Chromosomal organization of TOX2, a complex locus controlling host-selective toxin biosynthesis in Cochliobolus carbonum.

Race 1 isolates of the filamentous fungus Cochliobolus carbonum are exceptionally virulent on certain genotypes of maize due to production of a cyclic tetrapeptide, HC-toxin. In crosses between toxin-producing (Tox2+) and toxin-nonproducing (Tox2-) isolates, toxin production segregates in a simple 1:1 pattern, suggesting the involvement of a single genetic locus, which has been named TOX2. Earlier work had shown that in isolate SB111, TOX2 consists in part of two copies of a gene, HTS1, that encodes a 570-kD cyclic peptide synthetase and is lacking in Tox2- isolates. The genomic structure of TOX2 and the relationship between the two copies of HTS1 have now been clarified by using pulsedfield gel electrophoresis and physical mapping. In isolate SB111, both copies of HTS1 are on the largest chromosome (3.5 Mb), which is not present in the related Tox2- strain SB114. Two other genes known or thought to be important for HC-toxin biosynthesis, TOXA and TOXC, are also on the same chromosome in multiple copies. Other independent Tox2+ isolates also have two linked copies of HTS1, but in some isolates the size of the chromosome containing HTS1 is 2.2 Mb. Evidence obtained with Tox2+ -unique and with random probes is consistent with a reciprocal translocation as the cause of the difference in the size of the HTS1-containing chromosome among the Tox2+ isolates studied here. Physical mapping of the 3.5-Mb chromosome of SB111 that contains HTS1 using rare-cutting restriction enzymes and engineered restriction sites was used to map the chromosome location of the two copies of HTS1 and the three copies of TOXC. The results indicate that TOX2 is a complex locus that extends over more than 500 kb. The capacity to produce HC-toxin did not evolve by any single, simple mechanism.     

Reduced virulence caused by meiotic instability of the TOX2 chromosome of the maize pathogen Cochliobolus carbonum

The mechanisms by which pathogenic fungi evolve are poorly understood. Production of the host-selective cyclic peptide HC-toxin is controlled by a complex locus, TOX2, in the plant pathogen Cochliobolus carbonum. Crosses between toxin-producing (Tox2+) and toxin-nonproducing (Tox2-) isolates, as well as crosses between isolates in which the TOX2 genes were on chromosomes of different size, yielded progeny that had lost one or more copies of one or more of the TOX2 genes. Of approximately 200 progeny analyzed, eight (4%) had lost at least one TOX2 gene. All of them still had at least one functional copy of all of the known genes required for HC-toxin production (HTS1, TOXA, TOXC, and TOXE). Most deletion strains could be explained by simple chromosome breaks resulting in the loss of major contiguous portions (0.8 to 1.4 Mb) of the 3.5-Mb TOX2 chromosome, whereas others had more complicated patterns. All deletion strains had normal growth and were fertile, indicating that the 1.4 Mb of DNA contained no essential housekeeping genes. Most strains were also still virulent (Tox2+), but two had a novel phenotype of reduced virulence (RV), characterized by smaller lesions that expanded at a reduced rate and an inability to colonize plants systemically. Although the RV strains made no detectable HC-toxin in culture, the RV phenotype was dependent on the presence of a functional copy of HTS1, which encodes the central enzyme in HC-toxin biosynthesis. We propose that the RV strains still make a low level of HC-toxin, at least in planta, and that this is due to the loss of one or more genes that contribute to, but are not absolutely required for, HC-toxin synthesis.     

