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We previously reported that there were three copies of ATP1 coding for F1-α and two copies of ATP3 coding for F1-γ on the left and right arm of chromosome II, respectively. In this study, we present evidence that there are three closely linked copies of ATP2 encoding the β subunit of the F1F0-ATPase complex on the right arm of chromosome X in several laboratory strains, including Saccharomyces cerevisiae strain S288C, although it was reported by the yeast genome project that ATP2 is a single-copy gene. Chromosome X fragmentation, long-PCR, chromosome-walking and ATP2-disruption analysis using haploid wild-type strains and prime clone 70645 showed that the three copies of ATP2 are present on the right arm of chromosome X, like those of ATP1 on chromosome II. Each was estimated to be approximately 4 kb apart. We designated the ATP2 proximal to the centromere as ATP2a, the middle one as ATP2b and the distal one as ATP2c. The region containing the three ATP2s is composed of two repeated units of approximately 7 kb; that is, both ends ( ATP2a, ATP2c) accompanying the ATP2-neighboring ORFs are the same. A part of YJR119c, YJR120w, YJR122w (CAF17) and YJR123w (RP55), which were reported by the yeast genome project, are contained in the ATP2 repeated units; and the middle ATP2 of the three ATP2s, ATP2b, is located between the two repeated units. Expression of all three copies of ATP2 ( ATP2a, ATP2b, ATP2c) was confirmed because a single or double ATP2-disruptant could grow on glycerol, but a triple ATP2-disruptant could not. In addition, of the three copies of ATP1 and ATP2, even if only one copy of the ATP1 and ATP2 genes remained, the cells grew on glycerol. [ABSTRACT FROM AUTHOR]

The Alternaria alternata apple pathotype causes Alternaria blotch of susceptible apple cultivars through the production of a cyclic peptide, host-specific toxin, AM-toxin. We recently cloned a cyclic peptide synthetase gene, AMT, whose product catalyzes the production of AM-toxin and showed that it resides on chromosomes of 1.8 Mb or less, depending on the A. alternata apple pathotype strain. Reverse transcriptase (RT)-PCR, using primers specific to AMT, on laboratory sub-cultured strains previously shown to produce AM-toxin, identified one isolate that did not express the gene. A leaf necrosis bioassay confirmed an AM-toxin-minus phenotype. However, an original isolate of this strain which had not undergone sub-culture gave a positive result by both RT-PCR and bioassay. Contour-clamped homogeneous electric field electrophoresis and Southern hybridization demonstrated the loss of a 1.1-Mb chromosome in the non-toxin-producing isolate. Since this chromosome can be entirely lost without affecting growth, but is necessary for pathogenicity, we propose it is a conditionally dispensable chromosome. [ABSTRACT FROM AUTHOR]

The inheritance of telomere-associated restriction fragment length polymorphisms (tel-RFLP) and chromosome-length polymorphisms (CLPs) were criteria used for the identification of strains of Ustilago hordei that form a direct lineage. Teliospore collections of race 8 strains and strains reported to be derived from race 8 through inbreeding were used in these analyses. None of the race 8 strains examined in this study, representing three consecutive inbred generations, was polymorphic for any terminal BamHI and BglII chromosomal loci, nor did they have any apparent CLPs. Strains from a teliospore collection obtained in 1971 and designated 447, representing the first inbred generation of race 8 strains and a shift to increased virulence on cultivar Hannchen, had tel-RFLP arrays indistinguishable from the race 8 strains isolated in this study; and they had no obvious CLPs. Strains from the presumed second inbred generation, the 1279 teliospore line, which was pathogenic on six additional cultivars, had numerous CLPs and a tel-RFLP array that differed from race 8 strains at more than 50% of the terminal chromosomal BamHI and BglII restriction sites. The tel-RFLP array of each 1279 strain was identical and indistinguishable from the arrays of strains representing races 10 and 13, indicating that they share a direct lineage. A race 14 strain, also presumed to be derived from race 8 strains by inbreeding, had a unique tel-RFLP array and an electrophoretic karyotype that distinguished it from all other strains. The tel-RFLP arrays alone eliminate the 1279 and race 14 strains from being direct descendants from race 8 strains by inbreeding and suggest that this approach can identify a strain lineage among other inbred sexually reproducing fungi, or isolates that comprise different asexual clonal populations. [ABSTRACT FROM AUTHOR]

Electrophoretic karyotypes of ten strains of Mucor circinelloides f . lusitanicus were generated by contour-clamped homogeneous electric field (CHEF) gel-electrophoresis. Most of the strains analyzed showed polymorphisms, but a different main karyotype pattern could be correlated with each mating type. Genome structure was further analyzed by gene assignment to the chromosome-sized DNAs. Nonradioactive hybridization techniques identified the chromosomal localization of seven cloned genes. The hybridization patterns confirmed the similarity between the mating-type (−) strains and showed some heterogeneity among the mating-type (+) strains. Linkage was found between genes pyrF and chs3 in all the strains, between the gene leuA and the rDNA in all mating-type (−) strains and ATCC 1216b (+), and between chs2 and the rDNA in CBS 969.68 (+). This is the first time that gene linkage in M. circinelloides has been reported, but in some cases the linkage relationships obtained are strain-dependent. [ABSTRACT FROM AUTHOR]

