Unequal crossing-over and gene conversion at the amplified CUP1 locus of yeast

Summary Meiotic recombination was analyzed between two twelve-copy arrays of a gene amplification at theCUP1 locus ofSaccharomyces cerevisiae. Utilizing Southern analysis to identify spores with non-parental repeat arrays, we find that approximately 11% of a sample with 202 unselected tetrads possess at least one nonparental spore array. Both reciprocal and non-reciprocal changes are observed. The data suggest a model in which frequent mispairing among identical copies of the 2.0 kb repeat unit leads to the formation of unpaired loops containing integral numbers of repeat units. In this model, conversions involving the loops lead to non-reciprocal changes in arrays: about half are associated with reciprocal exchange, and net increases in repeat unit numbers occur about as frequently as net decreases. Thus, the known properties of gene conversion can account for all the segregations we observe.     

Mitochondrial genome diversity in soybean: repeats and rearrangements

Mitochondrial (mt) genome organization in soybean was examined at the molecular level. This study builds upon previous reports that four soybean cytoplasmic groups, Bedford, Arksoy, Lincoln, and soja-forage, are differentiated by polymorphisms detected with a 2.3 kb Hind III mtDNA probe [12]. The variation detected results from DNA alterations in a region within and around a 4.8 kb repeat. The Bedford-type cytoplasm is the only cytoplasm that contains copies of a 4.8 kb repeat in four different genomic environments, evidence that it is recombinationally active. The Lincoln- and Arksoy-type cytoplasms each contain two copies of the repeat, as well as unique fragments that appear to result from rare recombination events outside, but near, the repeat. The soja-forage-type cytoplasm contains no complete copies of the repeat, but does contain a unique truncated version of the repeat. Sequence analysis indicates that the truncation is a result of recombination across a 9 bp repeated sequence, CCCCTCCCC. The structural rearrangements that have occurred in the region surrounding the 4.8 kb repeat may provide a means to dissect species relationships and evolution within the subgenus soja.     

Analysis of putative DNA amplification genes in the element AUD1 of Streptomyces lividans 66

The amplifiable AUD1 element of Streptomyces lividans 66 consists of two copies of a 4.7 kb sequence flanked by three copies of a 1 kb sequence. The DNA sequences of the three 1 kb repeats were determined. Two copies (left and middle repeats) were identical: (1009 by in length) and the right repeat was 1012 bp long and differed at 63 positions. The repeats code for open reading frames (ORFs) with typical Streptomyces codon usage, which would encode proteins of about 36 kD molecular weight. The sequences of these ORFs suggest that they specify DNA-binding proteins and potential palindromic binding sites are found adjacent to the genes. The putative amplification protein encoded by the right repeat was expressed in Escherichia coli.     

Amplification of a gene for metallothionein by tandem repeat in a strain of cadmium-resistant yeast cells

Abstract In a cadmium-resistant strain of Saccharomyces cerevisiae , cells are protected against cadmium toxicity by the production of large amounts of cadmium-binding metallothionein, as occurs similarly in a copper-resistant strain. The apoprotein of the metallothionein is encoded by the CUP1 gene on chromosome VIII. The CUP1 gene is present as 8–10 copies in the cadmium-resistant strain as a result of tandem repeat of a 2.0-kb fragment of DNA that includes CUP1 , while the wild-type strain contains only a single copy of CUP1 . In the cadmium-resistant strain, some evidence for elongation of chromosome VIII with variations in length (maximum to 200 kb) was obtained. However, the elongation was not due to the tandem repeats of the CUP1 -containing region.     

Repeats and subrepeats in the intergenic spacer of rDNA from the nematode Meloidogyne arenaria

Summary Ribosomal DNA (rDNA) repeats of the plant-parasitic nematode Meloidogyne arenaria are heterogeneous in size and appear to contain 5S rRNA gene sequences. Moreover, in a recA ⁺ bacterial host, plasmid clones of a 9 kb rDNA repeat show deletion events within a 2 kb intergenic spacer (IGS), between 28S and 5S DNA sequences. These deletions appear to result from a reduction in the number of tandem 129 by repeats in the IGS. The loss of such repeats might explain how rDNA length heterogeneity, observed in the Meloidogyne genome, could have arisen. Each 129 by repeat also contains three copies of an 8 by subrepeat, which has sequence similarity to an element found in the IGS repeats of some plant rDNAs.     

