A meiotically reproducible chromosome length polymorphism in the ascomycete fungus Ophiostoma ulmi ( sensu lato )

We have followed the transmission of Ophiostoma ulmis.l. chromosome length polymorphisms (CLPs) into the F₂ generation to determine the reproducibility of a genome rearrangement culminating in the conversion of a 1.0 Mb chromosome into a 800 kb chromosome. The 1.0 Mb chromosome in strain CESS16K is thus far unique among O. ulmi s.l. wild-type strains, as no other wild-type strains have been observed with chromosomes smaller than 2.3 Mb. It has been previously shown that the 1.0 Mb chromosome is mitotically stable, carries at least one normally expressed gene, and is transmitted through meiosis. In this study, a series of crosses were performed to further elucidate the pattern of inheritance of the 1.0 Mb chromosome and the process of conversion of the 1.0 Mb species to 800 kb. In crosses where the 1.0 Mb chromosome was allowed to pair with itself or with the 800 kb chromosome, all progeny inherited a copy of the 1.0 Mb or 800 kb form, further demonstrating the A-type nature of these small chromosomes. When a cross was repeated between the strains CESS16K (1.0 Mb chromosome) and FG245B^r-O (no 1.0 Mb or 800 kb chromosome), the occurrence of a 800 kb chromosome was observed in 9% of the progeny. A reciprocal cross between an 800 kb strain and a strain with no 800 kb or 1.0 Mb chromosome was conducted, and a progeny strain containing a 1.0 Mb chromosome was recovered. The reproducibility and reciprocality of the 1.0 Mb to 800 kb chromosome conversion demonstrates that meiotic processes are responsible for this CLP, and that O. ulmi s.l. strains with various divergent genome architectures can remain sexually compatible. Received: 6 February 1996 / Accepted: 21 January 1997     

A Fine-Structure Map of Spontaneous Mitotic Crossovers in the Yeast Saccharomyces cerevisiae

Homologous recombination is an important mechanism for the repair of DNA damage in mitotically dividing cells. Mitotic crossovers between homologues with heterozygous alleles can produce two homozygous daughter cells (loss of heterozygosity), whereas crossovers between repeated genes on non-homologous chromosomes can result in translocations. Using a genetic system that allows selection of daughter cells that contain the reciprocal products of mitotic crossing over, we mapped crossovers and gene conversion events at a resolution of about 4 kb in a 120-kb region of chromosome V of Saccharomyces cerevisiae. The gene conversion tracts associated with mitotic crossovers are much longer (averaging about 12 kb) than the conversion tracts associated with meiotic recombination and are non-randomly distributed along the chromosome. In addition, about 40% of the conversion events have patterns of marker segregation that are most simply explained as reflecting the repair of a chromosome that was broken in G1 of the cell cycle.     

A meiotically reproducible chromosome length polymorphism in the ascomycete fungus Ophiostoma ulmi (sensu lato)

We have followed the transmission of Ophiostoma ulmis.l. chromosome length polymorphisms (CLPs) into the F₂ generation to determine the reproducibility of a genome rearrangement culminating in the conversion of a 1.0 Mb chromosome into a 800 kb chromosome. The 1.0 Mb chromosome in strain CESS16K is thus far unique among O. ulmi s.l. wild-type strains, as no other wild-type strains have been observed with chromosomes smaller than 2.3 Mb. It has been previously shown that the 1.0 Mb chromosome is mitotically stable, carries at least one normally expressed gene, and is transmitted through meiosis. In this study, a series of crosses were performed to further elucidate the pattern of inheritance of the 1.0 Mb chromosome and the process of conversion of the 1.0 Mb species to 800 kb. In crosses where the 1.0 Mb chromosome was allowed to pair with itself or with the 800 kb chromosome, all progeny inherited a copy of the 1.0 Mb or 800 kb form, further demonstrating the A-type nature of these small chromosomes. When a cross was repeated between the strains CESS16K (1.0 Mb chromosome) and FG245B^r-O (no 1.0 Mb or 800 kb chromosome), the occurrence of a 800 kb chromosome was observed in 9% of the progeny. A reciprocal cross between an 800 kb strain and a strain with no 800 kb or 1.0 Mb chromosome was conducted, and a progeny strain containing a 1.0 Mb chromosome was recovered. The reproducibility and reciprocality of the 1.0 Mb to 800 kb chromosome conversion demonstrates that meiotic processes are responsible for this CLP, and that O. ulmi s.l. strains with various divergent genome architectures can remain sexually compatible.     