There are at least ten plant diseases caused by Alternaria species in which host-specific toxins (HSTs) are responsible for fungal pathogenicity. Of these HST-producers, seven are considered distinct pathotypes of the species Alternaria alternata, and the remaining three are among other species of pathogenic Alternaria. Inter- and intra-specific variation among Alternaria taxa, including HST-producers, was determined by electrophoretic karyotyping using pulsed-field gel electrophoresis. A. alternata including seven pathotypes of A. alternata and eight non-pathogenic strains had 9–11 chromosomal bands with estimated sizes ranging from 0.4 to 5.7 Mb. In contrast, Alternaria species that are morphologically distinct from A. alternata had 8–10 bands with sizes between 0.9 and 5.7 Mb. Estimated genome sizes of A. alternata and other Alternaria species ranged from 28.8 to 33.6 Mb and 25.1 to 30.7 Mb, respectively. Other species of pathogenic Alternaria were difficult to differentiate from A. alternata on the basis of chromosome-size polymorphisms alone, but Southern analysis using rDNA as a probe could, in some cases, differentiate between them. These results were cytologically confirmed by 4′,6-diamidino-2-phenylindole (DAPI) staining and fluorescence in situ hybridization with a rDNA probe for mitotic metaphase chromosomes prepared by the germ-tube burst method. [ABSTRACT FROM AUTHOR]

Using a monoclonal antibody based ELISA, 600 pAN7-1 plasmid-tagged mutants of Penicillium paxilli were screened for paxilline accumulation and one paxilline-negative mutant, YI-20, was identified. A molecular analysis of this mutant showed that pAN7-1 was inserted at a single site but was present as 4–6 copies arranged in a head-to-tail tandem array. Rescue of flanking sequences and analysis of the corresponding genomic region revealed that YI-20 has an extensive deletion at the site of pAN7-1 integration. Probing of a CHEF gel with the same sequences showed that associated with the deletion is a rearrangement of chromosome Va. Targeted gene disruption of wild-type sequences adjacent to the site where pAN7-1 inserted, resulted in the generation of two additional paxilline-negative mutants; both were single crossovers with deletions extending outside the region mapped. Neither of these new mutants had a rearrangement of chromosome Va, suggesting that deletion of genes on this chromosome is responsible for the paxilline-negative phenotype. Telomeric fingerprinting of genomic digests of P. paxilli, combined with pulsed-field gel electrophoresis of chromosomal DNA, established that there are a minimum of eight chromosomes in this fungus. [ABSTRACT FROM AUTHOR]

We have investigated chromosomal segregation during meiosis in a cross between two polymorphic haploid laboratory strains of Saccharomyces cerevisiae, FL100 and GRF18. These two strains have large chromosome-length polymorphisms for chromosomes I and III allowing for easy scoring of parental chromosomes after meiotic segregation. Chromosome III in the FL100 strain was 35 kb shorter than chromosome III in GRF18, while FL100 chromosome I was 40 kb larger than chromosome I in GRF18. Segregation analysis of chromosomes I and III in 50 tetrads showed an apparent association between chromosomes I and III, whereas only the original parental association of chromosomes I and III was found in the spores. By hybridization with chromosome-specific probes we have shown that the polymorphisms are due to a large translocation from chromosome III onto chromosome I in FL100. The translocated fragment is larger than 80 kb and was mapped between Ty and HML. In nine tetrads analyzed, chromosome-length polymorphisms which did not segregate according to Mendelian law were observed. [ABSTRACT FROM AUTHOR]

Pulsed-field gel electrophoresis and a chromosome-specific cosmid DNA library were used to determine the karyotypes of wild-type Aspergillus nidulans isolates from around the world. Overall, little structural variation was found, with a few major exceptions. One isolate possessed a non-essential B-chromosome of about 1.0 million base pairs (mb). Another isolate had undergone a non-reciprocal translocation of about 1.6 mb of chromosome VI onto chromosome VIII. Other than these chromosomal differences, these isolates appeared phenotypically normal. To analyze its effects on meiosis, the translocation isolate was outcrossed with another wild-type derivative that had a 'normal' electrophoretic karyotype. This cross produced a range of phenotypes, including duplicated progeny that had a 'barren' phenotype similar to that described for Neurospora partial disomics. The duplication was somewhat vegetatively unstable. This is the first association of sterility with chromosomal duplication in A. nidulans. [ABSTRACT FROM AUTHOR]