The consensus land plant chloroplast gene order is present, with two alterations, in the moss Physcomitrella patens

Summary A restriction endonuclease cleavage site map for the enzymes ClaI and BglII, and a partial map for SacI, has been constructed for the chloroplast genome of the moss Physcomitrella patens (Hedw.) BSG. The plastid chromosome contains approximately 122 kb organized into small (21 kb) and large (82 kb) single-copy regions separated by two copies of a repeat sequence (9.4 kb) oriented in an inverted arrangement. Genes for 17 proteins and 2 ribosomal RNAs have been mapped using heterologous probes from corn, spinach, pea, and petunia. The general order and arrangement of the moss chloroplast genes are similar to the consensus land plant genome typified by that of spinach, with two major exceptions. First, there is an inversion of approximately 20 kb, bordered internally by psbA and atpH, and also containing the genes atpF and atpA. Second, rpl2 and rps19 have been relocated to a different position within the large single-copy region, adjacent to the 20 kb inversion.     

Comparison of the ribosomal RNA genes in four closely relatedCucurbitaceae

Cucurbitaceae are characterized by a high copy number for nuclear ribosomal RNA genes. We have investigated the genomic ribosomal DNA (rDNA) of four closely related species of this family with respect to structure, length heterogeneity, and evolution. InCucumis melo (melon) there are two main length variants of rDNA repeats with 10.7 and 10.55kb.Cucumis sativus (cucumber) shows at least three repeat types with 11.5, 10.5, and 10.2kb.Cucurbita pepo (zucchini) has two different repeat types with 10.0 and 9.3kb. There are also two different repeat types inCucurbita maxima (pumpkin) of about 11.2 and 10.5kb. Restriction enzyme mapping of the genomic rDNA of these four plants and of cloned repeats ofC. sativus shows further heterogeneities which are due to methylation or point mutations. By comparison of the restriction enzyme maps it was possible to trace some evolutionary events in the family ofCucurbitaceae. Some aspects of regulation and function of the middle repetitive rRNA genes (here between 2000 and 10000 copies) are discussed.     

Evolution and selection of Rhg1, a copy‐number variant nematode‐resistance locus

The soybean cyst nematode (SCN) resistance locus Rhg1 is a tandem repeat of a 31.2 kb unit of the soybean genome. Each 31.2‐kb unit contains four genes. One allele of Rhg1, Rhg1‐b, is responsible for protecting most US soybean production from SCN. Whole‐genome sequencing was performed, and PCR assays were developed to investigate allelic variation in sequence and copy number of the Rhg1 locus across a population of soybean germplasm accessions. Four distinct sequences of the 31.2‐kb repeat unit were identified, and some Rhg1 alleles carry up to three different types of repeat unit. The total number of copies of the repeat varies from 1 to 10 per haploid genome. Both copy number and sequence of the repeat correlate with the resistance phenotype, and the Rhg1 locus shows strong signatures of selection. Significant linkage disequilibrium in the genome outside the boundaries of the repeat allowed the Rhg1 genotype to be inferred using high‐density single nucleotide polymorphism genotyping of 15 996 accessions. Over 860 germplasm accessions were found likely to possess Rhg1 alleles. The regions surrounding the repeat show indications of non‐neutral evolution and high genetic variability in populations from different geographic locations, but without evidence of fixation of the resistant genotype. A compelling explanation of these results is that balancing selection is in operation at Rhg1.     

Homologous recombination and transposition generate chromosome I neopolymorphism during meiosis in Saccharomyces cerevisiae

We have studied the meiotic segregation of a chromosome length polymorphism (CLP) in the yeast Saccharomyces cerevisiae. The neopolymorphism frequently observed within the smallest chromosomes (I, VI, III and IX) is not completely understood. We focused on the analysis of the structure of chromosome I in 88 segregants from a cross between YNN295 and FL100trp. Strain FL100trp is known to carry a reciprocal translocation between the left arm of chromosome III and the right arm of chromosome I. PCR and Southern hybridization analyses were performed and a method for the rapid detection of chromosome I rearrangements was developed. Seven chromosome I types were identified among the 88 segregants. We detected 22 recombination events between homologous chromosomes I and seven ectopic recombination events between FL100trp chromosome III and YNN295 chromosome I. These recombination events occurred in 20 of the 22 tetrads studied (91%). Nine tetrads (41%) showed two recombination events. This showed that homologous recombination involving polymorphic homologues or heterologous chromosomes is the main source of neopolymorphism. Only one of the seven chromosome I variants resulted from a transposition event rather than a recombination event. We demonstrated that a Ty1 element had transposed within the translocated region of chromosome I, generating mutations in the 3′ LTR, at the border between U5 and PBS. Received: 7 May 1999 / Accepted: 14 February 2000     

