'Exceptional sons' from Drosophila melanogaster mothers carrying a balancer X chromosome

This study reports on exceptional males which are obtained by using Drosophila melanogaster mothers carrying the balancers In(1)FM6 or In(1)FM7 as one of their X chromosomes. The phenomenon was first observed in interspecific crosses between D. melanogaster females and males of its closest relatives which normally produce unisexual female hybrid progeny. Whereas hybrid sons from these crosses die as third instar larvae, the presence of the particular X balancers in the mother allows a low percentage of sons to survive. Similar sterile males are also observed among non-hybrid flies. Data are presented which suggest that the males thus generated could be hyperploid for part of their X chromosome as a result of a meiotic event in their mothers or else they could start life as female zygotes and change sex through a mitotic event at an early stage.     

Analysis of linkage relationships between genetic markers around the fragile X locus with special reference to the daughters of normal transmitting males

Summary Genetic linkage data from loci around the fragile X locus at Xq27.3 are analysed in the light of the hypothesis of Pembrey et al. (1985) concerning the generation of the fragile X mutation. Recombination between the four loci 52A, F9, fragile X, and ST14 is significantly decreased in meioses giving rise to the affected grandsons of normal transmitting males, when compared to families where there are no apparent normal transmitting males. There are at least two possible explanations for this phenomenon. Either the established fragile site at Xq27.3 promotes increased recombination in the distal part of the X chromosome as a secondary event, unrelated to the mechanism of formation of the fragile site itself, or an event involving recombination at or around Xq27.3 is the mechanism of formation of the full fragile X mutation, and the decreased recombination seen amongst flanking marker loci in meioses giving rise to the affected grandsons of normal transmitting males is the result of interference.     

Cytogenetic manifestations associated with the reversion, by gene amplification, at the HGPRT locus in V79 Chinese hamster cells

Some HGPRT spontaneous revertants were isolated from a mutant line (E2) of V79 Chinese hamster cells and phenotypically characterized. Dot-Blot hybridization with a 32P-labelled HGPRT probe revealed an increase in the number of HGPRT sequences in some of these revertants, suggesting the occurrence of gene amplification. Cytogenetic analysis performed in three of these revertants showed a characteristic abnormally banding region (ABR) on the elongated p arm of the X chromosome. In situ hybridization in one revertant (RHE2) showed that the amplified sequences reside on the p+ arm of the X chromosome in two different localizations. Because of the very probably clonal origin of the revertant, these features indicate that the amplified sequences might rearrange after their integration into the chromosome.     

Population genetic consequences of the fragile-X syndrome, based on the X-inactivation imprinting model.

We have examined the population genetic consequences of the model of Laird (Genetics 117:587-599, 1987) in which the fragile-X syndrome is caused by "imprinting" of a mutant chromosome. The imprinting event in this model results from a block to reactivation of an inactive X chromosome prior to oogenesis. If it is assumed that males carrying the imprinted chromosome never reproduce, the frequencies of females and males carrying the imprinted chromosome are expected to be equal. When a mutation-selection balance is established, there are expected to be somewhat more than twice as many females carrying the nonimprinted fragile X as carry the imprinted fragile-X chromosome, the excess depending on the fertility of fragile-X females. Nonpenetrant (transmitting) males, i.e., those with the nonimprinted fragile-X chromosome, are expected to be present at about the same frequency as are males with the syndrome. More than one-third of the nonimprinted chromosomes in the population are expected to be newly arisen in each generation. We have considered possible alternatives to the model of a mutation-selection balance. Nonimprinted carrier females would need to have 100% fertility excess to avoid postulating a high mutation rate to account for the very high prevalence of the syndrome.     

Spontaneous fusion in vivo between normal host and tumor cells: possible contribution to tumor progression and metastasis studied with a lectin-resistant mutant tumor.

