Characterization of expressed NBS-LRR resistance gene candidates from common bean.

A complex ancestral resistance (R) gene cluster, localized at the end of linkage group B4, and referred to as the B4 R gene cluster, has been previously genetically characterized. The B4 R gene cluster existed prior to the separation of the two major gene pools of cultivated common bean and contains several resistance specificities effective against the fungus Colletotrichum lindemuthianum. In this paper we report the molecular analysis of four expressed resistance gene candidates (RGCs) that map at the B4 R-cluster and co-localize with R-specificities or R-QTLs effective against C. lindemuthianum. These RGCs have been isolated from two genotypes that are representative of the two major gene pools of common bean: the BA8 and BA11 RGCs originating from the Mesoamerican BAT93 genotype, and the JA71 and JA78 RGCs originating from the Andean JaloEEP558 genotype. These RGCs encode NBS-LRR resistance-like proteins that are closely similar to the tomato I2 R-protein. Based upon sequence comparisons and genetic localization, we established that these four bean RGCs belong to two different subfamilies of R-sequences independently of their gene pool of origin. No feature discriminating the four RGCs according to their gene pool of origin has been observed yet. Comparative sequence analyses of the full-length RGCs and their flanking genomic sequences confirmed the ancestral origin of the B4 R-cluster.

Cloning and molecular characterization of three members of the NBS-LRR subfamily located in the vicinity of the Co-2 locus for anthracnose resistance in Phaseolus vulgaris.

Co-2 is one of the R-genes against anthracnose identified in common bean. A RAPD marker, cloned as PvH20, was previously shown to contain 6 imperfect leucine-rich-repeats and to reveal a family of related sequences in the vicinity of the Co-2 locus. Using PvH20 as probe, a genomic clone and 2 partial cDNAs were isolated. DNA sequencing revealed that the 6.1 kb genomic fragment contains sequences encoding both NBS-LRR (ORF1) and kinase-like (ORF2) products. The 2 partial cDNAs (cD7 and cD8) belong to the NBS-LRR subfamily as do most of the resistance genes cloned to date. The LRR domain of ORF1 is interrupted by 2 stop codons suggesting that it corresponds to a non-functional member of the multigene family and ORF2 appears to be a kinase pseudogene. The 3 NBS-LRR polypeptides share a high level of amino acid identity and represent different members of a related family. By genetic mapping ORF1, cD7, and cD8 were found to span a genetic distance of 3 cM: cD8 maps at 2 cM from Co-2 and 3 cM from ORF1, cD7 maps at 1 cM from ORF1 and co-segregates with Co-2, thus cD7 might be a putative candidate for the Co-2 R-gene.

A YAC contig map of Arabidopsis thaliana chromosome 3.

We have constructed a YAC contig map of Arabidopsis thaliana chromosome 3. From an estimated total size of 25 Mb, about 21 Mb were covered by 148 clones arranged into nine YAC contigs, which represented most of the low-copy regions of the chromosome. YAC clones were anchored with 259 molecular markers, including 111 for which linkage information was previously available. Most of the genetic map was included in the YAC coverage, and more than 60% of the genetic markers from the reference recombinant inbred line map were anchored, giving a high level of integration between the genetic and physical maps. The submetacentric structure of the chromosome was confirmed by physical data; 3R (the top arm of the linkage map) was about 12 Mb, and 3L (the bottom arm of the linkage map) was about 9 Mb. This YAC physical map will aid in chromosome walking experiments and provide a framework for large-scale DNA sequencing of chromosome 3.

Homing of a group II intron in yeast mitochondrial DNA is accompanied by unidirectional co-conversion of upstream-located markers.

Group II introns ai1 and ai2 of the Saccharomyces cerevisiae mitochondrial COXI gene encode proteins having a dual function (maturase and reverse transcriptase) and are mobile genetic elements. By construction of adequate donor genomes, we demonstrate that each of them is self-sufficient and practises homing in the absence of homing-type endonucleases encoded by either group I introns or the ENS2 gene. Each of the S. cerevisiae group II self-mobile introns was tested for its ability to invade mitochondrial DNA (mtDNA) from two related Saccharomyces species. Surprisingly, only ai2 was observed to integrate into both genomes. The non-mobility of ai1 was clearly correlated with some polymorphic changes occurring in sequences flanking its insertion sites in the recipient mtDNAs. Importantly, studies of the behaviour of these introns in interspecific crosses demonstrate that flanking marker co-conversion accompanying group II intron homing is unidirectional and efficient only in the 3' to 5' direction towards the upstream exon. Thus, the polar co-conversion and dependence of the splicing proficiency of the intron reported previously by us are hallmarks of group II intron homing, which significantly distinguish it from the strictly DNA-based group I intron homing and strictly RNA-based group II intron transposition.