Orthogonal field agarose-gel electrophoresis (OFAGE) of chromosomes from translocation-bearing and normal Neurospora crassa strains was utilized, first, to recover cosmids from a translocated region, and second, to map translocation breakpoints. Surprisingly, the right breakpoints in two independently derived, interstitial translocations, T(II → III) AR18 and T(II → VI)P2869, are within about 5.6 kbp of each other suggesting that this region of linkage group (LG) II may be fragile or otherwise subject to chromosome breakage. Mapping translocation breakpoints through OFAGE, or other similar methods, should allow for DNA sequencing across breakpoints that are not associated with mutant phenotypes or that are not within walking distance of cloned markers. [ABSTRACT FROM AUTHOR]

Pulsed-field gel electrophoresis (PFGE) of isolates of Pythium oligandrum with linear mitochondrial genomes revealed a distinct band in ethidium bromide-stained gels similar in size to values estimated by restriction mapping of mitochondrial DNA (mtDNA). Southern analysis confirmed that these bands were mtDNA and indicated that linear genomes were present in unit-length size as well as multimers. Isolates of this species with circular mtDNA restriction maps also had low levels of linear mono- and multimers. visualized by Southern analysis of PFGE gels. Examination of 17 additional species revealed similar results; three species had distinct linear mtDNA bands in ethidium bromide-stained gels while the remainder had linear mono- and multi-mers in lower amounts detected only by Southern analysis. Sequence analysis of an isolate of P. oligandrum with a primarily circular mitochondrial genomic map and a low amount of linear molecules revealed that the small unique region of the circular map (which corresponded to the terminal region of linear genomes) was flanked by palindromic intrastrand complementary sequences separated by a unique 194-bp sequence. Sequences with similarity to ATPase9 coding regions from other organisms were located adjacent to this region. Sequences with similarity to mitochondrial origins of replication and autonomously replicating sequences were also located in this region: their potential involvement in the generation of linear molecules is discussed. [ABSTRACT FROM AUTHOR]

We have investigated the mitotic and meiotic transmission of chromosome-length polymorphisms in Ophiostoma ulmi s.l., the causal agent of Dutch elm disease. The North-American aggressive (NAN) strain CESS16K has an atypical electrophoretic karyotype, carrying two chromosome-sized DNAs (chDNAs) that have not been observed in other members of the NAN biotype. Independent CESS16K chDNA preparations, even after repeated inoculation and recovery from the elm host, and analysis of 16 progeny strains after a cross between the NAN strains FG245B-O and CESS16K, demonstrated that these unique chDNAs are integral components of the CESS16K genome. Analysis of the progeny, by electrophoretic karyotyping and hybridizations with probes specific to individual chDNAs, presented evidence that genome rearrangements can occur as a consequence of meiosis. Even though novel electrophoretic karyotypes and a novel-sized chromosome were observed in the karyotypes of the progeny strains, the low level of reassortment between the chromosomes carrying length polymorphisms presented evidence that there are constraints to genome plasticity for this fungus. [ABSTRACT FROM AUTHOR]

Chromosomal DNA banding patterns were obtained for three strains of Torulaspora pretoriensis by contour-clamped homogenous-electric-field gel electrophoresis. Chromosomes were resolved into six or seven bands in the range of 800 to 2000 kb, and a polymorphism of these lengths was observed. By Southern-blot analysis, the three strains were shown to lack the DNA sequences homologous to the URA3, LEU2, TRP1, and HO genes of Saccharomyces cerevisiae. A uracil auxotrophic mutant derived from T. pretoriensis was transformed with three plasmids (YEp24, YRpHI, and YCp50) carrying the URA3 gene of S. cerevisiae by the lithium acetate method. [ABSTRACT FROM AUTHOR]

Cryptomonad algae and Chlorarachiniophyta are evolutionary chimaeras derived from the engulfment of an eukaryotic phototrophic endosymbiont by a eukaryotic host cell. Although much reduced, the endosymbiont's eukaryotic plasmatic compartment still contains a nucleus, the so-called nucleomorph. These nucleomorphs carry the smallest known eukaryotic genomes. We have characterized the genomes of several cryptomonads and a Chlorarachnion species by means of PFGE (pulsed-field gel electrophoresis). Hybridization studies with small subunit rDNA were used to identify the nucleomorph chromosomes. We also performed hybridization experiments with an hsp70 probe to estimate the distribution of this gene among the different algal species. The evolutionary, genetical, and physiological implications of our studies are discussed. A model on the possible function of the nucleomorph hsp70 gene products is presented. [ABSTRACT FROM AUTHOR]

Southern-blot hybridization analysis was used to identify and quantify chromosome-length polymorphisms for ten linkage groups of 14 races of Ustilago hordei. The bands identified by the probes were shown to vary as much as hundreds of kilobase pairs, but the magnitude of the variability was typically 5-15% of the average size of all bands to which a particular probe hybridized. A filamentous morphology mutant, recovered following heatshock treatment of a strain with the greatest number of chromosome bands, was shown to have suffered a 50-kb deletion in a 940-kb chromosome. The mutation to filamentous morphology, designated fil1-1, and the deletion, were shown to invariably cosegregate 2:2 with the wildtype (sporidial) morphology in an ordered tetrad. Genetic and physical analyses place the Fill locus and the deletion near the terminus of one arm of the 940-kb chromosome. These results suggest that deletions of this type may be one of the causes of chromosome-length polymorphisms observed in field isolates of U. hordei. [ABSTRACT FROM AUTHOR]