Drosophila gypsy insulator and yellow enhancers regulate activity of yellow promoter through the same regulatory element

There is ample evidence that the enhancers of a promoterless yellow locus in one homologous chromosome can activate the yellow promoter in the other chromosome where the enhancers are inactive or deleted, which is indicative of a high specificity of the enhancer–promoter interaction in yellow. In this paper, we have found that the yellow sequence from −100 to −69 is essential for stimulation of the heterologous eve (TATA-containing) and white (TATA-less) promoters by the yellow enhancers from a distance. However, the presence of this sequence is not required when the yellow enhancers are directly fused to the heterologous promoters or are activated by the yeast GAL4 activator. Unexpectedly, the same promoter proximal region defines previously described promoter-specific, long-distance repression of the yellow promoter by the gypsy insulator on the mod(mdg4) ^u1 background. These finding suggest that proteins bound to the −100 to −69 sequence are essential for communication between the yellow promoter and upstream regulatory elements.     

Deep sequencing of a candidate region harboring the SOX9 gene for the canine XX disorder of sex development

A disorder of sex development (DSD) in dogs with female sex chromosomes (78, XX), a lack of the SRY gene and the presence of testes or ovotestes is commonly diagnosed in numerous breeds. The molecular background of DSD is not fully recognized but has been linked to the copy number variation in the region harboring the SOX9 gene. We applied a genome‐wide association study and targeted next‐generation sequencing techniques to compare DSD and normal female dogs. The genome‐wide association study did not indicate a significant chromosome region. Targeted next‐generation sequencing of a 1.5‐Mb region on canine chromosome 9 harboring the SOX9 gene revealed two putatively DSD‐associated copy number variations 355 kb upstream and 691 kb downstream of SOX9, four blocks of low polymorphism and two blocks of an elevated heterozygosity. An initial next‐generation sequencing analysis showed an association with two SNPs, but validation in larger cohorts did not confirm this result. We identified a large homologous fragment (over 243.8 kb), named hfMAGI2, located upstream of SOX9, that overlaps a known copy number variation region. It shows a high sequence similarity with the 5′ flanking region of the MAGI2 gene located on canine chromosome 18 that encodes a protein involved in ovary formation during early embryonic development. Our study showed that the identified copy number variation region located upstream of the SOX9 gene contains potential regulatory sequences (long non‐coding RNA and hfMAGI2) and led to the assumption that a multiplication of this element may alter expression of the SOX9 gene, triggering the DSD phenotype.     

Microcloning of maize chromosome 9 by using a flow-sorting technique

We constructed a chromosome 9 lambda DNA library from flow-sorted maize chromosomes. Approximately 3 million maize chromosome 9 were collected with high purity by flow cytometric sorting of chromosomes isolated from an oat-maize chromosome 9 addition line based on the cytogram of fluorescent pulse area versus fluorescent pulse width. Chromosome 9 DNA was partially digested withBamH I, dephosphorylated, and ligated with arms ofBamH I-digested lambda DASH vector (Stratagene). A total of 2.0×10⁶ independent recombinants with an average insert size of 15 kb were obtained. For a 99% probability that every sequence of chromosome 9 is represented in at least one chimeric phage, 5.6×10⁴ cloned fragments are needed. This library covers the entire maize chromosome 9. Hybridizing cloned fragments with labeled maize genomic DNA showed that the high, middle, or low copy number DNA sequences presented in the different phage clones. This individual chromosome library is useful in plant genome mapping and gene isolation.     