Two homologous introns from related Saccharomyces species differ in their mobility.

We have studied gene conversion initiated by the ai3 intron of the Saccharomyces cerevisiae mitochondrial (mt) COXI gene and its homologous intron (S.cap.ai1) from Saccharomyces capensis. The approach used involved the measurement of intron transmission amongst the progeny of crosses between constructed recipient and donor strains. We found that the S. cerevisiae ai3 intron is extremely active as a donor in gene conversion, whereas its homologous S. capensis intron is not. We have established the sequence of S.cap.ai1 and compared its open reading frame (ORF) with that of I-SceIII encoded by the homologous S. cerevisiae intron. The two protein-coding intron sequences are almost identical, except that the S. capensis ORF contains an in-frame stop codon. This finding provides a strong indication that the 3' part of the S. cerevisiae intron ORF encoding I-SceIII (which should not be translated in the S. capensis intron) must be critical for function of mtDNA endonucleases to mediate intron mobility.

The C-terminal domain of yeast cytochrome b is essential for a correct assembly of the mitochondrial cytochrome bc1 complex.

Yeast mutants modifying the C-terminal region of mitochondrial cytochrome b were isolated and characterized. A nonsense mutation of the leucine codon 335 (TTA-->TAA), 50 residues before the normal C-terminus, blocks incorporation of heme into the apocytochrome b and prevents growth on non-fermentable substrates. The same defects were observed in a frameshift mutant (after codon 348, TAT-->TATT) in which the last 37 C-terminal residues are predicted to be replaced by a novel sequence of 33 amino acids. Function was regained in the nonsense mutant only by true back mutations restoring a protein of the wild-type sequence. The respiratory capacity was restored to wild-type levels in the frameshift mutant by a variety of single base subtractions located within a window of 24 bases before or after the original +T addition, these pseudo-reversions resulted in single or multiple (up to five) consecutive amino acid replacements between positions 346 and 354 and restored the wild-type sequence from position 355 to 385. These data, combined with hydropathy calculations and sequence comparisons, suggest that the C-terminal domain of cytochrome b forms a transmembrane segment essential for the correct assembly of the cytochrome bc1 complex.

Sequence of the mitochondrial gene encoding subunit I of cytochrome oxidase in Saccharomyces douglasii.

We have determined the complete sequence of the mitochondrial (mt) gene (COXI) coding for cytochrome oxidase subunit I of Saccharomyces douglasii. This gene is 7238 bp long and includes four introns. The salient feature of the S. douglasii COXI gene is the presence of two introns, Sd.ai1 and Sd.ai2, which have not been observed in S. cerevisiae genes. Both are group-I introns and are located at novel positions compared with the S. cerevisiae COXI. Interestingly, one of these introns (the second one) is inserted at the same position as intron 2 of COXI of Kluyveromyces lactis and also as intron 8 of the same gene in Podospora anserina. The ORFs contained in these three introns display a high degree of similarity. Comparisons of exonic and intronic sequences of the COXI of two Saccharomyces species reinforces our previous conclusions: the evolution of mt genes in yeast obeys different rules to those found in vertebrates.

The MRS1 gene of S. douglasii: co-evolution of mitochondrial introns and specific splicing proteins encoded by nuclear genes.

We have developed a rapid and simple methodology to locate yeast genes within cloned inserts, obtain partial sequence information, and construct chromosomal disruptions of these genes. This methodology has been used to study a nuclear gene from the yeast S. douglasii (a close relative of S. cerevisiae), which is essential for the excision of the mitochondrial intron aI1 of S. douglasii (the first intron in the gene encoding subunit I of cytochrome oxidase), an intron which is not present in the mitochondrial genome of S. cerevisiae. We have shown that this gene is the homologue of the S. cerevisiae MRS1 gene, which is essential for the excision of the mitochondrial introns bI3 and aI5 beta of S. cerevisiae, but is unable to assure the excision of the intron aI1 from the coxI gene of S. douglasii. The two genes are very similar, with only 13% nucleotide substitutions in the coding region, transitions being 2.5 times more frequent than transvertions. At the protein level there are 86% identical residues and 7% conservative substitutions. The divergence of the MRS1 genes of S. cerevisiae and S. douglasii, and the concomitant changes in the structure of their mitochondrial genomes is an interesting example of the co-evolution of nuclear and mitochondrial genomes.