Previous studies demonstrated that growth in DBA/2 mice of MDW4, a wheat germ agglutinin-resistant (WGAr) mutant of the highly metastatic MDAY-D2 DBA/2 mouse tumor, led to the emergence of WGA-sensitive (WGAs) revertants having higher ploidy levels at the site of inoculation as well as at distant visceral metastases. The results implied that MDW4 was nonmetastatic but progressed to become metastatic in vivo only after a cellular change took place which was accompanied by extinction of the WGAr phenotype and acquisition of a higher number of chromosomes. Results presented here provide strong and direct evidence for the underlying mechanism being spontaneous cell fusion in vivo between the MDW4 (WGAr) tumor cells and normal host cells, at least some of which are of bone marrow origin. Thus, growth of the H-2d MDW4 tumor cells in (C3H X DBA/2)F1 (H-2k X H-2d) or (C57BL/6 X DBA/2)F1 (H-2b X H-2d) mice led to the appearance of WGAs revertants bearing the H-2k or H-2b major histocompatibility complex antigens associated with the C3H or C57BL/6 parental strains, respectively. Similarly, WGAs revertants of MDW4 were found to express H-2k antigens after growth in CBA/HT6T6 (H-2k) leads to DBA/2 bone marrow radiation chimeras. Attempts to mimic the in vivo hybridization process were successful in that in vitro somatic cell fusion between an ouabain-resistant (OuaR), 6-thioguanine-resistant (Thgr) derivative of the MDW4 mutant and either normal bone marrow or spleen cells resulted in loss of the WGAr phenotype in the hybrids (thus showing its recessive character) and increased malignant properties in vivo. An analysis of spontaneous frequencies of re-expression of various drug resistance genetic markers in several hybrid metastatic cells was also consistent with chromosome segregation of the sensitive alleles. The results show that tumor progression and the emergence of metastatic cell variants could arise as a consequence of tumor X host cell fusion followed by chromosome segregation. We also discuss the possibility that this type of event may normally be a very rare one during the growth of tumors, the frequency of which can be artificially amplified by the use of certain classes of lectin-resistant mutants carrying particular cell surface alterations.     

Magnification of the Ribosomal Genes in Female DROSOPHILA MELANOGASTER

The genetically induced increase in the number of 18S + 28S ribosomal genes known as magnification has been reported to occur in male Drosophila but has not previously been observed in females. We now report that bobbed magnified (bbm) is recovered in progeny of female Drosophila carrying three different X bobbed (Xbb) chromosomes and the helper XYbb chromosome, which is a derivative of the Ybb- chromosome. Using different combinations of bb or bb+ X and Y chromosomes, we show that magnification in females requires both a deficiency in ribosomal genes and the presence of a Y chromosome: X/X females that are rDNA-deficient but do not carry a Y chromosome do not produce bbm; similarly, X/X/Y females that carry a Y chromosome but are not rDNA-deficient do not produce bbm. Bobbed magnified is only recovered from rDNA-deficient X/XY, X/X/Y or XX/Y females. We have also found that females carrying a ring Xbb chromosome together with the XYbb- chromosome do not produce bbm, indicating that ring X chromosomes are inhibited to magnify in females as in males. We postulate that the requirement for a Y chromosome is due to sequences on the Y chromosome that regulate or encode factor(s) required for magnification, or alternatively, affect pairing of the ribosomal genes.--These studies demonstrate that magnification is not limited to males but also occurs in females. Magnification in females is induced by rDNA-deficient conditions and the presence of a Y chromosome, and probably occurs by a mechanism similar to that in males.     

Heteromorphic X chromosomes in 46,XX males: Evidence for the involvement of X-Y interchange