Diversity of DcMaster-like elements of the PIF/Harbinger superfamily in the carrot genome

Transposable elements constitute a significant fraction of plant genomes. Both autonomous and non-autonomous elements of the DcMaster family, residing in the carrot genome, were described previously. DcMaster elements were classified as members of the PIF/Harbinger superfamily. In the present paper we report on the identification of other groups of DcMaster-like elements. Beside the previously described, relatively highly abundant miniature inverted repeat element (MITE) family Krak (ca. 3,600 copies, ca. 80% intra-family similarity), three other families, i.e. 1.1 kb long KrakL1 (ca. 3,000 copies, ca. 98% intra-family similarity), 1.2 kb long KrakL2 (a single identified element, 71.1% similar to KrakL1 consensus), and 2.2 kb long Midi (few elements, 99% intra-family similarity), were revealed. The fact that all members within each of the newly found families were almost identical in their sequence suggests their very recent activity. The families differed in terms of their similarity to the canonical DcMaster element. They also differed in copy number, from just a few copies of Midi to few thousand copies of Krak. A modification of the DcMaster Transposon Display (DcMTD) marker system, targeted specifically towards Krak elements, was used to investigate their insertion polymorphism. It was shown that Krak insertion sites, similarly to those of the DcMaster elements, were highly polymorphic between the wild and the cultivated Daucus carota.     

Evolution of a B2 tagged sequence from a long-range repeat family in the genus Mus

A long-range repeat family of more than 50 kb repeat size is clustered in Chromosomes (Chr) 1 of Mus musculus and M. spretus. In M. musculus this long-range repeat family shows considerable variation of copy-number frequency and contains coding regions for at least two genes. In an intron of a gene, which is part of the repeat, a B2 small interspersed repetitive element (SINE) is inserted at identical positions. The B2 element is present in all copies of the long-range repeat family; it was presumably a component of the ancestral single-copy precursor sequence that gave rise by amplification to the repeat family. Copies of the long-range repeat family vary with respect to the number of TAAA tandem repeats in the A-rich 3 end region of the B2 element. As inferred from polymerase chain reaction (PCR) data, presence and frequency of repeat number variants in the (TAAA)_n block are strain and species specific. The B2 element and its flanking regions were sequenced from two copies of the long-range repeat family. Sequence divergence between the two copies (only non-CG base substitutions and deletions/insertions) was determined to be 2.6%. Based on the drift rate in human Alu elements and a correction for the higher drift rates in rodents, and estimate for the divergence time of 1.7 million years was calculated. Since the long-range repeat family is present in M. musculus and M. spretus, it must have evolved by amplification before the separation of the two species about 1–4 million years ago.     

Analysis of putative DNA amplification genes in the element AUD1 of Streptomyces lividans 66

The amplifiable AUD1 element of Streptomyces lividans 66 consists of two copies of a 4.7 kb sequence flanked by three copies of a 1 kb sequence. The DNA sequences of the three 1 kb repeats were determined. Two copies (left and middle repeats) were identical: (1009 by in length) and the right repeat was 1012 bp long and differed at 63 positions. The repeats code for open reading frames (ORFs) with typical Streptomyces codon usage, which would encode proteins of about 36 kD molecular weight. The sequences of these ORFs suggest that they specify DNA-binding proteins and potential palindromic binding sites are found adjacent to the genes. The putative amplification protein encoded by the right repeat was expressed in Escherichia coli.     

