The quinate pathway is induced by quinate in the wild-type virulent Rhizoctonia solani isolate Rhs 1AP but is constitutive in the hypovirulent, M2 dsRNA-containing isolate Rhs 1A1. Constitutive expression of the quinate pathway results in downregulation of the shikimate pathway, which includes the pentafunctional arom gene in Rhs 1A1. The arom gene has 5,323 bp including five introns as opposed to a single intron found in arom in ascomycetes. A 199-bp upstream sequence has a GC box, no TATAA box, but two GTATTAGA repeats. The largest arom transcript is 5,108 nucleotides long, excluding the poly(A) tail. It contains an open reading frame of 4,857 bases, coding for a putative 1,618-residue pentafunctional AROM protein. A Kozak sequence (GCGCCATGG) is present between +127 and +135. The 5′-end of the arom mRNA includes two nucleotides (UA) that are not found in the genomic sequence, and are probably added post-transcriptionally. Size and sequence heterogeneity were observed at both 5′- and 3′-end of the mRNA. Northern blot and suppression subtractive hybridization analyses showed that presence of a low amount of quinate, inducer of the quinate pathway, resulted in increased levels of arom mRNA, consistent with the compensation effect observed in ascomycetes.

Summary

To provide for thorough sampling of the Neurospora crassa mitochondrial genome for evolutionary studies, recombinant plasmids containing each of the EcoRI digestion fragments of the genome were assembled and used to map the locations of 89 additional restriction endonuclease cleavage sites, representing 10 newly mapped enzymes and 2 previously unmapped HincII sites. Data used to locate new restriction sites were obtained from digestions of whole mitochondrial DNA, digestions of the cloned EcoRI mitochondrial DNA fragments and hybridizations between new restriction fragments and the cloned fragments. Length measurements of the total genome and of EcoRI fragment 1 are larger than commonly reported.

Summary

The RAD52 gene of Saccharomyces cerevisiae has previously been shown to be involved in both recombination and DNA repair. Here we report on the cloning of this gene. A plasmid containing a 5.9 kb yeast DNA fragment inserted into the BamH1 site of the YEp13 vector has been isolated and shown to complement the X-ray sensitive phenotype of the rad52-1 mutation. The rad52-1 cells containing the plasmid form larger colonies than similar cells having lost the plasmid. This plasmid has been shown not to complement either the U.V. sensitivity or the recombination defect of the E. coli recA mutation. From the insert various fragments have been subcloned into the YRp7 and YIp5 vectors. Integration events of two of the subclones have been genetically mapped to the chromosomal location of RAD52, indicating that the structural gene has been cloned. A 1.97 kb BamH1 fragment subcloned into YRp7 in one orientation complements the rad52-1 mutation, while the same fragment in the opposite orientation fails to complement. Various other subclones indicate that a BglII site, within the BamH1 fragment, is in the RAD52 gene. This BglII site has been deleted by Sl-nuclease digestion and the resulting deletion inactivates the RAD52 gene. BAL31 deletions from one end of a 1.9 kb Sal1-BamH1 fragment have been isolated; up to 0.9 kb can be deleted without loss of RAD52 activity, indicating that the RAD52 gene is approximately 1 kb or less in length.

Summary

We have derived a DNA-mediated transformation system for the yeast Yarrowia lipolytica based on the lithium acetate method Ito et al. (1983) developed for Saccharomyces cerevisiae. The first plasmid used, pLD25, contains the Y. lipolytica LEU2 gene (coding for the enzyme beta-isopropylmalate dehydrogenase, EC 1.1.1.85) on a 6.6 kb piece of DNA inserted into pBR322. The recipient strain ATCC 20688 contains the rarely reverting mutation leu2-35. The Y. lipolytica LEU2 gene complements leuB mutants in Escherichia coli and leu2 mutants in S. cerevisiae and it also hybridizes weakly to the S. cerevisiae LEU2 gene. Y. lipolytica transformation frequencies of up to 10⁴ Leu⁺ cells per microgram of DNA, per 10⁸ viable cells have been obtained from plasmds linearized with restriction enzymes. The more than 100-fold increase in transformation frequency obtained by using linearized DNA instead of intact plasmid resembles the situation seen in S. cerevisiae for site-directed integrative transformation (Orr-Weaver et al. 1981). The transformants were stable when grown in non-selective medium. We found that pLD25 integrated at the leu2 region when either linear or intact plasmid was used to transform Y. lipolytica.