Summary G- and R-banded chromosome preparations from eight of twelve 46,XX males, with no evidence of mosaicism or a free Y chromosome, were distinguished in blind trials from preparations from normal 46,XX females by virtue of heteromorphism of the short arm of one X chromosome. Photographic measurements on X chromosomes and on chromosome pair 7 in cells from twelve 46,XX males, eight 46,XX females, and four 46,XY males revealed a significant increase in the size of the p arm of one X chromosome in the group of XX males, independently characterised as being heteromorphic for Xp. No such differences were observed between X chromosomes of normal males and females or between homologues of chromosome pair 7 in all groups. The heteromorphism in XX males is a consequence of an alteration in shape (banding profile) and length of the tip of the short arm of one X chromosome, and the difference in size of the two Xp arms in these 46,XXp+ males ranged from 0.4% to 22.9%. From various considerations, including the demonstration of a Y-specific DNA fragment in DNA digests from nuclei of one of three XX males tested, it is concluded that the Xp+ chromosome is a product of Xp-Yp exchange. These exchanges are assumed to originate at meiosis in the male parent and may involve an exchange of different amounts of material. The consequences of such unequal exchange are considered in terms of the inheritance of genes located on Yp and distal Xp. No obvious phenotypic difference was associated with the presence or absence of Xp+. Thus, some males diagnosed as 46,XX are mosaic for a cryptic Y-containing cell line, and there is now excellent evidence that maleness in others may be a consequence of an autosomal recessive gene. The present data imply that in around 70% of 46,XX males, maleness is a consequence of the inheritance of a paternal X-Y interchange product.     

Genomic imprinting: male mice with uniparentally derived sex chromosomes

Although it has been known that there is an X-chromosome imprinting effect during early embryogenesis in female mammals, it remains unknown if parental origin of the X chromosome has an effect in males. Furthermore, it has not been possible to produce animals with normal sex chromosomes of uniparental origin to further evaluate such imprinting effects. We have devised a breeding scheme to produce male mice, designated XPYP males, in which both the X and Y chromosomes are paternally inherited. To our knowledge, these are the first mammals produced that have a normal sex chromosome constitution but with both sex chromosomes derived from one parent. Development and reproduction in these XPYP males and the sex ratio and chromosome constitution of their offspring appeared normal; thus there is no apparent effect in males of having both sex chromosomes derive from one parent or of having the X chromosome derived from an inappropriate parent. Although we have detected no X-chromosome imprinting effect in these males, evidence from other sources suggest that the X chromosome is parentally imprinted. Thus detection and definition of an imprint can depend on the assay used.     

Sex ratio and testis size in mice carrying Sxr and T(X;16)16H

Mice heterozygous for the T(X;16)16H translocation and carrying Sxr on their normal (inactive) X chromosome (ie, T16H/X Sxr individuals) may develop as males, females, or hermaphrodites. The proportion of males varied from 22% to 65% depending on the source of the normal X chromosome. A model is proposed, according to which relatively small variations in the spreading of inactivation from the X chromosome into the attached Sxr fragment produce large changes in the proportion of males. Testis weight in T16H/X Sxr males was found to be significantly smaller than in X/X Sxr males, irrespective of the source of the normal X chromosome.     

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.     

A Two-Stage Model for the Control of rDNA Magnification

Males of the genotype bb/Ybb- have been shown to produce both magnified (bbm+) and, less frequently, reduced (bbrl) X chromosomes. An analysis of the progeny of single magnifying bb/Ybb- males reveals that bbm+ revertants may be recovered either as rare single events or, more frequently, in large clusters. To analyze the role of the bb phenotype in the induction of rDNA magnification we have constructed a series of bb and bb+ derivatives of Ybb-. Males carrying an X chromosomal bb allele and one of these derivatives (bb/bbYbb- or bb/bb+Ybb-) produce small numbers (one to two) of bbm+ progeny at a frequency similar to that observed for bb/Ybb- males but do not produce large clusters of bbm+ revertants. In addition, bb/bb+Ybb- males produce essentially equal numbers of magnified (bbm+) and reduced (bbrl) X chromosomes. These data, together with a consideration of the growth properties of the male germline in Drosophila, suggest that magnification/reduction may occur at two different times during development. Those events that give rise to large clusters, and, thus, necessarily arise early in germ cell development, appear to be dependent on the bb phenotype. However, those events that give rise to single bbm+ chromosomes arise late in spermatogenesis, probably at meiosis, and are independent of the bb phenotype.     

