insertions are located at the position corresponding to the 5' end of the major plasmid transcript or 24 nucleotides downstream near a cognate of the sequence at the major 5' RNA end. The structure of the suppressive plasmids suggests that the tRNAs were inserted via an RNA intermediate. The 3' end of the wild-type plasmid transcript can itself be folded into a secondary structure which has tRNA-like characteristics, similar to the tRNA-like structures at the 3' ends of plant viral RNAs. This structure may play a role in replication of the plasmids by reverse transcription. Major transcripts of the suppressive plasmids begin at the 5' end of the inserted mitochondrial tRNA sequence and are present in 25- to 100-fold-higher concentrations than are transcripts of wild-type plasmids. Mapping of 5' RNA ends within the inserted mtDNA sequences identifies a short consensus sequence (PuNPuAG) which is present at the 5' ends of a subset of mitochondrial tRNA genes. This sequence, together with sequences immediately upstream in the plasmids, forms a longer consensus sequence, which is similar to sequences at transcription initiation sites in Neurospora mitochondrial DNA. The suppressive behavior of the plasmids is likely to be directly related to the insertion of tRNAs leading to overproduction of plasmid transcripts.

A mitochondrial retroplasmid integrates into mitochondrial DNA by a novel mechanism involving the synthesis of a hybrid cDNA and homologous recombination

1994 ◽  
Vol 14 (10) ◽  
pp. 6419-6432
Author(s):  
C C Chiang ◽  
J C Kennell ◽  
L A Wanner ◽  
A M Lambowitz
Keyword(s):  
Mitochondrial Dna ◽  
Homologous Recombination ◽  
Rrna Gene ◽  
Template Switching ◽  
Transcript Analysis ◽  
Wild Type ◽  
Mitochondrial Trna ◽  
Integration Mechanism ◽  
Circular Dnas ◽  
Simple Integration

The Mauriceville and Varkud mitochondrial plasmids of Neurospora spp. are closely related, small circular DNAs that propagate via an RNA intermediate and reverse transcription. Although the plasmids ordinarily replicate autonomously, they can also integrate into mitochondrial DNA (mtDNA), yielding defective mtDNAs that in some cases cause senescence. To investigate the integration mechanism, we analyzed four cases in which the Varkud plasmid integrated into the mitochondrial small rRNA gene, three in wild-type subcultures and one in a senescent mutant. Our analysis suggests that the integrations occurred by the plasmid reverse transcriptase template switching between the plasmid transcript and internal sequences in the mitochondrial small rRNA to yield hybrid cDNAs that circularized and recombined homologously with the mtDNA. The integrated plasmid sequences are transcribed, presumably from the mitochondrial small rRNA promoters, resulting in hybrid RNAs containing the 5' segment of the mitochondrial small rRNA linked head-to-tail to the full-length plasmid transcript. Analysis of additional senescent mutants revealed three cases in which the plasmid used the same mechanism to integrate at other locations in the mtDNA. In these cases, circular variant plasmids that had incorporated a mitochondrial tRNA or tRNA-like sequence by template switching integrated by homologous recombination at the site of the corresponding tRNA or tRNA-like sequence in mtDNA. This simple integration mechanism involving template switching to generate a hybrid cDNA that integrates homologously could have been used by primitive retroelements prior to the acquisition of a specialized integration machinery.

scholarly journals A mitochondrial retroplasmid integrates into mitochondrial DNA by a novel mechanism involving the synthesis of a hybrid cDNA and homologous recombination.

1994 ◽  
Vol 14 (10) ◽  
pp. 6419-6432 ◽  
Author(s):  
C C Chiang ◽  
J C Kennell ◽  
L A Wanner ◽  
A M Lambowitz
Keyword(s):  
Mitochondrial Dna ◽  
Homologous Recombination ◽  
Rrna Gene ◽  
Template Switching ◽  
Transcript Analysis ◽  
Wild Type ◽  
Mitochondrial Trna ◽  
Integration Mechanism ◽  
Circular Dnas ◽  
Simple Integration