Contour-clamped homogeneous electric field (CHEF) gel electrophoresis was used to separate chromosomal size DNA molecules of two Mucor circinelloides strains. Electrophoretic karyotypes revealed the presence of eight distinct bands for the M. circinelloides f. lusitanicus strain, and four, presumably multiple, bands for the M. circinelloides f. gryseo-cyanus strain. The approximate sizes of the resolved chromosomal DNA bands ranged from 2.3 to 8.1 Mb, giving estimated genome sizes of 38.7 and 32.6 Mb, respectively. Hybridisation techniques were used to assign the leuA gene to a chromosome. [ABSTRACT FROM AUTHOR]

Chromosome-substituted haploid segregants were selected from among the benomyl-induced progeny of an interspecific hybrid produced by polyethylene-glycol-induced fusion of protoplasts of an Aspergillus nidulans 'master strain' and an A. quadrilineatus auxotrophic mutant. These segregants were examined by RFLP, RAPD, and isoenzyme analysis. The A. nidulans ribosomal repeat unit was assigned to chromosome V, while the benA and the pyrG genes were assigned to linkage groups VIII and I, respectively, of A. nidulans. None of the other cloned genes tested ( gdhA, amdS and 25 s rRNA) showed polymorphism between the two parents. The method was also used to assign RAPD markers and isoenzyme bands of β-arylesterase, phosphatases, NAD-dependent malate dehydrogenase, and cellulase, to A. nidulans chromosomes and/or to their A. quadrilineatus equivalents. The isoenzyme and DNA sequences assigned to chromosomes could be used to saturate the genetic map of A. nidulans, or could serve as starting points for the construction of a genetic map of A. quadrilineatus. No method affording the same possibilities has been described so far in Aspergilli. This chromosome-assay method may be a useful alternative to pulsed-field-gel electrophoretic procedures for the assignment of molecular markers to chromosomes. [ABSTRACT FROM AUTHOR]

The hydrophobic protein cerato-ulmin (CU), produced by Ophiostoma ulmi, has been implicated in the pathogencity of this fungus on elm. Primers were designed based on the nucleotide sequence deduced from the published CU amino-acid sequence, and a DNA fragment of the cu gene was amplified using the polymerase chain reaction. The amplified cu fragment was used as a hybridization probe to identify and isolate the cu gene from a genomic DNA library of an aggressive isolate of O. ulmi (= O. novo-ulmi). The cu coding region is interrupted by two introns and encodes a 100 amino-acid prepro-CU polypeptide that is processed to a 75 amino-acid mature protein upon secretion. CU shows significant sequence similarity to hydrophobins secreted by certain other fungi. [ABSTRACT FROM AUTHOR]

Nine members, MEL2-MEL10, of the MEL gene family coding for α-galactosidase were physically mapped to the ends of the chromosomes by chromosome fragmentation. Genetic mapping of the genes supported the location of all the MEL genes in the left arm of their resident chromosomes. [ABSTRACT FROM AUTHOR]

Thirteen chromosomal-sized DNA bands of the cultivated mushroom Agaricus bisporus have been resolved using the method of clamped homogeneous electric field (CHEF) electrophoresis. Using chromosome size standards from Schizosaccharomyces pombe, Saccharomyces cerevisiae and Candida albicans, the estimated size of the chromosomal DNAs ranged from 3.5 to 1.2 megabase pairs (Mb). By Southern hybridization with homologous gene probes, the chromosomal location of cellulase and laccase genes have been mapped. In addition, rDNA has been assigned to chromosomal bands using a heterologous gene probe. Genomic rearrangement is suggested in the commercial heterokaryon, as indicated by the presence of non-comigrating homologous chromosomes, identified by a number of probes for particular DNA sequences. [ABSTRACT FROM AUTHOR]

Zygotes isolated by micromanipulation from crosses of Saccharomyces cerevisiae strains, one of which carries a kar mutation, give rise most frequently to cytoductant colonies showing the nuclear constitution of either one of the two haploid parental strains. In crosses of kar2-1 strains to wild-type, about 10% of the cytoductants of both mating types are homozygous autodiploids. There is evidence indicating that self-diploidization occurs by fusion between sibling nuclei in the heterokaryotic zygote. Here we describe this phenomenon and propose to take advantage of it for the construction of genotypically-defined diploids able to mate, and of polyploid strains, which are useful tools in genetic and cytological studies. [ABSTRACT FROM AUTHOR]