Sex Determination in Polyploids of Caenorhabditis Elegans

In Caenorhabditis elegans triploid animals with two X chromosomes (symbolized 3A;2X) are males. However, these triploid males can be feminized by making them mutant for recessive dosage compensation mutations, by adding X chromosome duplications or by microinjecting particular DNA sequences termed feminizing elements. None of these treatments affects diploid males. This study explores several aspects of these treatments in polyploids. The dosage compensation mutants exhibit a strong maternal effect, such that reduction of any of the dosage compensation gene functions in the mother leads to sex reversal of 3A;2X animals. Likewise, all X chromosome duplications tested cause both sex reversal and intersexual development of many 3A;2X animals. Microinjected feminizing element DNA does not cause extensive sex reversal, but does result in intersexual development in 3A;2X animals. Neither X chromosome duplications nor microinjected feminizing elements show the extreme maternal effect of the dosage compensation mutants, although there is indirect evidence for a maternal effect of the feminizing elements. In particular, very little feminizing element DNA needs to be microinjected in order to feminize triploid males, far less than what is needed for stable inheritance, implying that feminizing elements can work within the mother's gonad. However, even very high concentrations of microinjected feminizing elements do not affect sex determination in diploid males, suggesting that they are not part of the numerator of the X/A ratio. In addition, no pair of X chromosome duplications feminizes diploid males, suggesting that none of these duplications contains a numerator of the X/A ratio. Instead, I infer that an X-linked locus, as yet undefined, must be present in two copies for hermaphrodite development to ensue or that the two X chromosomes might interact.     

Reduced X-linked rare polymorphism in males in comparison to females of Drosophila melanogaster

Natural selection is assumed to act more strongly on X-linked loci than on autosomal loci because the fitness effect of a recessive mutation on the X chromosome is fully expressed in hemizygous males. Therefore, selection is expected to fix or remove recessive mutations on the X chromosome more efficiently than those on autosomes. However, the assumption that hemizygosity of the X chromosome selectively accelerates changes in allele frequency has not been confirmed directly. To examine this assumption, we investigated current natural selection on X-linked chemoreceptor genes in a natural population of Drosophila melanogaster by comparing nucleotide diversity, linkage disequilibrium (LD), and departure from the neutrality in 4 chemoreceptor genes on 100 X chromosomes each from female and male flies. The general pattern of nucleotide diversity and LD for the genes investigated was similar in females and males. In contrast, males harbored significantly fewer rare polymorphisms defined as singletons and doubletons. When all the gene sequences were concatenated, Tajima's D showed a significant departure from the neutrality in both females and males, whereas Fu and Li's F* value revealed departure only in males. These results suggest that some rare polymorphisms on the X chromosome from females are recessively deleterious and are removed by stronger purifying selection when transferred to hemizygous males.     

The contribution of the y chromosome to hybrid male sterility in house mice

Hybrid sterility in the heterogametic sex is a common feature of speciation in animals. In house mice, the contribution of the Mus musculus musculus X chromosome to hybrid male sterility is large. It is not known, however, whether F(1) male sterility is caused by X-Y or X-autosome incompatibilities or a combination of both. We investigated the contribution of the M. musculus domesticus Y chromosome to hybrid male sterility in a cross between wild-derived strains in which males with a M. m. musculus X chromosome and M. m. domesticus Y chromosome are partially sterile, while males from the reciprocal cross are reproductively normal. We used eight X introgression lines to combine different X chromosome genotypes with different Y chromosomes on an F(1) autosomal background, and we measured a suite of male reproductive traits. Reproductive deficits were observed in most F(1) males, regardless of Y chromosome genotype. Nonetheless, we found evidence for a negative interaction between the M. m. domesticus Y and an interval on the M. m. musculus X that resulted in abnormal sperm morphology. Therefore, although F(1) male sterility appears to be caused mainly by X-autosome incompatibilities, X-Y incompatibilities contribute to some aspects of sterility.     