Cloning of ribosomal RNA genes from an Indian isolate ofGiardia lamblia and the use of intergenic nontranscribing spacer regions in the differentiation ofGiardia from other enteric pathogens

The ribosomal RNA genes from an Indian isolate ofGiardia lamblia have been cloned and characterized with respect to size, composition and copy number. Southern blotting and rDNA cloning ofGiardia lamblia revealed that genes coding for ribosomal RNA (rRNA) are exceptionally small and are encoded within a 5.6 kb genome fragment repeat. The rDNA repeat unit of this isolate was found to be highly G-C rich like other human isolates and the physical map showed severalSmaI sites. There are 132 copies of the rDNA repeat unit per cell in a head to tail arrangement. Two fragments corresponding to intergenic (0.2 kb and 0.3 kb) region and one (0.8 kb) containing both an intergenic region and a small part of the small subunit ribosomal RNA (SS rRNA) have been identified within the rDNA. These were used in heterogeneity studies ofGiardia isolated from two geographic locations as well as in the analysis of cross reactivity with other enteric organisms. In Southern blots, all the three fragments were found to be highly specific for the differential diagnosis ofGiardia spp. from the other enteric pathogens. These findings should help in developing a sensitive and more specific method for the diagnosis of giardiasis over currently available techniques.     

Genetic mapping of mitochondrial markers by recombinational analysis in Chlamydomonas reinhardtii

The mitochondrial genome of Chlamydomonas reinhardtii is a 15.8 kb linear DNA molecule present in multiple copies. In crosses, the meiotic products only inherit the mitochondrial genome of the mating type minus (paternal) parent. In contrast mitotic zygotes transmit maternal and paternal mitochondrial DNA copies to their diploid progeny and recombinational events between molecules of both origins frequently occur. Six mitochondrial mutants unable to grow in the dark (dk^– mutants) were crossed in various combinations and the percentages of wild-type dk⁺ recombinants were determined in mitotic zygotes when all progeny cells had become homoplasmic for the mitochondrial genome. In crosses between strains mutated in the COB (apocytochrome ) gene and strains mutated in the COX1 (subunit 1 of cytochrome oxidase) gene, the frequency of recombination was 13.7% (± 3.2%). The corresponding physical distance between the mutation sites was 4.3 kb. In crosses between strains carrying mutations separated by about 20 bp, a recombinational frequency of 0.04% (± 0.02%) was found. Two other mutants not yet characterized at the molecular level were also used for recombinational studies. From these data, a linear genetic map of the mitochondrial genome could be drawn. This map is consistent with the positions of the mutation sites on the mitochondrial DNA molecule and thereby validates the method used to generate the map. The frequency of recombination per physical distance unit (3.2% ± 0.7% per kilobase) is compared with those obtained for other organellar genomes in yeasts and Chlamydomonas.     

Frequency of telomere repeat units in the Drosophila miranda genome

Cloned DNA fragments of Drosophila miranda which label all chromosome ends show a basic tandem repeat unit of 4.4 kb. The D. miranda telomere specific tandem repeats do not cross-hybridize with genomic D. melanogaster DNA which itself contains telomere repeat units of 3 kb. For a more detailed analysis of the functional criteria of telomere specific sequences we determined the repetition frequency of the tandem repeat units. As a low estimate we found a repetition frequency of 20 for female D. miranda DNA. This is on average equivalent to 2 telomere repeat units per chromosome end in the female D. miranda karyotype. However, a variable number of tandem repeat units per chromosome end would describe more closely the obtained differences in the labeling intensity between the individual chromosomes (X¹L-5). For the D. miranda male DNA we determined a repetition frequency of 90. The frequency difference of 70 copies between male and female DNA must be due to the Y-chromosome.     

Responder (Rsp) alleles in the segregation distorter (SD) system of meiotic drive in Drosophila may represent a complex family of satellite repeat sequences

