Characterization of a novel plasmid DNA found in mitochondria of N. crassa

Collins, Richard A.; Stohl, Lori L.; Cole, Michael; Lambowitz, Alan M.

doi:10.1016/0092-8674(81)90335-4

Cited by 126 publications

(51 citation statements)

References 42 publications

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“…As shown in Fig. 1, Mauriceville and Varkud plasmid monomers are circular double-stranded DNAs of 3.6 and 3.7 kb, respectively, and contain a single long open reading frame (ORF) of 710 amino acids (2,9,31). This ORF encodes an 81-kDa reverse transcriptase (RT) protein that is highly conserved between the two plasmids and presumably functions in their replication (24).…”

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

et al. 1994

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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.The Mauriceville plasmid and closely related Varkud plasmid, found in the mitochondria of some Neurospora strains, are novel retroelements that may be related to retroviral ancestors (23,38). The plasmids exist predominantly as small circular DNAs and concatemers but appear to replicate via an RNA intermediate and reverse transcription. As shown in Fig. 1, Mauriceville and Varkud plasmid monomers are circular double-stranded DNAs of 3.6 and 3.7 kb, respectively, and contain a single long open reading frame (ORF) of 710 amino acids (2, 9, 31). This ORF encodes an 81-kDa reverse transcriptase (RT) protein that is highly conserved between the two plasmids and presumably functions in their replication (24). The plasmid RT contains conserved amino acid sequence blocks 1 to 7, found in most RTs, as well as an additional sequence block, 2a, characteristic of the non-long terminal repeat (non-LTR) retroelements (14, 39). Among the non-LTR retroelements, the plasmid RT is most closely related to those encoded by bacterial retrons and group II introns (14, 39). As expected for a primitive retroelement, the plasmid lacks motifs characteristic of gag, RNase H, protease, integrase, or Zn2+ fingerlike domains, which are associated with the RTs of other retroelements (24,26,27,37).Biochemical experiments have provided insight into the replication mechan...

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

et al. 1994

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“…Nuclei were isolated by the procedure of Hautala et al (13), as modified by Collins et al (8). To isolate DNA, the nuclei were dissolved in a solution containing 1% sodium dodecyl sulfate, 100 mM NaCl, 2 mM EDTA, and 100 mM Trishydrochloride (pH 8.2), extracted twice with phenol-chloroform-isoamyl alcohol (50:48:2), and precipitated with etha- (22).…”

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“…To isolate DNA, the nuclei were dissolved in a solution containing 1% sodium dodecyl sulfate, 100 mM NaCl, 2 mM EDTA, and 100 mM Trishydrochloride (pH 8.2), extracted twice with phenol-chloroform-isoamyl alcohol (50:48:2), and precipitated with etha- (22). Southern hybridizations were as described previously (8,34), except that some hybridizations were carried out in 45% formamide.…”

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Transformation of Neurospora crassa with recombinant plasmids containing the cloned glutamate dehydrogenase (am) gene: evidence for autonomous replication of the transforming plasmid.

Grant¹,

Lambowitz²,

Rambosek³

et al. 1984

Mol. Cell. Biol.

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We have characterized Neurospora crassa transformants obtained with plasmid pJR2, which consists of the Neurospora glutamate dehydrogenase (am) gene cloned in pUC8 and an am'32 host strain which contains a deletion encompassing the cloned fragment. Every one of 33 transformants tested showed extreme meiotic instability: less than 1 or 2% am+ progeny were obtained in initial or successive backcrosses between am' transformants and am'32 or in intercrosses between am+ progeny. Furthermore, am+ progeny from backcrosses gave a high proportion of auxotrophic (am) mitotic segregants during vegetative growth. These results indicate that the am+ character is not stably integrated into chromosomal DNA in any of the transformants tested. Nuclear DNAs from six transformants were analyzed by Southern hybridization. All six transformants contained sequences homologous to pJR2. Four showed restriction fragments expected for unmodified pJR2, but most showed additional bands. Southern blots of undigested DNAs showed that the plasmid sequences are present predominantly in high-molecular-weight form (larger than 20 kilobases). Southern blots showed that auxotrophic (am) progeny from a backcross to am'32 had lost restriction bands corresponding to free plasmid but retained additional bands, apparently integrated into chromosomal DNA in a nonfunctional manner. Considered together, these results are most reasonably interpreted as follows: (i) recombinant plasmids containing the am+ gene can replicate autonomously in N. crassa, (ii) the free plasmids are present in oligomeric or modified form or both, and (iii) plasmid sequences also integrate at multiple sites in the deletion host but in a nonfunctional manner. An alternate interpretation-that tandem repeats of the plasmid are integrated into chromosomal DNA but eliminated during meiosis-cannot be completely excluded. However, stable integration of the am gene can be obtained under a variety of other conditions, viz., using the am gene cloned in a phage lambda vector (J. A. Kinsey and J. A. Rambosek, Mol. Cell. Biol. 4:117-122, 1984), using derivatives of pJR2, or using pJR2 to transform a frameshift mutant.

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“…The Mauriceville and Varkud strains of Neurospora harbor novel genetic elements termed mitochondrial retroplasmids (mRPs) (1,2). These mRPs exist predominantly as small circular DNA monomers of 3.6 and 3.7 kb for the Mauriceville (pMAU) and Varkud (pVAR) plasmids, respectively ( Fig.…”

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Characterization of an unusual tRNA-like sequence found inserted in a Neurospora retroplasmid

Mohr¹,

Wanner²,

Bertrand³

et al. 2000

Nucleic Acids Research

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We characterized an unusual tRNA-like sequence that had been found inserted in suppressive variants of the mitochondrial retroplasmid of Neurospora intermedia strain Varkud. We previously identified two forms of the tRNA-like sequence, one of 64 nt (TRL-64) and the other of 78 nt (TRL-78) containing a 14-nt internal insertion in the anticodon stem at a position expected for a nuclear tRNA intron. Here, we show that TRL-78 is encoded in Varkud mitochondrial (mt)DNA within a 7 kb sequence that is not present in Neurospora crassa wild-type 74A mtDNA. This 7-kb insertion also contains a perfectly duplicated tRNA Trp gene, segments of several mitochondrial plasmids and numerous GC-rich palindromic sequences that are repeated elsewhere in the mtDNA. The mtDNA-encoded copy of TRL-78 is transcribed and apparently undergoes 5′-and 3′-end processing and 3′ nucleotide addition by tRNA nucleotidyl transferase to yield a discrete tRNA-sized molecule. However, the 14 nt intron-like sequence in TRL-78, which is missing in the TRL-64 form, is not spliced detectably in vivo or in vitro. Our results show that TRL-78 is an unusual tRNA-like species that could be incorporated into suppressive retroplasmids by the same reverse transcription mechanism used to incorporate mt tRNAs. The tRNA-like sequence may have been derived from an intron-containing nuclear tRNA gene or it may serve some function, like tmRNA. Our results suggest that mt tRNAs or tRNA-like species may be integrated into mtDNA via reverse transcription, analogous to SINE elements in animal cells.

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Characterization of a novel plasmid DNA found in mitochondria of N. crassa

Cited by 126 publications

References 42 publications

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

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

Transformation of Neurospora crassa with recombinant plasmids containing the cloned glutamate dehydrogenase (am) gene: evidence for autonomous replication of the transforming plasmid.

Characterization of an unusual tRNA-like sequence found inserted in a Neurospora retroplasmid

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