Pisatin demethylase genes are on dispensable chromosomes while genes for pathogenicity on carrot and ripe tomato are on other chromosomes in Nectria haematococca

Studies on the wide-host-range fungus Nectria haematococca MP VI have shown a linkage between virulence on pea and five of nine PDA genes that encode the ability to detoxify the pea phytoalexin, pisatin. Most of the PDA genes are on chromosomes of approximately 1.6 megabases (Mb) and two of these genes, PDA1-2 and PDA6-1, have been demonstrated to reside on approximately 1.6-Mb chromosomes that can be lost during meiosis. Prior studies also have shown that the dispensable chromosome carrying PDA6-1 contains a gene (MAK1) necessary for maximum virulence on chickpea. The present study evaluated whether the other approximately 1.6-Mb chromosomes that carry PDA genes also are dispensable, their relationship to each other, and whether they contain genes for pathogenicity on hosts other than pea or chickpea. DNA from the PDA1-1 chromosome (associated with virulence on pea) and the PDA6-1 chromosome (associated with virulence on chickpea) were used to probe blots of contour-clamped homogeneous electric field (CHEF) gels of isolates carrying different PDA genes and genetically related Pda- isolates. All of the approximately 1.6-Mb PDA-bearing chromosomes hybridized with both probes, indicating that they share significant similarity. Genetically related Pda-progeny lacked chromosomes of approximately 1.6 Mb and there was no significant hybridization of any chromosomes to the PDA1-1 and PDA6-1 chromosome probes. When isolates carrying different PDA genes and related Pda- isolates were tested for virulence on carrot and ripe tomato, there was no significant difference in lesion sizes produced by Pda+ and Pda- isolates, indicating that genes for pathogenicity on these hosts are not on the PDA-containing chromosomes. These results support the hypothesis that the chromosomes carrying PDA genes are dispensable and carry host-specific virulence genes while genes for pathogenicity on other hosts are carried on other chromosomes.     

Behavior of chromosomes after meiosis inCoprinus cinereus

The movement was investigated of a specific chromosome in the F₁ progeny of the basidiomyceteCoprinus cinereus. We focussed our attention on the smallest chromosome of the 5302 strain. We first constructed a chromosome-smallest library and screened it with a chromosome-specific clone, pRC 1. The pRC 1 probe hybridized only with the smallest chromosome of the 5302 strain, and it detected one band of different mobility in two parental strains. In the F₁ progeny, the probe hybridized with one to three chromosomes. Most of the hybridized chromosomes in the F₁ progeny were positioned in terms of mobility between the hybridized chromosomes of the two parental strains. Therefore, they were probably generated by meiotic recombination between homologous chromosomes of different sizes.     

A Restriction Fragment Length Polymorphism Map and Electrophoretic Karyotype of the Fungal Maize Pathogen Cochliobolus Heterostrophus

A restriction fragment length polymorphism (RFLP) map has been constructed of the nuclear genome of the plant pathogenic ascomycete Cochliobolus heterostrophus. The segregation of 128 RFLP and 4 phenotypic markers was analyzed among 91 random progeny of a single cross; linkages were detected among 126 of the markers. The intact chromosomal DNAs of the parents and certain progeny were separated using pulsed field gel electrophoresis and hybridized with probes used to detect the RFLPs. In this way, 125 markers were assigned to specific chromosomes and linkages among 120 of the markers were confirmed. These linkages totalled 941 centimorgans (cM). Several RFLPs and a reciprocal translocation were identified tightly linked to Tox1, a locus controlling host-specific virulence. Other differences in chromosome arrangement between the parents were also detected. Fourteen gaps of at least 40 cM were identified between linkage groups on the same chromosomes; the total map length was therefore estimated to be, at a minimum, 1501 cM. Fifteen A chromosomes ranging from about 1.3 megabases (Mb) to about 3.7 Mb were identified; one of the strains also has an apparent B chromosome. This chromosome appears to be completely dispensable; in some progeny, all of 15 markers that mapped to this chromosome were absent. The total genome size was estimated to be roughly 35 Mb. Based on these estimates of map length and physical genome size, the average kb/cM ratio in this cross was calculated to be approximately 23. This low ratio of physical length to map distance should make this RFLP map a useful tool for cloning genes.     

