Identification of X Chromosome Regions in Caenorhabditis Elegans That Contain Sex-Determination Signal Elements

The primary sex-determination signal of Caenorhabditis elegans is the ratio of X chromosomes to sets of autosomes (X/A ratio). This signal coordinately controls both sex determination and X chromosome dosage compensation. To delineate regions of X that contain counted signal elements, we examined the effect on the X/A ratio of changing the dose of specific regions of X, using duplications in XO animals and deficiencies in XX animals. Based on the mutant phenotypes of genes that are controlled by the signal, we expected that increases (in males) or decreases (in hermaphrodites) in the dose of X chromosome elements could cause sex-specific lethality. We isolated duplications and deficiencies of specific X chromosome regions, using strategies that would permit their recovery regardless of whether they affect the signal. We identified a dose-sensitive region at the left end of X that contains X chromosome signal elements. XX hermaphrodites with only one dose of this region have sex determination and dosage compensation defects, and XO males with two doses are more severely affected and die. The hermaphrodite defects are suppressed by a downstream mutation that forces all animals into the XX mode of sex determination and dosage compensation. The male lethality is suppressed by mutations that force all animals into the XO mode of both processes. We were able to subdivide this region into three smaller regions, each of which contains at least one signal element. We propose that the X chromosome component of the sex-determination signal is the dose of a relatively small number of genes.     

Dosage Compensation in Drosophila: Nadp-Enzyme Activities and Cross-Reacting Material

The relationships between gene dosage, enzyme activities and CRM levels have been determined for G6PD and 6PGD. Enzyme activities and CRM levels were directly proportional and increased in genotypes carrying duplications of the respective structural genes. When a duplication consisting of the distal 45% of the X chromosome was used to duplicate Pgd+, 6PGD activity and CRM increased and G6PD activity decreased. When the proximal 55% of the X chromosome was duplicated, G6PD activity and CRM increased whereas 6PGD activity and CRM levels decreased. These observations support the model of dosage compensation of X-linked genes that invokes an autosomal activator in limited concentrations for which X-linked loci compete. The distal 45% of the X chromosome, when duplicated, caused a significant increase in NADP-malic enzyme activity and CRM levels, as if a structural gene for NADP-ME is sex-linked.     

Chromosome Rearrangements in CAENORHABDITIS ELEGANS

A method for selecting unlinked duplications of a part of the X chromosome of C. elegans is described. Five such duplications have been identified. One of them, Dp (X;V)1, is translocated to linkage group V, where it suppresses crossing over along the left half of linkage group V. Dp(X;V)1 homozygotes grow slowly and are sterile. The other four duplications are associated with chromosome fragments, as observed cytologically by fluorescence microscopy, and tend to be lost. Their frequency of loss is higher in strains homozygous for a mutation that promotes nondisjunction of X chromosomes. The recombination frequencies between two of these duplications and the X have been measured: the frequencies are at least 50 times less than for X-X recombination in the same region. The duplications may prove useful as balancers of recessive lethal mutations.     

Duplications of the X chromosome in males: evidence that most parts of the X chromosome can be active in two copies

Summary We have analysed two duplications of the X chromosome in male patients using chromosome replication and DNA methylation patterns as determinants of the functional status of the duplicated segments. In both cases, the large duplicated regions, Xq12-q22 and Xq26.3-qter, were not inactivated. A review of previously reported male cases revealed that these duplications were also not subject to inactivation. Taken together, the examined duplications cover almost the entire X chromosome except the pericentromeric region and Xq25–26. Thus, most regions of the X chromosome can be present in two functional copies without lethal consequences.     