Sex chromosome evolution in fish: the formation of the neo-Y chromosome in Eigenmannia (Gymnotiformes)

Chromosomes of a species of Eigenmannia presenting a X₁X₁X₂X₂:X₁X₂Y sex chromosome system, resulting from a Y-autosome Robertsonian translocation, were analyzed using the C-banding technique, chromomycin A₃ (CMA₃) and mithramycin (MM) staining and in situ digestion by the restriction endonuclease AluI. A comparison of the metacentric Y chromosome of males with the corresponding acrocentrics in females indicated that a C-band-positive, CMA₃/MM-fluorescent and AluI digestion-resistant region had been lost during the process of translocation, resulting in a diminution of heterochromatin in the males. It is hypothesized that the presence of a smaller amount of G+C-rich heterochromatin in the sex chromosomes of the heteromorphic sex when compared with the homomorphic sex may be associated with the sex determination mechanism in this species and may be a more widely occurring phenomenon in fish with differentiated sex chromosomes than was initially thought. Received: 1 April 1999; in revised form: 16 October 1999 / Accepted: 4 December 1999     

Double-strand breaks on artificial chromosomes in yeast

Yeast artificial chromosomes composed primarily of bacteriophage λ DNA exhibit very low levels of meiotic crossing over compared with similarly sized intervals of natural yeast DNA. When these recombinationally quiet chromosomes were augmented with a 12.5 kb insert of sequences from yeast chromosome VIII, genetic studies demonstrated that the artificial chromosomes had acquired recombination properties characteristic of this region of chromosome VIII. On authentic yeast chromosomes, most meiotic recombination events are initiated at sites where the DNA is cleaved to create a double-strand break (DSB). This report describes physical analyses that were carried out to examine the relationship between DSB sites and the recombination behavior of the artificial chromosomes. The results show that DSBs are rare on these artificial chromosomes, except for the 12.5 kb insert. Mapping of the DSB sites shows that their positions correlate with the previously determined positions of DSB sites on chromosome VIII. Deletion of two characterized chromosome VIII DSB sites from the 12.5 kb insert on the artificial chromosome resulted in the loss of the predicted DSB fragments and a reduction in crossing over between artificial chromosomes. Received: 15 May 1998; in revised form: 26 September 1999 / Accepted: 18 November 1999     

Cytological identification of two X-chromosome types in the wood lemming (Myopus schisticolor)

In the wood lemming (Myopus schisticolor) three genetic types of sex chromosome constitution in females are postulated: XX, X^*X and X^*Y (X^*=X with a mutation inactivating the male determining effect of the Y chromosome). Males are all XY. It is shown in the present paper that the two types of X chromosomes, X and X^*, exhibit differences in the G-band patterns of their short arms. In addition, it was demonstrated in unbanded chromosomes that the short arm in X^* is shorter than in X. The origin of these differences is still obscure; but they allow to identify and to distinguish the individual types of sex chromosome constitution, as of XX versus X^*X females and of X^*Y females versus XY males, on the basis of G-banded chromosome preparations from somatic cells.     

Novel genes influencing the expression of the yellow locus and mdg4 (gypsy) in Drosophila melanogaster

Summary We used a system with a mobilized Stalker transposable element, sometimes in combination with P-M hybrid dysgenesis, in the search for new mutations interfering with the y ² mutation induced by mdg4 (gypsy) insertion into the yellow locus. A novel gene, modifier of mdg4, was detected in chromosome 3. The mutation mod(mdg4) either enhanced or suppressed phenotypic changes in different mutations induced by mdg4 insertions. Thus, mod(mdg4) seems to be involved in the control of mdg4 expression. Six other loci designated as enhancers of yellow were also detected. The e(y) ⁿ (with n from 1–6) mutations enhanced the expression of several y mutations induced by different insertions into the yellow locus. The major change is a damage of bristle and hair pigmentation which is not suppressed by su(Hw) mutations. On the other hand, e(y) ⁿ alleles do not interact with mdg4 induced mutations in other loci. All e(y) ⁿ genes are located in different regions of the X chromosome. One may speculate that e(y) ⁿ genes are involved in trans-regulation of the yellow locus and possibly of some other loci.     