Identification of new pisatin demethylase genes (PDA5 and PDA7) in Nectria haematococca and non-Mendelian segregation of pisatin demethylating ability and virulence on pea due to loss of chromosomal elements

Previous studies have shown that high virulence on pea in Nectria haematococca Mating Population VI is linked to the ability to detoxify the pea phytoalexin, pisatin, via demethylation (Pda). To test this linkage further, a highly virulent Pda(+) isolate (34-18) was used as the recurrent parent in backcrosses to Pda(-) isolates, but most of the progeny were low in virulence on pea, and tetrad analysis gave conflicting ratios for the genetic control of Pda. Southern analysis of 34-18 and progeny showed that 34-18 carries a gene similar to PDA1 (PDA1-2), two new PDA genes, PDA5 and PDA7, and that all three genes can be lost during meiosis. Southern analysis of electrophoretic karyotypes showed that PDA1-2 is on a 1.5-Mb dispensable chromosome in 34-18 and that PDA5 and PDA7 are on a 4.9-Mb chromosome in 34-18 but are found on variably sized chromosomes in progeny. Loss of PDA5 or PDA7 in progeny was not generally associated with morphological phenotypes, except in progeny from some crosses between PDA5 parents. Loss of PDA5 was associated with growth abnormalities in these crosses, suggesting that in some genetic backgrounds at least a portion of the PDA5/PDA7 chromosome is essential for normal growth. All highly virulent progeny had PDA1-2 or a combination of PDA5 and PDA7 while isolates that lacked the three genes were low in virulence, supporting the hypothesis that Pda, or genes linked to PDA genes, are necessary for virulence on pea. However, low virulence isolates with PDA genes were also identified, suggesting that there are pathogenicity genes that can segregate independently of PDA genes.     

Regulation of cyclic peptide biosynthesis and pathogenicity in Cochliobolus carbonum by TOXEp, a novel protein with a bZIP basic DNA-binding motif and four ankyrin repeats

HC-toxin is an epoxide-containing cyclic tetrapeptide that is a critical virulence determinant in the pathogenic interaction between the filamentous fungus Cochliobolus carbonum and maize. HC-toxin exerts a potent cytostatic effect on plant and animal cells by inhibiting histone deacetylase. The biosynthesis of HC-toxin by C. carbonum is controlled by a complex genetic locus, TOX2, that contains multiple, duplicated copies of genes encoding export and biosynthetic enzymes. A new gene in the TOX2 complex, TOXE, has now been isolated. Mutation of TOXE by targeted gene disruption has no effect on growth and sporulation but abolishes HC-toxin production and pathogenicity. TOXE is required for the expression of three genes with a known or putative role in HC-toxin production, but is not required for expression of HTS1, which encodes the large, multifunctional peptide synthetase that is the central enzyme in HC-toxin biosynthesis. At its N-terminus, TOXEp has a bZIP basic DNA binding domain, but it does not contain any discernible leucine zipper or helix-loop-helix. At its carboxy terminus, TOXEp contains four ankyrin repeats. In having these two common regulatory motifs in a single polypeptide, TOXEp appears to represent a novel class of regulatory protein. TOXE is present only in HC-toxin-producing (Tox2⁺) isolates of C. carbonum. Most Tox2⁺ isolates have two copies; in strain SB111, one copy of TOXE is on the same 3.5-Mb chromosome that contains all of the other genes known to be involved in HC-toxin biosynthesis, and the second copy of TOXE is on a 0.7-Mb chromosome.     

Identification and characterization of DNA markers associated with a locus conferring virulence on barley in the plant pathogenic fungus Cochliobolus sativus