A Cytogenetic Analysis of Cyclic Nucleotide Phosphodiesterase Activities in Drosophila

The genome of Drosophila melanogaster has been surveyed for chromosomal regions which exert a dosage effect on the activities of cAMP phosphodiesterase or cGMP phosphodiesterase. Two regions increase cAMP phosphodiesterase activity when present as duplications. A region of the X chromosome increases cAMP phosphodiesterase activity when duplicated and decreases that activity when deficient. This region has been delimited to chromomeres 3D3 and 3D4, with 3D4 being the most probable locus, and may contain a structural gene for cAMP phosphodiesterase. A region on the third chromosome, 90E-91B, increases cAMP phosphodiesterase activity when duplicated but has no effect on the activity when deficient. Two regions increase cGMP phosphodiesterase activity when present as duplications. A region of the X chromosome, 5D-9C, increases cGMP phosphodiesterase activity when duplicated, but smaller duplications covering this region fail to show such an increase, indicating that a single locus is not responsible for the increase observed for the larger duplication. A region of the third chromosome, 88C-91B, also increases cGMP phosphodiesterase activity when duplicated. Smaller duplications covering this region show smaller increases than that observed for the larger duplication, suggesting that at least three loci between 88C and 91B contribute to the observed increase by that region. Deficiencies covering region 88C-91B do not affect cGMP phosphodiesterase activity. No locus for a presumptive structural gene for cGMP phosphodiesterase has been found. Limitations of the use of segmental aneuploidy in locating structural genes for enzymes are discussed.     

Recombination between Small X Chromosome Duplications and the X Chromosome in Caenorhabditis Elegans

Twelve new X chromosome duplications were identified and characterized. Eight are translocated to autosomal sites near four different telomeres, and four are free. Ten include unc-1(+), which in wild type is near the left end of the X chromosome, and two of these, mnDp72(X;IV) and mnDp73(X;f), extend rightward past dpy-3. Both mnDp72 and mnDp73 recombined with the one X chromosome in males in the unc-1-dpy-3 interval at a frequency 15- to 30-fold higher than was observed for X-X recombination in hermaphrodites in the same interval. Recombinant duplications and recombinant X chromosomes were both recovered. Recombination with the X chromosome in the unc-1-dpy-3 interval was also detected for five other unc-1(+) duplications, even though their right breakpoints lie within the interval. In hermaphrodites, mnDp72 and mnDp73 promoted meiotic X nondisjunction and recombined with an X chromosome in the unc-1-dpy-3 interval at frequencies comparable to that found for X-X recombination; mnDp72(X;IV) also promoted trisomy for chromosome IV. A mutation in him-8 IV was identified that severely reduced recombination between the two X chromosomes in hermaphrodites and between mnDp73 and the X chromosome in males. Recombination between the X chromosome and duplications of either the right end of the X or a region near but not including the left end was rare. We suggest that the X chromosome has one or more elements near its left end that promote meiotic chromosome pairing.     

Molecular analysis of male-viable deletions and duplications allows ordering of 52 DNA probes on proximal Xq.

While performing a systematic search for chromosomal microdeletions in patients with clinically complex X-linked syndromes, we have observed that large male-viable deletions and duplications are clustered in heterochromatic regions of the X chromosome. Apart from the Xp21 band, where numerous deletions have been found that encompass the Duchenne muscular dystrophy gene, an increasing number of deletions and duplications have been observed that span (part of) the Xq21 segment. To refine the molecular and genetic map of this region, we have employed 52 cloned single-copy DNA sequences from the Xcen-q22 segment to characterize two partly overlapping tandem duplications and two interstitial deletions on the proximal long arm of the human X chromosome. Together with a panel of somatic cell hybrids that had been described earlier, these four rearrangements enabled us to order the 52 probes into nine different groups and to narrow the regional assignment of several genes, including those for tapetochoroidal dystrophy and anhidrotic ectodermal dysplasia.     

Duplication of an Xp segment that includes the ZFX locus causes sex inversion in man

Summary Two 46,XY females with tandem duplications of an X short arm segment were studied by cytogenetic and Southern blot analysis. The results show that the duplicated segment in each case included the Xp21.2–Xp22.2 interval, resulting in a double dose of ZFX on the single active X chromosome. The results from our two cases, in conjunction with those reported by other workers, lead us to conclude that the duplication is the reason for the sex inversion. If ZFY and ZFX are indeed sex-determining gene loci, these findings favour a model of sex determination characterized by antagonistic interaction between these genes.     

