Robert A. Akins

A protein required for splicing group I introns in Neurospora mitochondria is mitochondrial tyrosyl-tRNA synthetase or a derivative thereof

The nuclear cyt-18 mutants of Neurospora crassa are defective in splicing a number of group I introns in mitochondria. Here, cloning and sequencing of the cyt-18 gene show that it contains an open reading frame having significant homology to bacterial tyrosyl-tRNA synthetases. Biochemical and genetic experiments lead to the conclusions that the cyt-18 gene encodes mitochondrial tyrosyl-tRNA synthetase, that mutations in this gene inhibit splicing directly, and that mitochondrial tyrosyl-tRNA synthetase or a derivative of this protein is related to the soluble activity that functions in splicing the mitochondrial large rRNA intron and possibly other group I introns. Analysis of partial revertants provides direct evidence that the cyt-18 gene encodes a protein or proteins with two activities, splicing and aminoacylation, that can be partially separated by mutation. Our findings may be relevant to the evolution of introns and splicing mechanisms in eukaryotes.

Mitochondrial Plasmids of Neurospora: Integration into Mitochondrial DNA and Evidence for Reverse Transcription in Mitochondria

The Mauriceville (3.6 kb) and Varkud (3.8 kb) mitochondrial plasmids of Neurospora are closely related, closed circular DNAs whose nucleotide sequences and genetic organization suggest relationships to mitochondrial introns and retrotransposons. Here we isolated mutants whose growth is impaired as a result of malevolent behavior of these plasmids. All 12 mutants contain variant plasmids that are suppressive relative to mtDNA, and ten also contain defective mtDNAs. All the suppressive plasmids contain small insertions, generally including a mitochondrial tRNA sequence, at or near the major 5 RNA start site. The structure of the suppressive plasmids suggests that they were generated via an RNA intermediate and a reverse transcription step. At least three of the mutants contain defective mtDNAs into which mitochondrial plasmid sequences have integrated. Sequences at the plasmid-mtDNA junctions are also consistent with integration via an RNA intermediate.

Function of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing requires an idiosyncratic domain not found in other synthetases.

Neurospora mitochondrial tyrosyl-tRNA synthetase (mt TyrRS), which is encoded by nuclear gene cyt-18, functions in splicing group I introns. Analysis of intragenic partial revertants of the cyt-18-2 mutant and in vitro mutants of the cyt-18 protein expressed in E. coli showed that splicing activity of the cyt-18 protein is dependent on a small N-terminal domain that has no homolog in bacterial or yeast mt TyrRSs. This N-terminal splicing domain apparently acts together with other regions of the protein to promote splicing. Our findings support the hypothesis that idiosyncratic sequences in aminoacyl-tRNA synthetase may function in processes other than aminoacylation. Furthermore, they suggest that splicing activity of the Neurospora mt TyrRs was acquired after the divergence of Neurospora and yeast, and they demonstrate one mechanism whereby splicing factors may evolve from cellular RNA binding proteins.

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

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.

Nucleotide sequence of the Varkud mitochondrial plasmid of Neurospora and synthesis of a hybrid transcript with a 5' leader derived from mitochondrial RNA

The Mauriceville and Varkud mitochondrial plasmids of Neurospora are closely related, closed circular DNAs (3.6 and 3.7 kb, respectively; 1 kb = 10(3) bases or base-pairs), whose characteristics suggest relationships to mitochondrial DNA introns and retrotransposons. Here, we characterized the structure of the Varkud plasmid, determined its complete nucleotide sequence and mapped its major transcripts. The Mauriceville and Varkud plasmids have more than 97% positional identity. Both plasmids contain a 710 amino acid open reading frame that encodes a reverse transcriptase-like protein. The amino acid sequence of this open reading frame is strongly conserved between the two plasmids (701/710 amino acids) as expected for a functionally important protein. Both plasmids have a 0.4 kb region that contains five PstI palindromes and a direct repeat of approximately 160 base-pairs. Comparison of sequences in this region suggests that the Varkud plasmid has diverged less from a common ancestor than has the Mauriceville plasmid. Two major transcripts of the Varkud plasmid were detected by Northern hybridization experiments: a full-length linear RNA of 3.7 kb and an additional prominent transcript of 4.9 kb, 1.2 kb longer than monomer plasmid. Remarkably, we find that the 4.9 kb transcript is a hybrid RNA consisting of the full-length 3.7 kb Varkud plasmid transcript plus a 5 leader of 1.2 kb that is derived from the 5 end of the mitochondrial small rRNA. This and other findings suggest that the Varkud plasmid, like certain RNA viruses, has a mechanism for joining heterologous RNAs to the 5 end of its major transcript, and that, under some circumstances, nucleotide sequences in mitochondria may be recombined at the RNA level.