Chromosomal DNA of Australian field-isolates of the phytopathogenic ascomycete Leptosphaeria maculans was resolved by pulsed-field gel electrophoresis. All isolates examined had highly variable karyotypes. Ascospores (sexual spores) derived from single pseudothecia (sexual fruiting bodies) isolated from Brassica napus (oilseed rape) stubble were analyzed. In two tetrads four distinct karyotypes were observed, with only one chromosomal DNA band in common to all the members of each tetrad. Although isolates had highly variable karyotypes, two overall patterns were present. In one pattern there were at least 12 chromosomal DNA bands, the largest being greater than 2.2 Mb in size; in the other there were more than 15 chromosomal DNA bands, the largest being about 2.0 Mb. The chromosomal DNA preparations included mitochondrial DNA which migrated as a diffuse band between 0.10 and 0.15 Mb in size, and DNA molecules of 8 and 9 kb in size. [ABSTRACT FROM AUTHOR]

Primary gene amplification, i.e., mutation from one gene copy to multiple gene copies per genome, is important in genomic evolution, as a means of producing anti-cancer drug resistance, and is associated with the progression of tumor malignancy. Primary amplification has not been studied in normal eukaryotic cells because amplifications are extremely rare in these cells. A system has been developed to phenotypically identify co-amplifications of the ADH4 and CUP1 genes of Saccharomyces cerevisiae and 21 independent spontaneous amplifications have been isolated. [ABSTRACT FROM AUTHOR]

Four new meiotic recombination genes were previously isolated by selecting for mutations that rescue the meiotic lethality of rad52 spo13 strains. One of these genes, REC114, is described here, and the data confirm that REC114 is a meiosis-specific recombination gene with no detectable function in mitosis. REC114 is located on chromosome XIII approximately 4,9 cM from CIN4. The nucleotide sequence reveals an open reading frame of 1262 bp, consensus intron splice sites close to the 3′ end, and indicates that the second exon codes for only seven amino acids. In the promoter region, a URS1 consensus sequence (TGGGCGGCTA), identical to the URS1 found in the promoter of SPO16, is present 93 bp upstream of the translation start site. Northern-blot hybridization demonstrates that REC114 is transcribed only during meiosis and that it is not expressed in the absence of the IME1 gene product, even when IME2 is constitutively expressed. [ABSTRACT FROM AUTHOR]

The organization of the mitochondrial genome in the IR36 indica variety of rice was analyzed using constant-field and CHEF gel electrophoresis. The minimum complexity of the genome is estimated to be 300 kb. There was no evidence for a master circular molecule of this size. The genome exists in vivo in multiple subgenomic circular molecules or mitochondrial chromosomes. Using several rare-cutting restriction enzymes a restriction map was constructed for a 117-kb circular chromosome. Six genetic loci have been placed on this chromosome; cox1 and atp1 have been assigned precise positions while four additional genes ( rrn26, cob2, atp6 and rrn18) were mapped to restriction fragments. [ABSTRACT FROM AUTHOR]

Southern blot and nucleotide sequence analysis of Phanerochaete chrysosporium BKM-F-1767 genomic clones indicate that this wood-degrading fungus contains at least six genes with significant homology to the Trichoderma reesei cellobiohydrolase I gene ( cbh1). Using pulsed-field gel electrophoresis to separate P. chrysosporium chromosomes, the six cellulase genes were found to hybridize to at least three different chromosomes, one of which is dimorphic. The organization of these genes was similar in another P. chrysosporium strain, ME 446. It is clear that, unlike T. reesei, the most well-studied cellulolytic fungus, P. chrysosporium contains a complex, cbh1-like gene family. [ABSTRACT FROM AUTHOR]

The map position of three 1,3- β-glucanase-encoding genes in S. cerevisiae has been determined following conventional meiotic and mitotic mapping combined with recombinant DNA techniques. EXG1, EXG2 and SSG1 were localized to chromosomes XII, IV and XV, respectively, by hybridizing the cloned genes to Southern blots of chromosomes sepaated by pulsed-field gel electrophoresis, in conjunction with the rad52-1-dependent chromosome-loss mapping technique. Meiotic tetrad analyses further localized the EXG1 gene 6.1 centimorgans centromere-proximal to CDC25 on the right arm of chromosome XII. EXG2 was positioned between LYS4 and GCN2 on the right arm of chromosome IV, at distances of 6.2 centimorgans from LYS4 and 4.9 centimorgans from GCN2. Finally, the SSG1 locus mapped on the right arm of chromosome XV, about 8.2 centimorgans to the centromere-proximal side of HIS3. [ABSTRACT FROM AUTHOR]