Fertility,sex determination,and the X chromosome

Because of its function, the X chromosome has a special status in mammalian genomes, with the specific occurrence of genes that influence both female and male fertility. Long ago, the XO karyotype (Turner syndrome) was associated with infertility, proving the correlation between normal X chromosome dosage and normal female fertility. Nevertheless, the search for specific X-borne fertility genes was not completely successful and suggested, instead, that female X-linked fertility, for example, depends upon groups of X-linked genes. Conversely, X-linked hyperfertility has been observed in sheep, where a mutation in BMP15 leads to a hyperfertile phenotype, but only in the heterozygous state. Many male fertility genes map to the X chromosome, consistent with a genetic model developed in the early 1990s. Ironically, NR0B1 (formerly DAX1), once presented as the paradigm of genes responsible for ovarian development and function, is probably one of these male fertility factors and is active in the maintenance of spermatogenesis. Indeed, duplications of this gene on the human X chromosome lead to XY sex reversal, as NR0B1 is able to counterbalance the effect in humans. Nevertheless, invalidation experiments in mice demonstrate the effect of this factor on male germ-cell production.     

Heterokaryon Incompatibility Genes in NEUROSPORA CRASSA Detected Using Duplication-Producing Chromosome Rearrangements

Evidence is presented for five or six previously undetected heterokaryon incompatibility (het) loci, bringing to about ten the number of such genes known in Neurospora crassa. The genes were detected using chromosome duplications (partial diploids), on the basis of properties previously known for het genes in duplications. Duplications homozygous for het genes are usually normal in growth and morphology, whereas those heterozygous are strikingly different. The heterozygotes are inhibited in their initial growth, produce brown pigment on appropriate medium, and later "escape" from their inhibition, as a result of somatic events, to produce wild-type growth. - Five normal-sequence strains were crossed to 14 duplication-producing chromosome rearrangements, and the duplication progeny were examined for properties characteristic of duplications heterozygous for known het genes. Each cross produced duplications for a specific region of the genome, depending on the rearrangement. Normal-sequence strains were wild types from nature, chosen from diverse geographic locations to serve as sources of genetic variation. - The duplication method was very effective. Most of the longer duplications uncovered het genes. The genes are: het-5 (on linkage group IR, in the region covered by duplications produced using rearrangement T (IR LEADS TO VIR)NM103), het-6 (on IIL, covered by T(IIL LEADS TO VI)P2869 and T(IIL LEADS TO IIIR)AR18 duplications), het-7 (tentatively assigned to IIIR, T(IIIR LEADS TO VIL)D305), het-8 (VIL, T(VIL LEADS TO IR)T39M777), het-9 (VIR LEADS TO IVR)AR209), and het-10 (VIIR, T(VIIR LEADS TO IL)5936.     

Genetic and Segregation Analysis of Escherichia coli Strains Containing a Tandem Duplication of the trpD-purB Region of the Chromosome

Genetic and segregation analysis of Escherichia coli strains containing a partial duplication of the trp operon reveal that the 2.5-min-long region trpD-purB is duplicated in tandem in the chromosome. The adjacent loci cysB and fabD are not duplicated. Although one copy of the duplicated region is longer than the maximum size of bacteriophage P1kc transducing fragments, the frequency at which the duplicated segment trpDCBA is transferred by transduction to tonB-trp deletion strains is equal to that observed for transfer of the normal trp operon. This suggests that three-point recombination events believed to account for transduction of long duplications occur as frequently as two-point recombination events believed to account for normal transduction. Cotransduction frequencies of trpDCBA with the duplicated loci tonB, galU, tyrT, and hemA are very similar to those for the trp operon with the same loci. This indicates that normal genetic linkage is maintained during the three-point recombination event. However, purB, which is normally unlinked to trp by transduction, is closely linked to trpDCBA and thus must be near the repeat point of the duplication. Transduction tests with point mutations in the trp operon indicated that the repeat point occurs near the normal boundary between trpE and trpD. Segregation analysis of heterogenotes constructed from tonB-trp deletion strains shows that the frequency at which a marker is lost is approximately proportional to its distance from the repeat point. This finding is consistent with a random, singlesite crossover event during segregation. Several observations indicate that non-reciprocal genetic exchange also occurs between copies of the duplication. Analysis of heterogenotes containing dadR1 and dadR(+) demonstrate that the mutant allele is transdominant.     