X chromosome inactivation and micronuclei in normal and Turner individuals

Studies on aneuploidy have shown that the X is the most frequently lost chromosome in females, and that the number of X chromosome-positive micronuclei increases with age in women. Recently, we showed that the inactive X chromosome is incorporated preferentially in micronuclei. The objectives of the current study were, firstly, to determine the incidence of X chromosome incorporation into micronuclei in males and, secondly, to determine the incidence of X chromosome incorporation into micronuclei of females with Turner syndrome. Blood samples were obtained from 18 male newborns and 35 normal adult males ranging in age from 22 to 79 years and from seven women with non-mosaic Turner syndrome aged 11–39 years. Isolated lymphocytes were cultured in the presence of cytochalasin B and 2000 binucleated cells per subject were scored for micronuclei. Cells were then hybridized with the biotinylated X centromere-specific probe, pBamX7, and visualized with fluorescein-conjugated avidin. All micronucleated cells were relocated and evaluated for the presence or absence of the X chromosome. Of the 335 micronuclei observed, 6.6% (22/335) contained an X chromosome. Analysis of variance shows a statistically significant increase, for both males and Turner females, in the number of X chromosome-positive micronuclei with age (P < 0.001). These data also show that the X chromosome is included in micronuclei from males more often than would be expected by chance (P < 0.005; χ² analysis, 15 df). Here we show that there is a tenfold difference in the frequency of X chromosome-positive micronuclei in 46,XX females compared to 46,XY males and 45,X females, providing further support to our previous finding that the X chromosome in micronuclei is the inactive chromosome. Received: 29 April 1997 / Accepted: 9 May 1997     

Tsix-deficient X chromosome does not undergo inactivation in the embryonic lineage in males: implications for Tsix-independent silencing of Xist

Differential induction of the X-linked non-coding Xist gene is a key event in the process of X inactivation occurring in female mammalian embryos. Xist is negatively regulated in cis by its antisense gene Tsix through modification of the chromatin structure. The maternal Xist allele, which is normally silent in the extraembryonic lineages, is ectopically activated when Tsix is disrupted on the same chromosome, and subsequently the maternal X chromosome undergoes inactivation in the extraembryonic lineages even in males. However, it is still unknown whether the single Tsix-deficient X chromosome (XDeltaTsix) in males is also inactivated in the embryonic lineage. Here, we show that both male and female embryos carrying a maternally derived XDeltaTsix could survive if the extraembryonic tissues were complemented by wild-type tetraploid cells. In addition, Xist on the XDeltaTsix was properly silenced and methylated at CpG sites in adult male somatic cells. These results indicate that the embryonic lethality caused by the maternal XDeltaTsix is solely attributable to the defects in the extraembryonic lineages. XDeltaTsix does not seem to undergo inactivation in the embryonic lineage in males, suggesting the presence of a Tsix-independent silencing mechanism for Xist in the embryonic lineage.     

Male-Sex-Ratio Trait in Drosophila Pseudoobscura: Frequency of Autosomal Aneuploid Sperm

Males with the SR X chromosome show the "sex-ratio" (sr) phenotype in which they produce almost entirely daughters. The few sons (about 1%) are invariably sterile X/O males and result entirely from nullo-XY sperm. The "male-sex-ratio" (msr) phenotype is a modified form of sr in which SR/Y males produce a higher frequency of sterile X/O sons. The msr trait is due to the presence of the SR X-chromosome in males which are also homozygous for one or more autosomes from the L116 strain. Here the frequency of nullo-3 and diplo-3 sperm from msr males was measured by crossing to a compound-3 strain and found to be 13.8% and 3.2%, respectively, of the total viable sperm. The sr males produced very low levels of nullo-3 sperm at a frequency not different from control X/Y males and a slightly elevated frequency of diplo-3 sperm over X/Y males. The msr males were found to have only 12% the fecundity of sr males and in matings to cause a high frequency of brown inviable eggs. These results indicate that high rates of autosomal aneuploidy are not restricted to chromosome 3 but also occur for chromosomes 2, 4 and 5. The overall frequency of autosomal aneuploid sperm is estimated to be approximately 50%. Microscopic studies of meiosis in testes from msr males indicates meiotic nondisjunction and meiotic chromosome loss are responsible for the msr phenotype. Last, microscopic studies of sperm cysts from msr males reveal high levels of spermiogenic failure.