In D. melanogaster males carrying Segregation Distorter (SD) second chromosomes, sperm receiving sensitive alleles of the Responder (Rsp) locus are subject to high rates of dysfunction. The Rsp region is located in 2R immediately adjacent to the centromere in heterochromatic band 39, and covers roughly 600 kb of material, of which approximately 85 kb is comprised of several hundred copies of a 240-bp satellite DNA sequence. Cytological observations as well as molecular analysis of rearrangements which bisect h39 indicate that sensitivity of the Rsp target to SD action is also subdivisible, and sensitivities of the component pieces appear to be correlated with copy number of the 240 bp repeat. In an attempt to examine possible higher order sequence structure for these blocks, PCR using single primers derived from a canonical repeat was used to identify potential reversals of direction of tandem arrays; that is, head-to-head or tail-to-tail junctions. Surprisingly, for two different Rsp alleles, only a single such reversal product for each was identified, differing in size and sequence between alleles. Sequencing of PCR products identified diverged copies of the canonical repeats that would not have been found using the levels of DNA stringency employed in earlier studies. Examination of Southern digests and slot-blots for DNA quantification indicates that adding the estimated numbers of such diverged copies to the canonical repeat copies discovered earlier is potentially sufficient to account for the entire 600 kb Rsp region. This adds strength to the hypothesis that this extended family of repeats is in fact the target of SD-mediated sperm dysfunction. Implications of these results for understanding the evolution of repetitive DNA are also discussed.     

Cloning and analysis of CUT1, a cutinase gene from Magnaporthe grisea

Summary A gene from Magnaporthe grisea was cloned using a cDNA clone of the Colletotrichum gloeosporioides cutinase gene as a heterologous probe; the nucleotide sequence of a 2 kb DNA segment containing the gene has been determined. DNA hybridization analysis shows that the M. grisea genome contains only one copy of this gene. The predicted polypeptide contains 228 amino acids and is homologous to the three previously characterized cutinases, showing 74% amino acid similarity to the cutinase of C. gloeosporioides. Comparison with previously determined cutinase sequences suggests that the gene contains two introns, 115 and 147 bp in length. The gene is expressed when cutin is the sole carbon source but not when the carbon source is cutin and glucose together or glucose alone. Levels of intracellular and extracellular cutinase activity increase in response to growth in the presence of cutin. The activity level is higher in a transformant containing multiple copies of the cloned gene than in the parent strain. Non-denaturing polyacrylamide gels stained for esterase activity show a single major band among intracellular and extracellular proteins from cutin-grown cultures that is not present among intracellular and extracellular proteins prepared from glucose-grown or carbon-starved cultures. This band stains more intensely in extracts from the multicopy transformant than in extracts from the parent strain. We conclude that the cloned DNA contains a M. grisea gene for cutinase, which we have named CUT1.     

Strain variation in the katG region of Mycobacterium tuberculosis

Southern blot analysis of chromosomal DNA from clinical isolates of Mycobacterium tuberculosis using cosmid DNA probes revealed extensive strain variation in the katG region of the genome. In addition to deletion of the katG gene itself in some isoniazid-resistant strains, adjacent DNA fragments were missing or altered in a range of drug-sensitive and drug-resistant isolates. A species-specific 2 kb Kpnl fragment located 10 kb upstream of katG in M. tuberculosis H37Rv hybridized to fragments of differing size in different clinical isolates and was characterized in detail. Sequence analysis of this fragment showed that it comprised three tandem copies of a novel 75 bp repeat element flanked by multiple copies of the previously described 10 bp major polymorphic tandem repeat of M. tuberculosis (MPTR). The copy number of the 75 bp repeat was found to vary between strains, allowing application of a poly-merase chain reaction amplification strategy for strain differentiation. These results indicate that the katG region of the M. tuberculosis genome is highly variable and unstable. The presence of repetitive sequences may contribute to instability in this region of the genome.     

Application of Ty1 for cloned gene insertion: amplification of a large regulated expression cassette in Saccharomyces cerevisiae

A large cassette, 4.6 × 10³ bases (4.6 kb) in length, containing an inducible expression system (the yeast CUP1 promoter fused to the Escherichia coli lacZ structural gene) and a bacterial neomycin-resistance gene (neo) has been cloned into the noncoding region of a GAL1-regulated Ty1 retrotransposon. Galactose was used to induce retrotransposition in Saccharomyces cerevisiae, and cells containing integrations were selected by resistance to the aminoglycoside G418. Integrations of neo and CUP1p-lacZ were verified, and -galactosidase activity was confirmed. Analysis via Southern blots demonstrated integrations at various chromosomal locations, and the number of insertions obtained ranged from one to five after three rounds of induction. Therefore, the packaging limit of Ty1 virus-like particles for RNA is at least 10.3 kb and Ty1 can transpose foreign genes as large as 4.6 kb, demonstrating the practical application of Ty1 for the insertion of large regulated expression cassettes.