Cochliobolus sativus is a plant pathogenic fungus that causes spot blotch on barley and wheat. Virulence of a pathotype-2 isolate (ND90Pr) on barley cultivar Bowman was previously determined to be controlled by a single locus. To identify DNA markers associated with this virulence locus, amplified fragment length polymorphism (AFLP) analysis was conducted on 104 progeny isolates derived from a cross between isolates ND90Pr (exhibiting high virulence on Bowman) and ND93-1 (exhibiting low virulence on Bowman). Among 115 AFLP markers identified, 14 were linked to the virulence locus VHv1 in isolate ND90Pr, six of which co-segregated with VHv1. Two (E-AG/M-CA-207 and E-AG/M-CG-121) of the six co-segregating AFLP markers were cloned and used to probe genomic DNAs from the fungal parents and progeny. Both markers hybridized only with DNAs from ND90Pr and the virulent progeny. These two cloned markers were also used as probes to survey field isolates of C. sativus collected from different regions of the world and again only hybridized to DNAs from isolates that had the same virulence phenotype as ND90Pr. The results of this study indicate that E-AG/M-CA-207 and E-AG/M-CG-121 are closely linked to VHv1 and are unique to isolates carrying the virulence locus. Development of a linkage group, coupled with the identification of closely linked molecular markers, will facilitate the cloning of the virulence gene VHv1 in C. sativus by map-based cloning.     

Regulation of cyclic peptide biosynthesis and pathogenicity in Cochliobolus carbonum by TOXEp, a novel protein with a bZIP basic DNA-binding motif and four ankyrin repeats

HC-toxin is an epoxide-containing cyclic tetrapeptide that is a critical virulence determinant in the pathogenic interaction between the filamentous fungus Cochliobolus carbonum and maize. HC-toxin exerts a potent cytostatic effect on plant and animal cells by inhibiting histone deacetylase. The biosynthesis of HC-toxin by C. carbonum is controlled by a complex genetic locus, TOX2, that contains multiple, duplicated copies of genes encoding export and biosynthetic enzymes. A new gene in the TOX2 complex, TOXE, has now been isolated. Mutation of TOXE by targeted gene disruption has no effect on growth and sporulation but abolishes HC-toxin production and pathogenicity. TOXE is required for the expression of three genes with a known or putative role in HC-toxin production, but is not required for expression of HTS1, which encodes the large, multifunctional peptide synthetase that is the central enzyme in HC-toxin biosynthesis. At its N-terminus, TOXEp has a bZIP basic DNA binding domain, but it does not contain any discernible leucine zipper or helix-loop-helix. At its carboxy terminus, TOXEp contains four ankyrin repeats. In having these two common regulatory motifs in a single polypeptide, TOXEp appears to represent a novel class of regulatory protein. TOXE is present only in HC-toxin-producing (Tox2⁺) isolates of C. carbonum. Most Tox2⁺ isolates have two copies; in strain SB111, one copy of TOXE is on the same 3.5-Mb chromosome that contains all of the other genes known to be involved in HC-toxin biosynthesis, and the second copy of TOXE is on a 0.7-Mb chromosome. Received: 20 April 1998 / Accepted: 21 September 1998     

A decarboxylase encoded at the Cochliobolus heterostrophus translocation-associated Tox1B locus is required for polyketide (T-toxin) biosynthesis and high virulence on T-cytoplasm maize

Genes at two unlinked loci (Tox1A and Tox1B) are required for production of the polyketide T-toxin by Cochliobolus heterostrophus race T, a pathogenic fungus that requires T-toxin for high virulence to maize with T-cytoplasm. Previous work indicated that Tox1A encodes a polyketide synthase (PKS1) required for T-toxin biosynthesis and for high virulence. To identify genes at Tox1B, a wild-type race T cDNA library was screened for genes missing in the genome of a Tox1B deletion mutant. The library was probed, first with a 415-kb NotI restriction fragment from the genome of the Tox1B mutant, then with the corresponding 560-kb fragment from the genome of wild type. Two genes, DEC1 (similar to acetoacetate decarboxylase-encoding genes) and RED1 (similar to genes encoding members of the medium-chain dehydrogenase/reductase superfamily), were recovered. Targeted disruption of DEC1 drastically reduced both T-toxin production and virulence of race T to T-cytoplasm maize, whereas specific inactivation of RED1 had no apparent effect on T-toxin production (as determined by bioassay) or on virulence. DEC1 and RED1 map within 1.5 kb of each other on Tox1B chromosome 6;12 and are unique to the genome of race T, an observation consistent with the hypothesis that these genes were acquired by C. heterostrophus via a horizontal transfer event.     