Summary

Romanomennis culicivorax, an obligate parasitic nematode of mosquitos, possesses an unusually large mitochondrial genome. Individuals are monomorphic for one of several mitochondrial DNA (mtDNA) size variants ranging from 26–32 kb. In this report, we demonstrate that the mitochondrial genome size differential in three isofemale lineages is due to the presence of mtDNA sequences amplified to different copy numbers within each mtDNA molecule. Restriction enzyme analysis and DNA sequencing studies reveal that each mitochondrial genome contains one of two 3.0 kb repeat types that differ by approximately 30 bp. This difference is primarily due to a short (23 bp) imperfect tandem duplication present within the larger of two polymorphic repeating units. The 3.0 kb reiterated DNA sequences are present as direct, tandem repeats and as inverted portions of the same sequence located elsewhere in the genome. Based on mtDNA analysis of an independently reared R. culicivorax culture, we conclude that events resulting in mitochondrial genome rearrangement occurred in natural field populations prior to propagation within the laboratory.

Summary

The chloroplast psbB, psbF, petB, and petD genes are cotranscribed and give rise to many overlapping RNAs. The mechanism and significance of this mode of expression are of interest, particularly because the accumulation of the psb and pet gene products respond differently to both light and, in C4 species such as maize, developmental signals. We present an analysis of the maize psbB, psbF, petB, and petD genes and intergenic regions. The genes are organized similarly in maize (a C4 species) and in several C3 species. Functional class II-like introns interrupt the 5′ ends of petB and petD. Both spliced and unspliced RNAs accumulate; these encode alternative forms of the petB and petD proteins, differing at their N-termini. Promoter-like elements between psbF and petB, and biased codon usage suggest that the differential regulation of the psb and pet genes might be achieved at both the transcriptional and translational levels.

Summary

A 243 by intron was found within the 23S rRNA gene of the unicellular green alga Chlorella ellipsoidea. This intron is A+T-rich (63.7%) compared with the 23S rRNA (50.5%) and is located in domain II of the 23S rRNA. In contrast to rRNA introns so far known, this intron is considerably small and does not posses features of group I introns in spite of its possible folded secondary structure; this is a new type rRNA intron. The complete nucleotide sequence of the 23S rRNA gene (2,965 bp) was also compared with that of tobacco chloroplasts and E. coli.

Summary

We have cloned and sequenced a fragment of watermelon mitochondrial DNA (mtDNA) which contains a gene homologous to mitochondrial URF-1 (Unidentified Reading Frame-1) of vertebrates, Drosophila yakuba and Aspergillus nidulans. URF-1 is thought to encode a component of the respiratory chain NADH dehydrogenase. Two coding regions in the watermelon gene are separated by approximately 1,450 by of untranslatable DNA. These two exons encode the central portions of URF-1, and are highly conserved. We postulate that three additional exons, selected by their map location and amino acid homology to other URF-1 sequences, encode the remainder of the polypeptide. This is the first description of a plant mitochondrial gene with multiple introns.

Summary

The mitochondrial and nuclear genomes of Coprinus stercorarius and C. cinereus were compared to assess their evolutionary relatedness and to characterize at the molecular level changes that have occurred since they diverged from a common ancestor. The mitochondrial genome of C. stercorarius (91.1 kb) is approximately twice as large as that of C. cinereus (43.3 kb). The pattern of restriction enzyme recognition sites shows both genomes to be circular, but reveals no clear homologies; furthermore, the order of structural genes is different in each species. The C. stercorarius mitochondrial genome contains a region homologous to a probe derived from the yeast mitochondrial var1 gene, whereas its nuclear genome does not. By contrast, the C. cinereus nuclear, but not mitochondrial, genome contains a region homologous to the var1 probe. Only a small fraction of either the nuclear or mitochondrial genomes, perhaps corresponding to the coding sequences, is capable of forming duplexes in interspecies solution reassociations, as measured by binding to hydroxylapatite. Those sequences capable of reassociating were found to have approximately 15% divergence for the mitochondrial genomes and 7%–15% divergence for the nuclear genomes, depending on the conditions of reassociation.

Summary

We have isolated several Tetrahymena thermophila chromosomal DNA fragments which function as autonomously replicating sequences (ARS) in the heterologous Saccharomyces cerevisiae and Schizosaccharomyces pombe selection systems. The Tetrahymena ARS sequences were first isolated in S. cerevisiae and were derived from non-ribosomal micro- and macronuclear DNA. Sequence analysis of the ARS elements identified either perfect or close matches with the 11 by S. cerevisiae ARS core consensus sequence. Subcloning studies of two Tetrahymena ARS elements defined functional regions ranging in size from 50 to 300 bp. Testing of the ARS elements in S. pombe revealed that most of the T. thermophila inserts confer ARS function in both yeasts, at least in the sense of promoting a high transformation frequency to plasmids which contain them. However, the actual sequences responsible for ARS activity were not always identical in the two yeasts.