Localization of the active gene of aldolase on chromosome 16, and two aldolase A pseudogenes on chromosomes 3 and 10

Summary Southern blot analysis of human genomic DNA hybridized with a coding region aldolase A cDNA probe (600 bases) revealed four restriction fragments with EcoRI restriction enzyme: 7.8 kb, 13 kb, 17 kb and >30 kb. By human-hamster hybrid analysis (Southern technique) the principal fragments, 7.8 kb, 13 kb, >30 kb, were localized to chromosomes 10, 16 and 3 respectively. The 17-kb fragment was very weak in intensity; it co-segregated with the >30-kb fragment and is probably localized on chromosome 3 with the >30-kb fragment. Analysis of a second aldolase A labelled probe protected against S1 nuclease digestion by RNAs from different hybrid cells, indicated the presence of aldolase A mRNAs in hybrid cells containing only chromosome 16. Under the stringency conditions used, the EcoRI sequences detected by the coding region aldolase A cDNA probe did not correspond to aldolase B or C. The 7.8-kb and >30-kb EcoRI sequences, localized respectively on chromosomes 10 and 3, correspond to aldolase A pseudogenes, the 13-kb EcoRI sequence localized on chromosome 16 corresponds to the aldolase active gene. The fact that the aldolase A gene and pseudogenes are located on three different chromosomes supports the hypothesis that the pseudogenes originated from aldolase A mRNAs, copied into DNA and integrated in unrelated chromosomal loci.     

The Sec-1 locus on the short arm of chromosome 1R of rye (Secale cereale)

This paper describes a detailed sequence analysis of the ω-secalin gene array at theSec-1 locus on the short arm of chromosome 1 of rye. The analysis shows that the genes are separated by 8 kb of spacer sequence and that the gene/spacer units are arranged in a head to tail fashion. The boundaries of the array are identified, and a fragment containing the majority of the genes in the array is separated by PFG analysis. The sequence data of one 9.2 kb gene unit have been determined, and because of the similarity of the gene units within the array these data provide a detailed sequence analysis of 140 kb of theSec-1 locus. Fluorescence in situ hybridization, using lambda clones isolated for the structural analysis, identifies the position of the array on the rye chromosomes relative to the 5S rRNA genes. Edited by: W. Hennig     

Detection of single-copy genes and chromosome rearrangements in Petunia hybrida by fluorescence in situ hybridization

DNA sequences homologous to single-copy genes were labelled with biotinylated dUTP or digoxygenin-labelled dUTP and hybridized to chromosome spreads. The hybridization signals were visualized with fluorescent avidin- or antibody-conjugates. This method allowed the detection of DNA targets on metaphase chromosomes as small as 1.4 kb. The hybridization signals were identified as fluorescent spots on both sister chromatids. Using an 18S rDNA probe as marker to identify chromosomes II and III it was possible to assign single-copy genes to these chromosomes. In the line V30 the endogenous chalcone synthase gene (chsA) was mapped at the distal end of the short arm of chromosome 5. The cDNA probe for this single-copy gene was 1.4 kb. In contrast, in the lines Mitchell and V26 chsA was localized at the distal end of the long arm of chromosome 3, suggesting that a chromosomal rearrangement had taken place. In a transformed Petunia uidA, transgenes were detected using a 2.7 kb probe. One transgene was mapped on one of the homologues of chromosome II proximal to the ribosomal genes. This homologue could be distinguished from the other by having the ribosomal genes at the distal end of the long arm. Using multicolour fluorescence in situ hybridization it was shown that it is possible to detect the endogenous chsA genes and both transgenes simultaneously.     