Molecular karyotyping and chromosome length polymorphism in Cochliobolus sativus

Fungi are known to have variable genomes that can generate new virulence types capable of attacking important crop plants. To assess chromosome length polymorphisms in the barley spot blotch pathogen (Cochliobolus sativus), we analyzed the karyotypes of 16 isolates using contour-clamped homogeneous electric field (CHEF) electrophoresis. The collection of isolates studied were from diverse regions of the world (USA, Canada, Japan, Brazil, Uruguay, and Poland) and included representatives comprising the three known C. sativus pathotypes of 0, 1, and 2. Under two different running conditions, the number of CHEF bands observed ranged from 8 to 13 with a size range of 0.85 to 3.80 mega-bases (Mb). Each of the 16 isolates showed a unique banding pattern, except for two North Dakota isolates ND90Pr and ND91-Bowman, which were very similar. Single-copy DNA probes, previously assigned to each of the 15 chromosomes identified in reference isolate ND93-1, were hybridized to Southern blots of CHEF-separated chromosomes and revealed highly polymorphic chromosomes among isolates. Chromosomal rearrangements (translocations, deletions, duplications) were found in several isolates. DNA markers previously found linked to VHv1, a gene in pathotype 2 isolates conferring virulence on barley cultivar Bowman, also were used as probes in hybridizations with the CHEF blots. The results showed that the chromosome carrying the virulence gene in pathotype 2 isolates is larger than its counterpart without the gene in other isolates. This suggests that the genomic region carrying the virulence locus VHv1 is unique to pathotype 2 isolates. This study provides useful information on genome structure and divergence, which is essential for advancing our understanding of the genetics and biology of C. sativus.     

Plasmodium falciparum: analysis of karyotype polymorphism using chromosome-specific probes

Chromosome size variation in Plasmodium falciparum has been examined using a double heterogenous pulse field gradient electrophoresis apparatus and a series of chromosome-specific probes. In the 11 different isolates analyzed the chromosomal markers always hybridized to the corresponding chromosome, indicating that translocations do not significantly contribute to chromosome size variations. Furthermore, despite probes specific for chromosomes 5 and 6 no evidence was obtained to support the hypothesis of a chromosome duplication involving these chromosomes. The double heterogenous electric field combined with longer pulse times allowed the genome to be resolved into a larger number of chromosomal bands and as a result permitted the more precise mapping of cloned genes.     

Transfer of a supernumerary chromosome between vegetatively incompatible biotypes of the fungus Colletotrichum gloeosporioides.

Two biotypes (A and B) of Colletotrichum gloeosporioides infect the tropical legumes Stylosanthes spp. in Australia. These biotypes are asexual and vegetatively incompatible. However, field isolates of biotype B carrying a supernumerary 2-Mb chromosome, thought to originate from biotype A, have been reported previously. We tested the hypothesis that the 2-Mb chromosome could be transferred from biotype A to biotype B under laboratory conditions. Selectable marker genes conferring resistance to hygromycin and phleomycin were introduced into isolates of biotypes A and B, respectively. A transformant of biotype A, with the hygromycin resistance gene integrated on the 2-Mb chromosome, was cocultivated with phleomycin-resistant transformants of biotype B. Double antibiotic-resistant colonies were obtained from conidia of these mixed cultures at a frequency of approximately 10(-7). Molecular analysis using RFLPs, RAPDs, and electrophoretic karyotypes showed that these colonies contained the 2-Mb chromosome in a biotype B genetic background. In contrast, no double antibiotic colonies developed from conidia obtained from mixed cultures of phleomycin-resistant transformants of biotype B with biotype A transformants carrying the hygromycin resistance gene integrated in chromosomes >2 Mb in size. The results demonstrated that the 2-Mb chromosome was selectively transferred from biotype A to biotype B. The horizontal transfer of specific chromosomes across vegetative incompatibility barriers may explain the origin of supernumerary chromosomes in fungi.     