Involvement of tyrosyl-tRNA synthetase in splicing of group I introns in neurospora crassa mitochondria: Biochemical and immunochemical analyses of splicing activity

We reported previously that mitochondrial tyrosyl-tRNA synthetase, which is encoded by the nuclear gene cyt-18 in Neurospora crassa, functions in splicing several group I introns in N. crassa mitochondria (R. A. Akins and A. M. Lambowitz, Cell 50:331-345, 1987). Two mutants in the cyt-18 gene (cyt-18-1 and cyt-18-2) are defective in both mitochondrial protein synthesis and splicing, and an activity that splices the mitochondrial large rRNA intron copurifies with a component of mitochondrial tyrosyl-tRNA synthetase. Here, we used antibodies against different trpE-cyt-18 fusion proteins to identify the cyt-18 gene product as a basic protein having an apparent molecular mass of 67 kilodaltons (kDa). Both the cyt-18-1 and cyt-18-2 mutants contain relatively high amounts of inactive cyt-18 protein detected immunochemically. Biochemical experiments show that the 67-kDa cyt-18 protein copurifies with splicing and synthetase activity through a number of different column chromatographic procedures. Some fractions having splicing activity contain only one or two prominent polypeptide bands, and the cyt-18 protein is among the few, if not only, major bands in common between the different fractions that have splicing activity. Phosphocellulose columns resolve three different forms or complexes of the cyt-18 protein that have splicing or synthetase activity or both. Gel filtration experiments show that splicing activity has a relatively small molecular mass (peak at 150 kDa with activity trailing to lower molecular masses) and could correspond simply to dimers or monomers, or both, of the cyt-18 protein. Finally, antibodies against different segments of the cyt-18 protein inhibit splicing of the large rRNA intron in vitro. Our results indicate that both splicing and tyrosyl-tRNA synthetase activity are associated with the same 67-kDa protein encoded by the cyt-18 gene. This protein is a key constituent of splicing activity; it functions directly in splicing, and few, if any, additional components are required for splicing the large rRNA intron.

Differentiation promoting factors induced in Acanthamoeba by inhibitors of mitochondrial macromolecule synthesis

Abstract Acanthamoeba castellanii (line OS4) differentiates into a cyst when treated with mitochondrial inhibitors berenil, ethidium bromide, erythromycin, and chloramphenicol. The process is a function of cell density at the time of inhibition: Early log phase cells yield 0–20% cysts, whereas mid-log phase cells yield up to 55, 65, or 95% cysts when treated with chloramphenicol or erythromycin, ethidium bromide, or berenil, respectively. Medium from encysting mid-log phase cells contains an encystment enhancing activity (EEA) which, when transferred to low-density early log phase cultures, causes high levels of encystment. EEA has a nominal molecular weight of between 1000 and 10,000. It is resistant to nucleases, proteases, amylase, boiling, and 1 N NaOH, 25°C, but is inactivated by snake venom phosphodiesterase, autoclaving, and acidic or harsher alkaline treatments. Other lines, OS1 and OS5, which do not encyst well at any cell density in response to the four inhibitors or to EEA, nevertheless produce EEAs to which OS4 can respond. OS4 has been adapted to growth in a chemically defined medium; when starved for glucose and acetate, the cells produce a phosphodiesterase-sensitive EEA. A similar activity can be obtained from mitochondria treated with berenil in vitro. Several lines of evidence suggest that EEAs from the various sources are the same. We propose that they may be of mitochondrial origin and may act as developmental effectors under conditions unfavorable for cell growth.

Analysis of large deletions in the Mauriceville and Varkud mitochondrial plasmids of Neurospora

SummaryThe Mauriceville and Varkud mitochondrial plasmids are closely related, closed-circular DNAs (3.6 and 3.7 kb, respectively) that have characteristics of mtDNA introns and retroid elements. Both plasmids contain a 710 amino acid open reading frame (ORF) that encodes an 81 kDa protein having reverse transcriptase activity. Here, we analyzed two mutant plasmids, V5-36 and M3-24, that have undergone relatively large deletions (approximately 0.35 and 0.5 kb, respectively). Both deletions occur downstream of the long ORF in a noncoding region of the plasmids that contains a direct repeat of 160 bp and a cluster of five PstI-palindromes, a repetitive sequence element in Neurospora mtDNA. In V5-36, the deletion end points are at the bases of two hairpin structures that are centered around PstI-palindromes and flank the deleted region. In M3-24, the deletion junction contains an extra T-residue that is not encoded in the plasmid. In both plasmids, the deletion end points do not correspond to homologous or directly repeated sequences of more than one nucleotide, whose pairing could account for the deletion junction. The characteristics of the deletion end points can be accounted for either by illegitimate recombination, possibly following double strand breaks at cruciform structures, or by interruption of reverse transcription followed by reinitiation downstream. The finding that the deletions encompass the 160 bp direct repeat and all five PstI-palindromes indicates that neither are required for propagation of the plasmids and supports the hypothesis that PstI-palindromes are selfish DNA elements that inserted into a nonessential region of the plasmid.

Factors regulating the encystment enhancing activity (EEA) of Acanthamoeba castellanii

Summary: An extracellular encystment enhancing activity (EEA) greatly stimulates cyst formation by Acanthamoeba castellanii under several different conditions. The activity appears when conditions are suboptimal for growth. EEA does not induce encystment by itself, but enhances differentiation initiated by other factors. EEA titres and their relationship to differentiation are described for encystment induced by: (1) berenil: (2) glucose starvation; and (3) total nutrient starvation. Extracellular EEA was required for maximum encystment by low density cultures in (1) and (2), but not in (3). It was required during the period of visible cyst wall formation rather than for earlier events in encystment. The effectiveness of EEA was reduced under some conditions by spontaneous changes in cellular sensitivity, and by extracellular inhibitors.

Mitochondrial Plasmids of Neurospora and other Filamentous Fungi

There is no generally agreed-to definition of a mitochondrial plasmid, and the different definitions used by various investigators have created some confusion in the literature. We propose that small DNA species present in mitochondria be classified as either defective mitochondrial DNAs (mtDNAs) or mitochondrial plasmids, depending on their origin. We define defective mtDNAs as those small DNA species that derived from the standard mtDNA by deletion and/or rearrangement of mtDNA sequences. Such mtDNAs are defective in the sense that they contain some, but not all, the information required for formation of functional mitochondria.