The Mauriceville and Varkud mitochondrial plasmids of Neurospora spp. are closely related, small circular DNAs that propagate via an RNA intermediate and reverse transcription. Although the plasmids ordinarily replicate autonomously, they can also integrate into mitochondrial DNA (mtDNA), yielding defective mtDNAs that in some cases cause senescence. To investigate the integration mechanism, we analyzed four cases in which the Varkud plasmid integrated into the mitochondrial small rRNA gene, three in wild-type subcultures and one in a senescent mutant. Our analysis suggests that the integrations occurred by the plasmid reverse transcriptase template switching between the plasmid transcript and internal sequences in the mitochondrial small rRNA to yield hybrid cDNAs that circularized and recombined homologously with the mtDNA. The integrated plasmid sequences are transcribed, presumably from the mitochondrial small rRNA promoters, resulting in hybrid RNAs containing the 5' segment of the mitochondrial small rRNA linked head-to-tail to the full-length plasmid transcript. Analysis of additional senescent mutants revealed three cases in which the plasmid used the same mechanism to integrate at other locations in the mtDNA. In these cases, circular variant plasmids that had incorporated a mitochondrial tRNA or tRNA-like sequence by template switching integrated by homologous recombination at the site of the corresponding tRNA or tRNA-like sequence in mtDNA. This simple integration mechanism involving template switching to generate a hybrid cDNA that integrates homologously could have been used by primitive retroelements prior to the acquisition of a specialized integration machinery.

scholarly journals A Truncation Mutant of the 95-Kilodalton Subunit of Transcription Factor IIIC Reveals Asymmetry in Ty3 Integration

2001 ◽  
Vol 21 (22) ◽  
pp. 7839-7851 ◽  
Author(s):  
Michael Aye ◽  
Sandra L. Dildine ◽  
Jonathan A. Claypool ◽  
Sabine Jourdain ◽  
Suzanne B. Sandmeyer
Keyword(s):  
Transcription Initiation ◽  
Protein Complexes ◽  
Proteolytic Processing ◽  
Trna Genes ◽  
Wild Type ◽  
Long Terminal Repeats ◽  
Truncation Mutant ◽  
Two Hybrid ◽  
Orientation Bias

ABSTRACT Position-specific integration of the retroviruslike element Ty3 near the transcription initiation sites of tRNA genes requires transcription factors IIIB and IIIC (TFIIIB and TFIIIC). Using a genetic screen, we isolated a mutant with a truncated 95-kDa subunit of TFIIIC (TFIIIC95) that reduced the apparent retrotransposition of Ty3 into a plasmid-borne target site between two divergently transcribed tRNA genes. Although TFIIIC95 is conserved and essential, no defect in growth or transcription of tRNAs was detected in the mutant. Steps of the Ty3 life cycle, such as protein expression, proteolytic processing, viruslike particle formation, and reverse transcription, were not affected by the mutation. However, Ty3 integration into a divergent tDNA target occurred exclusively in one orientation in the mutant strain. Investigation of this orientation bias showed that TFIIIC95 and Ty3 integrase interacted in two-hybrid and glutathioneS-transferase pulldown assays and that interaction with the mutant TFIIIC95 protein was attenuated. The orientation bias observed here suggests that even for wild-type Ty3, the protein complexes associated with the long terminal repeats are not equivalent in vivo.

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.

Two novel mutations in the human coagulation factor VII promoter

2003 ◽  
Vol 90 (08) ◽  
pp. 194-205 ◽  
Author(s):  
Anita Kavlie ◽  
Leena Hiltunen ◽  
Vesa Rasi ◽  
Hans Prydz
Keyword(s):  
Transcription Initiation ◽  
Consensus Sequence ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Luciferase Reporter ◽  
Bleeding Tendency ◽  
Wild Type ◽  
Luciferase Reporter Gene ◽  
Coagulation Factor Vii ◽  
Transversion Mutation

SummaryThe factor VII genes of five unrelated Finnish female patients, F1-F5, with moderate bleeding tendency, were screened for mutations using single strand conformational polymorphisms and DNA sequencing. Heterozygous shifts were detected in exons 5 and 8 for patient F1, and sequencing confirmed the presence of the silent dimorphism H115H, the polymorphism R353Q and the mutation A294V. The patient F1 was also heterozygous for a novel –59T/G transversion mutation in the Hepatocyte nuclear factor 4-binding site. The remaining four patients carried a –32A/C transversion mutation located in a footprint (–51 to –32) covering the major transcription initiation start site (–51). There was also a consensus sequence match to an initiator response-like binding element covering –51. Two patients were homozygous and two heterozygous for this mutation. Plasma FVII:Ag and FVII:C levels were reduced in parallel. A strong reduction in binding affinity of a specific nuclear protein to the –32C-containing oligonucleotide was found by electrophoretic mobility shift assays on nuclear extracts from HepG2 cells. EDTA caused no reduced binding. A minimal promoter (–191 to +15) containing the wild-type sequence or the –32A/C or –59T/G mutations was cloned in front of the firefly luciferase reporter gene and transiently transfected into Hep3B cells. Reduced activities [23.0 ± 3.1% (–32C), 55.4 ± 6.3% (–59G), 100% (wild-type construct)] were found for the mutated promoters. Southwestern blotting and UV crosslinking analysis showed binding of three proteins (20, 20 and 50 kDa) to the putative initiator response element. The –32A/C mutant oligonucleotide bound two proteins.

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