Construction of a yeast artificial chromosome library of pepper (Capsicum annuum L.) and identification of clones from the Bs2 resistance locus

A yeast artificial chromosome (YAC) library was constructed using high-molecular-weight DNA isolated from pepper (Capsicum annuum L.) leaf protoplasts. Insert DNA was prepared by partial digestion using EcoRI and subjected to electrophoretic fractionation before in-gel ligation to the pJS97/98 YAC vector. Prior to transformation of yeast spheroplasts, ligation products were subjected to a second electrophoretic size selection. The library consists of about 19 000 clones with an average insert size of 500 kb, thus representing approximately three haploid genome equivalents. Three PCR-based markers tightly linked to the pepper Bs2 resistance gene were used to assess the utility of this library for positional cloning. Three YAC clones containing pepper genomic DNA from the Bs2 resistance locus were isolated from the library. The clones ranged in size from 270 kb to 1.2 Mb and should prove useful for the cloning of the Bs2 gene. Received: 15 January 1999 / Accepted: 11 May 1999     

Genomic in situ hybridization (GISH) analyses of Thinopyrum intermedium, its partial amphiploid Zhong 5, and disease-resistant derivatives in wheat

Genomic in situhybridization (GISH) to root-tip cells at mitotic metaphase, using genomic DNA probes from Thinopyrum intermedium and Pseudoroegneria strigosa, was used to examine the genomic constitution of Th. intermedium, the 56-chromosome partial amphiploid to wheat called Zhong 5 and disease-resistant derivatives of Zhong 5, in a wheat background. Evidence from GISH indicated that Th. intermedium contained seven pairs of St, seven J^S and 21 J chromosomes; three pairs of Th. intermedium chromosomes with satellites in their short arms belonging to the St, J, J genomes and homoeologous groups 1, 1, and 5 respectively. GISH results using different materials and different probes showed that seven pairs of added Th. intermedium chromosomes in Zhong 5 included three pairs of St chromosomes, two pairs of J^S chromosomes and two pairs of St-J^S reciprocal tanslocation chromosomes. A pair of chromosomes, which substituted a pair of wheat chromosomes in Yi 4212 and in HG 295 and was added to 21 pairs of wheat chromosomes in the disomic additions Z1, Z2 and Z6, conferred BYDV-resistance and was identical to a pair of St-J^S tanslocation chromosomes (StJ^S) in Zhong 5. The StJ^S chromosome had a special GISH signal pattern and could be easily distinguished from other added chromosomes in Zhong 5; it has not yet been possible to locate the BYDV-resistant gene(s) of this translocated chromosome either in the St chromosome portion belonging to homoeologous group 2 or in the J^S chromosome portion whose homoeologous group relationship is still uncertain. Among 22 chromosome pairs in disomic addition line Z3, the added chromosome pair had satellites and belonged to the St genome and homoeologous group 1. Disomic addition line Z4 carried a pair of added chromosomes which was composed of a group-7 J^S chromosome translocated with a wheat chromosome; this chromosome was different to 7 Ai-1, but was identical to 7 Ai-2. The leaf rust and stem rust resistance genes were located in the distal region of the long arm, whereas the stripe rust resistance gene(s) was located in the short arm or in the proximal region of the long arm of 7 Ai-2. A pair of J^S-wheat translocation chromosomes, which originated from the WJ^S chromosomes in Z4, was added to the disomic addition line Z5; the added chromosomes of Z5 carried leaf and stem rust resistance but not stripe rust resistance; Z5 is a potentially useful source for rust resistance genes in wheat breeding and for cloning these novel rust-resistant genes. GISH analysis using the St genome as a probe has proved advantageous in identifying alien Th. intermedium in wheat. Received: 17 May 1999 / Accepted: 22 June 1999     

A microcolinearity study at the earliness per se gene Eps-A m 1 region reveals an ancient duplication that preceded the wheat–rice divergence