A eukaryotic alanine racemase gene involved in cyclic peptide biosynthesis

The cyclic tetrapeptide HC-toxin is an essential virulence determinant for the plant pathogenic fungus Cochliobolus carbonum and an inhibitor of histone deacetylase. The major form of HC-toxin contains the D-isomers of Ala and Pro. The non-ribosomal peptide synthetase that synthesizes HC-toxin has only one epimerizing domain for conversion of L-Pro to D-Pro; the source of D-Ala has remained unknown. Here we present the cloning and characterization of a new gene involved in HC-toxin biosynthesis, TOXG. TOXG is present only in HC-toxin-producing (Tox2(+)) isolates of C. carbonum. TOXG is able to support D-Ala-independent growth of a strain of Escherichia coli defective in D-Ala synthesis. A C. carbonum strain with both of its copies of TOXG mutated grows normally in culture, and although it no longer makes the three forms of HC-toxin that contain D-Ala, it still makes a minor form of HC-toxin that contains Gly in place of D-Ala. The addition of D-Ala to the culture medium restores production of the D-Ala-containing forms of HC-toxin by the toxG mutant. The toxG mutant has only partially reduced virulence. It is concluded that TOXG encodes an alanine racemase whose function is to synthesize D-Ala for incorporation into HC-toxin.     

Comparative chromosome painting of chicken autosomal paints 1-9 in nine different bird species

In a Zoo-FISH study chicken autosomal chromosome paints 1 to 9 (GGA1-GGA9) were hybridized to metaphase spreads of nine diverse birds belonging to primitive and modern orders. This comparative approach allows tracing of chromosomal rearrangements that occurred during bird evolution. Striking homologies in the chromosomes of the different species were noted, indicating a high degree of evolutionary conservation in avian karyotypes. In two species, the quail and the goose, all chicken paints specifically labeled their corresponding chromosomes. In three pheasant species as well as in the American rhea and blackbird, GGA4 hybridized to chromosome 4 and additionally to a single pair of microchromosomes. Furthermore, in the pheasants fission of the ancestral galliform chromosome 2 could be documented. Hybridization of various chicken probes to two different chromosomes or to only the short or long chromosome arm of one chromosome pair in the species representing the orders Passeriformes, Strigiformes, and Columbiformes revealed translocations and chromosome fissions during species radiation. Thus comparative analysis with chicken chromosome-specific painting probes proves to be a rapid and comprehensive approach to elucidate the chromosomal relationships of the extant birds.     

The Cochliobolus carbonum SNF1 gene is required for cell wall-degrading enzyme expression and virulence on maize

The production of cell wall-degrading enzymes (wall depolymerases) by plant pathogenic fungi is under catabolite (glucose) repression. In Saccharomyces cerevisiae, the SNF1 gene is required for expression of catabolite-repressed genes when glucose is limiting. An ortholog of SNF1, ccSNF1, was isolated from the maize pathogen Cochliobolus carbonum, and ccsnf1 mutants of HC toxin-producing (Tox2(+)) and HC toxin-nonproducing (Tox2(-)) strains were created by targeted gene replacement. Growth in vitro of the ccsnf1 mutants was reduced by 50 to 95% on complex carbon sources such as xylan, pectin, or purified maize cell walls. Growth on simple sugars was affected, depending on the sugar. Whereas growth on glucose, fructose, or sucrose was normal, growth on galactose, galacturonic acid, maltose, or xylose was somewhat reduced, and growth on arabinose was strongly reduced. Production of HC toxin was normal in the Tox2(+) ccsnf1 mutant, as were conidiation, conidial morphology, conidial germination, and in vitro appressorium formation. Activities of secreted beta-1,3-glucanase, pectinase, and xylanase in culture filtrates of the Tox2(+) ccsnf1 mutant were reduced by 53, 24, and 65%, respectively. mRNA expression was downregulated under conditions that induced the following genes encoding secreted wall-degrading enzymes: XYL1, XYL2, XYL3, XYL4, XYP1, ARF1, MLG1, EXG1, PGN1, and PGX1. The Tox2(+) ccsnf1 mutant was much less virulent on susceptible maize, forming fewer spreading lesions; however, the morphology of the lesions was unchanged. The Tox2(-) ccsnf1 mutant also formed fewer nonspreading lesions, which also retained their normal morphology. The results indicate that ccSNF1 is required for biochemical processes important in pathogenesis by C. carbonum and suggest that penetration is the single most important step at which ccSNF1 is required. The specific biochemical processes controlled by ccSNF1 probably include, but are not necessarily restricted to, the ability to degrade polymers of the plant cell wall and to take up and metabolize the sugars produced.     