An electrophoretic karyotype of Trichoderma longibrachiatum (reesei) was obtained using contourclamped homogeneous electric field (CHEF) gel electrophoresis. Seven chromosomal DNA bands were separated in the wild-type T. longibrachiatum strain QM6a. The sizes of the chromosomal DNA bands ranged from 2.8 to 6.9 Mb, giving an estimated total genome size of about 33 Mb. The electrophoretic karyotype of the strain QM6a was compared to three hyper-cellulolytic mutant strains, QM9414, RutC30 and VTT-D-79125. The chromosome pattern of the mutant QM9414 was quite similar to that of the wild-type QM6a except that the smallest chromosome differed somewhat in size. The VTT-D-79125 and RutC30 strains, which have undergone several mutagenesis steps, showed striking differences in their karyotype compared to the initial parent. The chromosomal DNA bands were identified using the previously characterized T. longibrachiatum genes ( egl1, egl2, cbh1, cbh2, pgk1, rDNA) and random clones isolated from a genomic library. In all strains the cellulase genes cbh1, cbh2 and egl2 were located in the same linkage group (chromosome II in the wild-type), while the main endoglucanase, egl1, hybridized to another chromosomal DNA band (chromosome VI in the wild-type). [ABSTRACT FROM AUTHOR]

The junctions between X and Y′ subtelomeric repeats in Saccharomyces cerevisiae usually contain a stretch of telomere sequences, (GT). Two of three cloned X-Y′ junctions from strain YP1 have a replacement of about 200 bp of X, the internal telomere sequence, and 49 bp of Y′ by a 292 bp sequence. The first 227 bp of this insertion sequence are 100% identical to the fourth intron of cytochrome b. The rest of the insertion has homology to an unknown dispersed nuclear sequence. Recombination among subtelomeric regions can explain the nuclear distribution of this sequence and why telomeres can trap and maintain sequences that would otherwise be lost. [ABSTRACT FROM AUTHOR]

The temperature-conditional pet mutant, ts379, of Saccharomyces cerevisiae fails to synthesize mitochondrial ATP synthase subunit 9 at the restrictive temperature due to mutation of a single nuclear locus, AEP2. The inability to synthesize subunit 9 correlates with a lowered accumulation of the cognate oli1 mRNA indicating that the AEP2 product is involved in oli1 transcript maturation or stabilization. The AEP2 gene has been isolated in this study from a wild-type yeast genomic library by genetic complementation of ts379 at the restrictive temperature. A 1740 nucleotide open-reading frame was observed that encodes a basic, hydrophilic protein of 67534 Da which possesses a putative mitochondrial address signal. Disruption of chromosomal DNA within this reading frame produced a non-conditional respiratory mutant unable to synthesize subunit 9, identifying the AEP2 gene. Hybridization analyses indicate that AEP2 is located on chromosome XIII and produces a 2.1 kb poly(A) transcript. Two additional open-reading frames were found in close proximity to that of AEP2. The three open-reading frames shared no significant homology with entries in several data bases. [ABSTRACT FROM AUTHOR]

The gene coding for glyceraldehyde-3-phosphate dehydrogenase ( gpd) has been isolated from a genomic library of the filamentous fungus Curvularia lunata. The coding region of this gene consists of 1014 nucleotides and is interrupted by four introns. The gpd gene product shows a high degree of sequence identity with the corresponding proteins of various species belonging to both taxonomically related (e.g., Aspergillus nidulans), as well as more divergent, taxa. Using contour-clamped homogeneous electric field (CHEF) gel electrophoresis eight distinct chromosomal bands have been resolved, with two bands migrating as doublets and one as a triplet. Thus, the total number of chromosomes of C. lunata appears to be 12. The size of the chromosomes ranges from about 1.4 Mb to 4.0 Mb allowing an estimation of the genome to be approximately 29.7 Mb. By hybridization of fractionated chromosomes the gpd gene and the rDNA locus have been localized on individual chromosomes. [ABSTRACT FROM AUTHOR]

In CYH2/cyh2 heterozygous diploids of the yeast Saccharomyces cerevisiae resistance is dominant over sensitivity at low (0.5-5 μg/ml) cycloheximide (cyh) concentrations. The cyh-resistant haploid strain MMY1 confers relatively high (10 μg/ml) cyh-resistance to heterozygous diploids constructed by mating this strain with cyh-sensitive haploid strains. We present here a genetic and biochemical study of strain MMY1. Analysis of tetrads obtained from a MMY1 heterozygous diploid showed that two unlinked nuclear mutations, determining high-and low-cycloheximide resistance, were present in MMY1. From a genomic library of this strain, constructed in vector YCp50, two plasmids (pRC1 and pRC13) have been isolated which, respectively, confer high-and low-resistance phenotypes to cyh-sensitive S. cerevisiae strains. The restriction maps of pRC1 and pRC13 are totally unrelated. This finding suggests that the genes harboring the two mutations encoding cyh-resistance from MMY1 were cloned in plasmids pRC1 and pRC13, respectively. Pulse field gel electrophoresis showed that the DNA insert of pRC1 maps at either chromosome VII or XV, whereas that from pRC13 maps at chromosome XI. This latter gene appears to define a previously unreported locus and has been named cyh5. By restriction and nucleotide sequencing analysis, the cyh gene present in pRC1 has been shown to correspond to cyh2, which maps at chromosome VII. These results suggest that the dominant cyh-resistance phenotype conferred by MMY1 in heterozygous diploids is promoted by the presence of both cyh2 and cyh5. A 2.3 kb NcoI fragment, containing cyh2 from pRC1, has been inserted in vectors YCp50, YEp13 and YIp5. By selecting at discriminating cyh concentrations, the resulting constructs efficiently transform a variety of haploid and diploid yeasts of both laboratory and industrial origin. [ABSTRACT FROM AUTHOR]

