Showing papers on "Dosage compensation published in 1988"

Evidence that sisterless-a and sisterless-b are two of several discrete "numerator elements" of the X/A sex determination signal in Drosophila that switch Sxl between two alternative stable expression states.

Abstract: The primary signal for Drosophila sex determination is the number of X chromosomes relative to the number of sets of autosomes. The present report shows that the numerator of this X/A signal appears to be determined by the cumulative dose of a relatively limited number of discrete X-linked genetic elements, two of which are sisterless-a and sisterless-b. This discovery regarding the nature of the sex determination signal grew out of previous studies of both the likely X/A signal target (the feminizing switch gene, Sex-lethal) and two positive regulators of that target gene (sis-a and daughterless). Combinations of genetic perturbations in these three genes had been shown to have synergistic effects. A model proposed in part to account for these interactions generated a large variety of strong predictions for sex-specific synergistic interactions that would be diagnostic for X/A numerator elements and could distinguish them from other components of the sex determination system. All these predictions, as well as other predictions for X/A numerator elements, are shown here to be fulfilled. The most compelling observations involve sexually reciprocal viability effects of duplications of wild-type genes: combinations of sis-a(+), sis-b(+) and/or Sxl(+) duplications are lethal to males but rescue females from the otherwise lethal effects of changes in other components of the sex determination machinery. The many interactions described here illustrate an important principle that may seem counter-intuitive: perturbations of the sex determination signal for Drosophila generally will not appear to affect adult sexual phenotype. This principle follows from the fact that Sxl is involved in dosage compensation as well as sex determination, and from important aspects of the nature and timing of Sxl's regulation both by the X/A signal and by Sxl's own products (positive autoregulation). These factors mask potential effects on adult sexual differentiation by causing the premature death of cells and/or individuals. The fact that the vast array of results presented here conform to this principle is strong evidence in favor of a ``binary state'' model for Sxl regulation by the X/A signal. This model is favored over an alternative ``multiple state'' hypothesis that was proposed by others in a different study of the X/A signal. In that same study it was concluded that region 3E8-4F11 of the X chromosome contained especially potent X/A numerator elements. Here the behavior of sis genes is shown to differ from that of genes in region 3E8-4F11 in nearly every important respect. It now seems that this region cannot contain X/A numerator elements. Evidence presented here does indicate, however, that other X/A numerator elements besides sis-a and sis-b must exist. The nature of the interactions between genetic components of the sex determination machinery should make identification of these other elements relatively straightforward.

The William Allan memorial award address: X-chromosome inactivation and the location and expression of X-linked genes.

Abstract: Genes lies au chromosome X: identite et localisation, expression, inactivation, reactivation

The maternal sex determination gene daughterless has zygotic activity necessary for the formation of peripheral neurons in Drosophila.

Abstract: The daughterless (da) gene is known to have separate maternal and zygotic functions: Maternally supplied daughterless activity is required for proper sex determination and dosage compensation in female embryos, whereas loss of zygotically supplied da+ activity causes embryonic lethality in both male and female embryos. We have found that the zygotic da+ activity is necessary for neural development: The use of neuron-specific antibodies and beta-galactosidase-marked X chromosomes has revealed that in both male and female embryos deletions or strong mutations of the da gene remove all peripheral neurons and associated sensory structures without disrupting the epithelium from which they derive. Partial da+ function causes partial removal of peripheral neurons. Our results indicate that da+ is required for the formation of peripheral neurons and their associated sensory structures.

Molecular characterization of daughterless, a Drosophila sex determination gene with multiple roles in development.

Abstract: The daughterless (da) gene in Drosophila acts both maternally and zygotically to provide essential functions during development. Maternal da+ expression is required by embryos for the regulation of sex determination and dosage compensation. Zygotic da+ expression is required throughout development--early for the formation of the peripheral nervous system and perhaps for the proper functioning of genes in heterochromatic regions of the genome; during larval stages for growth and differentiation of the future adult epidermis; and in the somatic part of the gonad of adult females for proper egg membrane synthesis. Here we describe the cloning of da by the transposon tagging approach as well as some aspects of the molecular characterization of wild-type and mutant alleles. Despite the multiple developmental roles of da, the organization and expression of this gene appear relatively simple at this level of analysis. The gene codes for two transcripts, present in both sexes and at all stages of development. The nucleotide sequence of a nearly full-length cDNA predicts a protein product of 710 amino acids that shares sequence similarity with the His-Pro repeat of the Drosophila genes bicoid and paired. Two partial-loss-of-function da mutations (one of which is temperature sensitive) appear to be caused by DNA insertions in the 5'-untranslated region of the gene.