Recombination of 4p16 DNA markers in an unusual family with Huntington disease

The Huntington disease (HD) mutation has been localized to human chromosome 4p16, in a 6-Mb region between the D4S10 locus and the 4p telomere. In a report by Robbins et al., a family was identified in which an affected individual failed to inherit three alleles within the 6-Mb region originating from the parental HD chromosome. To explain these results, it was suggested that the HD locus (HD) lies close to the telomere and that a recombination event took place between HD and the most telomeric marker examined, D4S90. As a test of this telomere hypothesis, we examined six members of this family, five of whom are affected with HD, for the segregation of 12 polymorphic markers from 4p16, including D4S169, which lies within 80 kb of the 4p telomere. We separated, in somatic cell hybrids, the chromosomes 4 from each family member, to determine the phase of marker alleles on each chromosome. We excluded nonpaternity by performing DNA fingerprint analyses on all six family members, and we found no evidence for chromosomal rearrangements when we used high-resolution karyotype analysis. We found that two affected siblings, including one of the patients originally described by Robbins et al., inherited alleles from the non-HD chromosome 4 of their affected parents, throughout the 6-Mb region. We found that a third affected sibling, also studied by Robbins et al., inherited alleles from the HD chromosome 4 of the affected parent, throughout the 6-Mb region. Finally, we found that a fourth sibling, who is likely affected with HD, has both a recombination event within the 6-Mb region and an additional recombination event in a more centromeric region of the short arm of chromosome 4. Our results argue against a telomeric location for HD and suggest that the HD mutation in this family is either associated with DNA predisposed to double recombination and/or gene conversion within the 6-Mb region or is in a gene that is outside this region and that is different from that mutated in most other families with HD.     

Genetic control of X chromosome inactivation in mice: definition of the Xce candidate interval

In early mammalian development, one of the two X chromosomes is silenced in each female cell as a result of X chromosome inactivation, the mammalian dosage compensation mechanism. In the mouse epiblast, the choice of which chromosome is inactivated is essentially random, but can be biased by alleles at the X-linked X controlling element (Xce). Although this locus was first described nearly four decades ago, the identity and precise genomic localization of Xce remains elusive. Within the X inactivation center region of the X chromosome, previous linkage disequilibrium studies comparing strains of known Xce genotypes have suggested that Xce is physically distinct from Xist, although this has not yet been established by genetic mapping or progeny testing. In this report, we used quantitative trait locus (QTL) mapping strategies to define the minimal Xce candidate interval. Subsequent analysis of recombinant chromosomes allowed for the establishment of a maximum 1.85-Mb candidate region for the Xce locus. Finally, we use QTL approaches in an effort to identify additional modifiers of the X chromosome choice, as we have previously demonstrated that choice in Xce heterozygous females is significantly influenced by genetic variation present on autosomes (Chadwick and Willard 2005). We did not identify any autosomal loci with significant associations and thus show conclusively that Xce is the only major locus to influence X inactivation patterns in the crosses analyzed. This study provides a foundation for future analyses into the genetic control of X chromosome inactivation and defines a 1.85-Mb interval encompassing all the major elements of the Xce locus.