Wheat flowering is controlled by numerous genes, which respond to environmental signals such as photoperiod and vernalization. Earliness per se (Eps) genes control flowering time independently of these environmental cues and are responsible for the fine tuning of flowering time. We recently mapped the Eps-A ^m 1 gene on the end of Triticum monococcum chromosome arm 1A^mL. As a part of our efforts to clone Eps-A ^m 1 we developed PCR markers flanking this gene within a 2.7 cM interval. We screened more than one thousand gametes with these markers and identified 27 lines with recombination between them. Recombinant lines were used to generate a high-density map and to investigate the microcolinearity between wheat and rice in this region. We mapped ten genes from a 149 kb region located at the distal part of rice chromosome 5 (cdo393 – Ndk3) on a 3.7 cM region on wheat chromosome one. This region is part of an ancient duplication between rice chromosomes 5 and 1. Genes present in both rice chromosomes were less similar to each other than to the closest wheat orthologues, suggesting that this duplication preceded the divergence between wheat and rice. This hypothesis was supported by the presence of 18 loci duplicated both in rice chromosomes 5 and 1 and in the colinear wheat chromosomes from homoeologous groups 1 and 3. Independent gene deletions in wheat and rice lineages explain the alternations of colinearity between rice chromosome 5 and wheat chromosomes 1 and 3. Colinearity between the end of rice chromosome 5 and wheat chromosome 1 was also interrupted by a small inversion, and several non-colinear genes. These results suggest that the distal region of the long arm of wheat chromosome 1 was involved in numerous changes that differentiated wheat and rice genomes. This comparative study provided sufficient markers to saturate the Eps-A ^m 1 gene region and to precisely map this gene within a 0.9 cM interval flanked by the VatpC and Smp loci. Sequences obtained in this study: DQ196178, DQ196179, DQ196180, DQ196181, DQ196182, DQ196183, DQ196184, DQ196185, DQ196186, DQ196187, DQ196488, DQ198537, DQ308530, DQ308531, DQ308532, DQ308533, DQ308534, DQ308535, DQ308536, DQ308537, DQ308538, DQ308539, DQ308540     

Structural and mutational analysis of KCNQ2, the major gene locus for benign familial neonatal convulsions

Mutations in the voltage-gated potassium channel gene KCNQ2 on chromosome 20q13.3 are responsible for benign familial neonatal convulsions (BFNC), a rare monogenic idiopathic epilepsy. Here we report the determination of the detailed genomic structure of KCNQ2, and use of this information in mutational analysis. There are at least 18 exons, occupying more than 50 kb of genomic DNA. Several formerly unknown polymorphisms and splice variants as well as a new single base pair deletion mutation of unusual localization are described. In addition to facilitating more effective mutation detection among BFNC patients, the results presented here provide the basis for analysing the role of KCNQ2 in other types of epilepsy. Received: 24 November 1998 / Accepted: 8 January 1999     

Late replication patterns in adult and embryonic mice carrying Searle's X-autosome translocation

Bromodeoxyuridine-dye technique analysis of X chromosome DNA synthesis in female adult and fetal mice carrying the balanced form of the T(X; 16) 16H translocation demonstrated that the structurally normal X chromosome was late replicating (and hence presumably inactive) in 93% of the adult cells and 99% of the 9-day embryo cells, with the X¹⁶ chromosome late replicating in the remaining cells. We conclude from these results that in T16H/+ females either there is preferential inactivation of the normal X chromosome or that, if inactivation is random, cell selection takes place before 9 days of development. Two 9-day female embryos with an unbalanced karyotype were also studied; both had two late-replicating chromosomes in most of their cells, one being the chromosome 16^X, the other a normal X chromosome. These results, together with the presence of a late-replicating X¹⁶ chromosome in T16H/+ adult and fetal mice, support the concept that more than one inactivation center is present on the X chromosome of the mouse because the X¹⁶ and the 16^x chromosomes can be late replicating.