Two homologous mitochondrial introns from closely related Saccharomyces species differ by only a few amino acid replacements in their Open Reading Frames: one is mobile, the other is not.

We have undertaken a comprehensive study of the gene conversion of all the mitochondrial introns of Saccharomyces capensis. The approach used involved the measurements of intron transmission amongst the progeny of crosses between a recipient strain (Saccharomyces cerevisiae intronless mitochondria) and various donor strains (Saccharomyces capensis, with various combinations of mitochondrial introns). We have shown that the S. capensis second intron (bi2 of cytochrome b gene) is extremely active as a donor in gene conversion whereas its homologous S. cerevisiae intron is not. Determination of sequence of the S. capensis intron demonstrates that it differs from that of the homologous S. cerevisiae intron (bi2) by a very small number of nucleotide substitutions.

Incipient mitochondrial evolution in yeasts. I. The physical map and gene order of Saccharomyces douglasii mitochondrial DNA discloses a translocation of a segment of 15,000 base-pairs and the presence of new introns in comparison with Saccharomyces cerevisiae.

We have determined the physical and genetic map of the 73,000 base-pair mitochondrial genome of a novel yeast species Saccharomyces douglasii. Most of the protein and RNA-coding genes known to be present in the mitochondrial DNA of Saccharomyces cerevisiae have been identified and located on the S. douglasii mitochondrial genome. The nuclear genomes of the two species are thought to have diverged some 50 to 80 million years ago and their nucleo-mitochondrial hybrids are viable but respiratorily deficient. The mitochondrial genome of S. douglasii displays many interesting features in comparison with that of S. cerevisiae. The three mosaic genes present in both genomes are quite different with regard to their structure. The S. douglasii COXI gene has two new introns and is missing the five introns of the S. cerevisiae gene. The S. douglasii cytochrome b gene has one new intron and lacks two introns of the S. cerevisiae gene. Finally, the L-rRNA gene of S. douglasii, like that of S. cerevisiae, has one intron of which the structure is different. Another salient feature of the S. douglasii mitochondrial genome reported here is that the gene order is different in comparison with S. cerevisiae mitochondrial DNA. In particular, a segment of approximately 15,000 base-pairs including the genes coding for COXIII and S-rRNA has been translocated to a position between the genes coding for varl and L-rRNA.

Incipient mitochondrial evolution in yeasts. II. The complete sequence of the gene coding for cytochrome b in Saccharomyces douglasii reveals the presence of both new and conserved introns and discloses major differences in the fixation of mutations in evolution.

We have determined the complete sequence of the mitochondrial gene coding for cytochrome b in Saccharomyces douglasii. The gene is 6310 base-pairs long and is interrupted by four introns. The first one (1311 base-pairs) belongs to the group ID of secondary structure, contains a fragment open reading frame with a characteristic GIY ... YIG motif, is absent from Saccharomyces cerevisiae and is inserted in the same site in which introns 1 and 2 are inserted in Neurospora crassa and Podospora anserina, respectively. The next three S. douglasii introns are homologous to the first three introns of S. cerevisiae, are inserted at the same positions and display various degrees of similarity ranging from an almost complete identity (intron 2 and 4) to a moderate one (intron 3). We have compared secondary structures of intron RNAs, and nucleotide and amino acid sequences of cytochrome b exons and intron open reading frames in the two Saccharomyces species. The rules that govern fixation of mutations in exon and intron open reading frames are different: the relative proportion of mutations occurring in synonymous codons is low in some introns and high in exons. The overall frequency of mutations in cytochrome b exons is much smaller than in nuclear genes of yeasts, contrary to what has been found in vertebrates, where mitochondrial mutations are more frequent. The divergence of the cytochrome b gene is modular: various parts of the gene have changed with a different mode and tempo of evolution.