Whole chromosomes from the fungal phytopathogen Leptosphaeria maculans were separated by transverse alternating field electrophoresis. The chromosome complements from several isolates of both highly and weakly virulent strains were compared. Small variations in chromosome size and apparent number were detected among isolates of the same strain. However, dramatic differences in both chromosome number and size were found when isolates of the highly virulen strain were compared to those of the weakly virulent. Highly virulent isolates had 6-8 distinct bands whereas weakly virulent isolates had 12-14. The genome sizes were estimated to be at least 8.6x10 base pairs for the virulent strain and 1.6x10 base pairs for the weakly virulent strain. The major differences found in the chromosome complements of the two strains, in combination with results of other biochemical, morphological, and genetic studies, indicate that they are distinct species. [ABSTRACT FROM AUTHOR]

Transverse alternating field electrophoresis (TAFE) was used to compare the electrophoretic karyotypes of seven Septoria tritici isolates sampled from a single population. Isolates were selected based on differences at 12 DNA restriction fragment length polymorphism (RFLP) loci. Significant differences in electrophoretic karyotype existed among the isolates. Isolates had 14-16 bands, believed to correspond to chromosomes, ranging in size from approximately 330 to 3500 kb. Homologous chromosomes were identified by hybridization of anonymous single-locus and multiplelocus nuclear DNA sequences to Southern blots of TAFE gels. Length differences of up to 20% existed among homologous chromosomes. Densitometry and probe hybridization data showed that several bands contained two chromosomes. Probe pSTS192 hybridized to two chromosomes in each isolate, supporting previous data which suggested that this probe hybridized to two unlinked RFLP loci. Hybridization with probe pSTL53 provided additional evidence of partial diploidy at an RFLP locus in one isolate. [ABSTRACT FROM AUTHOR]

Cochliobolus heterostrophus was transformed with a plasmid (pH1S) containing a bacterial gene ( hygB), which confers resistance to the antibiotic hygromycin B when under control of an 838-bp fragment of promoter 1 from C. heterostrophus. The plasmid integrated at either homologous (52% single copy, 33% tandemly repeated copies) or ectopic (4% single copy, 11% tandemly repeated copies) sites on different chromosomes, resulting in four distinct configurations of integrated DNA. All four configurations were highly stable during mitotic growth; virtually no loss of integrated DNA was detected after five subcultures on nonselective medium or after seven cycles of pathogenesis on maize, the normal host of this fungus. However, deletion of integrated DNA was detected after eight or more disease cycles. The frequency of deletion depended on the configuration of the recombinant chromosome. A single copy of pH1S integrated at an homologous site was flanked by direct repeats of the target sequence and was least stable; up to 50% of the population lacked integrated DNA after 12 disease cycles. A single copy integrated at an ectopic site had no repeated DNA directly associated with it and was the most stable; no deletions were detected after 12 disease cycles. Tandemly repeated copies of pH1S integrated at either homologous or ectopic sites appeared to have intermediate stability; 2-18% of each population lost at least one copy after 12 disease cycles, although in no case were all copies deleted. Cytosine residues of integrated DNA were methylated during mitotic growth, but this had no apparent effect on the expression of hygB. [ABSTRACT FROM AUTHOR]

Six chromosomal DNA bands of Schizophyllum commune have been resolved using transverse alternating field electrophoresis. The estimated sizes of the chromosomal DNAs ranged from 5.1 to 1.2 megabase pairs (Mb), the total genome size being approximately 35-36 Mb. Chromosomal length polymorphisms were found between the two S. commune isolates examined. The DNA bands corresponding to the two chromosomes containing the A and B mating-type loci were identified in hybridization experiments using probes specific to their respective linkage groups. The utility of eluted chromosomal DNAs as hybridization probes to select clones from genomic libraries, and the use of these clones in transformation experiments to identify genes of interest, are discussed. [ABSTRACT FROM AUTHOR]

Isolates representing two types (A and B) of Colletotrichum gloeosporioides, which cause different anthracnose disease on the tropical pasture legumes Stylosanthes spp. in Australia, were found to have distinct electrophoretic karyotypes. Type A isolates had five large maxi-chromosomes (2-6 Mb) and eight to ten smaller mini-chromosomes (270-600 kb), while Type B isolates had three maxi chromosomes (4.7->6 Mb) and three to five mini-chromosomes (330-1200 kb). Extensive chromosomal polymorphisms for both length and number were observed in the mini-chromosomes amongst isolates within both types. Results indicate that chromosomal rearrangements may have a role in generating variation in this pathogen. [ABSTRACT FROM AUTHOR]