Dosage Regulation of the Active X Chromosome in Human Triploid Cells

In mammals, dosage compensation is achieved by doubling expression of X-linked genes in both sexes, together with X inactivation in females. Up-regulation of the active X chromosome may be controlled by DNA sequence–based and/or epigenetic mechanisms that double the X output potentially in response to autosomal factor(s). To determine whether X expression is adjusted depending on ploidy, we used expression arrays to compare X-linked and autosomal gene expression in human triploid cells. While the average X:autosome expression ratio was about 1 in normal diploid cells, this ratio was lower (0.81–0.84) in triploid cells with one active X and higher (1.32–1.4) in triploid cells with two active X''s. Thus, overall X-linked gene expression in triploid cells does not strictly respond to an autosomal factor, nor is it adjusted to achieve a perfect balance. The unbalanced X:autosome expression ratios that we observed could contribute to the abnormal phenotypes associated with triploidy. Absolute autosomal expression levels per gene copy were similar in triploid versus diploid cells, indicating no apparent global effect on autosomal expression. In triploid cells with two active X''s our data support a basic doubling of X-linked gene expression. However, in triploid cells with a single active X, X-linked gene expression is adjusted upward presumably by an epigenetic mechanism that senses the ratio between the number of active X chromosomes and autosomal sets. Such a mechanism may act on a subset of genes whose expression dosage in relation to autosomal expression may be critical. Indeed, we found that there was a range of individual X-linked gene expression in relation to ploidy and that a small subset (∼7%) of genes had expression levels apparently proportional to the number of autosomal sets.     

A complex genetic basis to X-linked hybrid male sterility between two species of house mice

The X chromosome plays a central role in the evolution of reproductive isolation, but few studies have examined the genetic basis of X-linked incompatibilities during the early stages of speciation. We report the results of a large experiment focused on the reciprocal introgression of the X chromosome between two species of house mice, Mus musculus and M. domesticus. Introgression of the M. musculus X chromosome into a wild-derived M. domesticus genetic background produced male-limited sterility, qualitatively consistent with previous experiments using classic inbred strains to represent M. domesticus. The genetic basis of sterility involved a minimum of four X-linked factors. The phenotypic effects of major sterility QTL were largely additive and resulted in complete sterility when combined. No sterility factors were uncovered on the M. domesticus X chromosome. Overall, these results revealed a complex and asymmetric genetic basis to X-linked hybrid male sterility during the early stages of speciation in mice. Combined with data from previous studies, we identify one relatively narrow interval on the M. musculus X chromosome involved in hybrid male sterility. Only a handful of spermatogenic genes are within this region, including one of the most rapidly evolving genes on the mouse X chromosome.     

Determining the order of genes,centromeres, and rearrangement breakpoints inNeurospora by tests of duplication coverage

Nontandem segmental duplications provide a useful alternative to conventional recombination mapping for determining gene order in a haploid organism such asNeurospora. When an insertional or terminal rearrangement is crossed by Normal sequence, a class of progeny is produced that have a precisely delimited chromosome segment duplicated. In such Duplication progeny, a recessive gene in the Normal-sequence donor chromosome may or may not be masked (“covered”) by its dominant wild-type allele in the translocation-sequence recipient chromosome. Coverage depends upon whether the gene in question is left or right of the rearrangement breakpoint. The recessive gene will be heterozygous and covered (not expressed) if its locus is within the duplicated segment, but it will be haploid and expressed if the locus is outside the segment. Not only genes but also centromeres can be mapped by means of duplications, because genes included in. the same viable duplication must reside in the same chromosome arm. - Numerous sequences in the current genetic maps ofN. crassa have been determined using duplications. Gene order in the albino region and in the centromere region of linkage group I provide examples. Over 50 insertional or terminal rearrangements are available from which nontandem duplications of defined content can be obtained at will; collectively these cover about 75% of the genome. - Intercrosses between partially overlapping chromosome rearrangements also produce Duplication progeny containing two copies of regions between the breakpoints. The 180 mapped reciprocal translocations and inversions include numerous overlapping combinations that can be used for duplication mapping.