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.

A transformation system utilizing Agrobacterium tumefaciens was developed for targeted gene disruption in Monilinia fructicola, a fungal pathogen that causes brown rot disease of stone fruits. Transformation with a vector containing the neomycin phosphotransferase II (nptII) cassette flanked with 4 kb cutinase gene (Mfcut1) flanking sequences resulted in an average of 13 transformants per 10⁵ spores. When assayed by PCR and DNA blot analyses, more than 50% of the transformants recovered had integrated in the targeted Mfcut1 locus. Both target-gene-specific and non-specific integrations carried direct (head-to-tail) repeat T-DNA integrations. Sequence analysis of these T-DNA integrations revealed that 26 bp of the T-DNA right border were missing at the junctions between direct repeats in all cases. The recombination event during non-specific T-DNA integration in this fungus was unlike that reported in Agrobacterium-mediated transformation in plants.

Summary

Five of 21 red algal genera were found to contain circular dsDNA plasmids, typically of two or more sizes per species. Clones of the two plasmids (GL4.4 and GL3.5 kbp), characterizing all isolates of Gracilariopsis lemaneiformis, do not cross-hybridize with each other, with the nuclear, plastid or mitochondrial genomes of G. lemaneiformis, or with any DNA genomes of the other red algae examined. Clones of both plasmids hybridized with discrete bands on Northern blots of total RNA and poly(A)⁺ RNA. Sequencing of the G. lemaneiformis 3.5 kbp plasmid revealed two potential open reading frames which, when used to probe Northern blots, confirmed the presence of specific transcripts. These autonomously replicating plasmids are present in high copy number per cell and in constant proportion to each other. Their constancy suggests a function of significance to the species. Red algal plasmids may provide useful vectors for transforming economically important red algal species.

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

We have developed a novel system to examine conversion, exchange and mispairing involving a nontandem duplication of the ade8 locus in yeast by monitoring the segregation of heterozygous markers between the duplicated sequence. Plasmid Yrp 17 carries the yeast selectable markers URA3⁺ and TRP1⁺. Yrpl7 derivatives with a 4 kb insert carrying ade8-18 were used to clone the mutations trpl-1 and ura3-1 by gap repair. Integrants of the resulting plasmids at the Ade8 locus were crossed to yield diploid hybrids with a non-tandem duplication of Ade8 and heterozygosity for the plasmid markers between the duplicated sequences. 1192 complete, unselected asci were analyzed and 270 exhibiting recombination of the markers contributed by the plasmid were analyzed by Southern transfers to detect changes in plasmid sequences. Twenty-seven tetrads had unequal homologous exchanges and five had unequal sister-chromatid exchanges. Seven tetrads carry an additional copy of the integrated plasmid and ten are missing one. We propose that these two classes represent conversions of the entire 11 kb plasmid, which occur after misalignment and formation of an unpaired loop. Mispairing is a frequent event, and occurs in approximately fifty percent of all meioses. The system described provides a means to determine the meiotic rules of conversion, exchange and pairing for duplicated DNA sequences.

C to U editing ofapt9, nad3, andcox2 mRNAs was investigated in maize seedlings at various developmental stages as well as in suspension-cultured cells. Heterogeneïty of mRNAs that result from incomplete editing was analyzed for each gene and from five tissues or developmental conditions. The editing status of approximately 30 cDNA clones was determined by digestion with a restriction enzyme that discriminates between unedited and edited DNA sequences. Theatp9 and splicedcox2 cDNAs were essentially completely edited in all samples examined. Analysis of three editing sites ofnad3 cDNAs indicated that incompletely edited cDNAs were detected in all tissues and treatments with a temporal increase in the overall editing status, from 50% at 3 days to about 75% at 7 days. These results indicate that incompletely edited mRNAs are prevalent for some plant mitochondrial genes, and can change with developmental or growth conditions.

Summary

Prior cytological observations using DAPI/epifluorescence microscopy have suggested that the method could be used to rapidly screen plant species for their potential mode of plastid DNA transmission. Cytoplasmic DAPI-DNA aggregates were observed in generative cells of germinated pollen of Medicago sativa (alfalfa), a species known genetically to display biparental transmission, but not in Antirrhinum majus (snapdragon), a species known to be maternal for plastid transmission. If, as suggested, these aggregates are plastid DNA nucleoids, then M. sativa pollen should contain plastid DNA detectable by molecular biology methods and A. majus pollen should not. Total DNA was isolated from germinated pollen and analyzed by Southern blot hybridization. A clone containing part of the rbcL gene from the garden pea plastome was used as a probe for plastid DNA. This probe hybridized with a restriction fragment from M. sativa pollen DNA, but not detectably with A. majus pollen DNA, thereby corroborating the identification of the cytoplasmic DAPI-DNA aggregates in M. sativa pollen as plastid DNA, and confirming the cytologically determined absence of plastic DNA in A. majus pollen.

Identification of the soybean mitochondrial atpA open reading frame (atpA ORF) was based on sequence similarity with atpA genes in other plant mitochondria and partial protein sequencing. The atpA reading frame ends with four tandem UGA codons which overlap four tandem AUG codons initiating an unidentified reading frame, orf214. The atpA-orf214 region is found in multiple sequence contexts in soybean mitochondrial DNA (mtDNA), which can be attributed to the presence of two recombination repeats. A 1-kb repeat spans 600 nucleotides (nt) of atpA N-terminal coding region and 400 nt of upstream sequence. Its four configurations correspond to two full-length atpA-orf214 genes and two truncated pseudogenes. A 2-kb repeat lies 3 kb downstream from the 1-kb repeat. Restriction maps of cosmid clones suggest that a 10-kb segment containing both repeats is itself duplicated in the mt genome. With two recombination repeats present in a total of three copies per genome, soybean mtDNA is expected to consist of a complex population of subgenomic molecules. Transcription of the atpA loci was analysed by Northern blotting and S1 nuclease protection. The atpA genes express multiple transcripts with one major 3′ end and heterogeneous 5′ sequences extending several kb upstream of the atpA coding region. The atpA gene and orf214 are co-transcribed on all major transcripts. The pseudogenes do not express stable RNAs.