Although Saccharomyces cerevisiae strains generally have similar chromosomal band patterns as revealed by pulsed field gel electrophoresis, individual bands often move slightly differently from one strain to the other. Surveying strains from our stock collection, we found that nearly all the bands of a certain pair of strains differed in their mobility. Some of these chromosome length polymorphisms segregated in a 2:2 ratio, indicating that they resulted from single structural alterations (i.e. additions or deletions). One of these was mapped on the right arm of chromosome 1. Others did not segrate in a simple 2:2 ratio. That is, there were progenies which had bands not present in either parent. We suggest that these new bands are the products of recombination between homologous chromosomes having two or more structural alterations. [ABSTRACT FROM AUTHOR]

Electrophoretic analysis reveals that the nucleus of the ultra-small eukaryotic alga Cyanidioschyzon merolae contains approximately 11.7×10 base pairs (11.7 Mb) of DNA. This compact genome is fragmented into 15 small chromosomes ranging in size from 410 to 1700 kb. The migratory behaviour of chloroplast DNA is consistent with the presence of a circular plastid genome of about 170 kb. The conformation of mitochondrial DNA resembles that in yeasts and fungi and is predominantly linear and heterogenous in size. [ABSTRACT FROM AUTHOR]

The GGP1 gene encodes the only GPI-anchored glycoprotein (gp115) that has been purified todate in the budding yeast Saccharomyces cerevisiae. It is a single-copy gene whose deduced amino-acid sequence shares no significant homology to any other known protein. In this paper we report a Southern hybridization analysis of genomic DNA from different eukaryotic organisms to identify homologues of the GGP1 gene. We have analyzed DNA prepared from a unicellular green alga ( Chlamydomonas eugametos), from two distantly related yeast species ( Candida cylindracea and Schizosaccharomyces pombe), and from the common bean Phasoleus vulgaris. The moderate stringency of the experimental conditions and the high specificity of the probes used indicate that a single-copy of GGP1-related sequences exists in all these eukaryotic organisms. The chromosomal localization of the GGP1 gene in S. cerevisiae has also been determined. [ABSTRACT FROM AUTHOR]

The genome of the cellulolytic fungus Penicillium janthinellum BIOURGE was resolved completely by rotating field electrophoresis. The gel pattern revealed 8-10 different chromosomes. On the basis of data for yeast chromosome size standards from Schizosaccharomyces pombe and Saccharomyces cerevisiae, chromosome sizes in the range from 2.0 to 8 mb were estimated. By Southern hybridization with heterologous probes, the chromosomal locations of rDNA, the elongation factor EF1, actin and ubiquitin have all been mapped. In addition, first results with heterologous and homologous probes for cellulolytic genes from Trichoderma reesei and P. janthinellum are presented. In contrast to the other probes, signals obtained with cellulase genes are clearly distributed over several chromosomes. [ABSTRACT FROM AUTHOR]

A number of A. chrysogenum strains from a lineage improved in cephalosporin C production were analysed by contoured-clamped homogenous electric field gel electrophoreis (CHEF). Although antibiotic titre was increased across the lineage, chromosome rearrangements were only observed at two points in it. In one member of the lineage the chromosomal changes included those which altered the size of the chromosome on which the isopenicillin N synthetase gene ( pcbC) was located. It is proposed that chromosome changes are a chance event in an industrial strain improvement programme. [ABSTRACT FROM AUTHOR]

Fractionation of intact chromosomes of the ascomycete Podospora anserina by contour-clamped homogenous electric field (CHEF) gel electrophoresis resulted in the resolution of five distinct chromosomal bands. Two of these bands migrated as doublets. Using chromosomal standards from Schizosaccharomyces pombe we estimated the size of the P. anserina genome to about 33.4 megabases (Mb). By heterologous hybridization of fractionated chromosomes the rDNA locus was identified on one of the two chromosomes migrating at about 4.9 Mb. [ABSTRACT FROM AUTHOR]

Chromosomal DNA molecules from the plant pathogenic fungus Phoma tracheiphila were separated by orthogonal-field-alternation gel electrophoresis (OFAGE). Intact chromosomal DNA was obtained by a protocol which involves embedding fungal hyphae in low-gelling agarose followed by enzymatic digestion of the cell wall and subsequent lysis of the other cell components by proteinase k and sarcosyl. The electrophoretic karyotype revealed the presence of twelve distinct bands. [ABSTRACT FROM AUTHOR]

Chromosome samples for pulsed-field electrophoresis have been prepared without first generating protoplasts. The technique involves treatment of intact, agarose-solidified cell material with protease in the presence of EDTA and SDS. Saccharomyces cerevisiae, Ustilago hordei, Tilletia caries, and T. controversa karyotypes are clearly resolved with this technique. Colonies of U. hordei and S. cerevisiae removed from the surface of agarsolidified media and prepared for PFGE by this abbreviated method also yield well resolved karyotypes. [ABSTRACT FROM AUTHOR]