Nonrandom X chromosome inactivation in B cells from carriers of X chromosome-linked severe combined immunodeficiency

Abstract: X chromosome-linked severe combined immunodeficiency (XSCID) is characterized by markedly reduced numbers of T cells, the absence of proliferative responses to mitogens, and hypogammaglobulinemia but normal or elevated numbers of B cells. To determine if the failure of the B cells to produce immunoglobulin might be due to expression of the XSCID gene defect in B-lineage cells as well as T cells, we analyzed patterns of X chromosome inactivation in B cells from nine obligate carriers of this disorder. A series of somatic cell hybrids that selectively retained the active X chromosome was produced from Epstein-Barr virus-stimulated B cells from each woman. To distinguish between the two X chromosomes, the hybrids from each woman were analyzed using an X-linked restriction fragment length polymorphism for which the woman in question was heterozygous. In all obligate carriers of XSCID, the B-cell hybrids demonstrated preferential use of a single X chromosome, the nonmutant X, as the active X. To determine if the small number of B-cell hybrids that contained the mutant X were derived from an immature subset of B cells, lymphocytes from three carriers were separated into surface IgM positive and surface IgM negative B cells prior to exposure to Epstein-Barr virus and production of B-cell hybrids. The results demonstrated normal random X chromosome inactivation in B-cell hybrids derived from the less mature surface IgM positive B cells. In contrast, the pattern of X chromosome inactivation in the surface IgM negative B cells, which had undergone further replication and differentiation, was significantly nonrandom in all three experiments [logarithm of odds (lod) score greater than 3.0]. These results suggest that the XSCID gene product has a direct effect on B cells as well as T cells and is required during B-cell maturation.

Genetic evidence that the sans fille locus is involved in Drosophila sex determination.

Abstract: Females homozygous for sans fille1621 (= fs(1)1621) have an abnormal germ line. Instead of producing eggs, the germ-line cells proliferate forming ovarian tumors or excessive numbers of nurse cells. The Sex-lethal gene product(s) regulate the branch point of the dosage compensation and sex determination pathways in the soma. The role of Sex-lethal in the germ line is not clear but the germ line of females homozygous for female sterile Sex-lethal alleles or germ-line clones of loss-of-function alleles are characterized by ovarian tumors. Females heterozygous for sans fille1621 or Sex-lethal are phenotypically wild type with respect to viability and fertility but females trans-heterozygous for sans fille1621 and Sex-lethal show ovarian tumors, somatic sexual transformations, and greatly reduced viability.

Sex determination in Drosophila: the X-chromosomal gene liz is required for Sxl activity.

Abstract: In Drosophila, females require products of the gene Sxl for sex determination, dosage compensation and fertility. I show here that the X-chromosomal gene liz, located in 4F1 to 4F11 and previously called fs(1)1621, provides maternal and zygotic functions necessary for Sxl activity in germ line and soma. In XX animals, the mutation SxlM1 which was reported to express the female-specific functions of Sxl constitutively can rescue all phenotypes resulting from lack of liz product. XY animals carrying SxlM1 and lacking maternal or zygotic liz activity survive as males with some female traits. A stock was constructed in which the females are liz SxlM1/liz SxlM1 and males liz SxlM1/Y. This shows that SxlM1 is not truly expressed constitutively in animals with an X:A ratio of 0.5, but requires activity of liz for initiation or maintenance.

Effect of ageing on reactivation of the human X-linked HPRT locus.

Abstract: In mammals, X-chromosome dosage compensation is achieved by inactivating one X chromosome in female cells1. To test the hypothesis2 that genes on the silent X chromosome reactivate as a consequence of ageing, we examined the X-linked hypoxanthine phosphoribosyltransferase (HPRT) locus in 41 women who are heterozygous for mutations at this locus, leading to severe deficiency of the enzyme (Lesch-Nyhan syndrome3). We find that heterozygotes who are more than 10 yr old have an excess of HPRT+ skin fibroblast clones (59% rather than the 50% expected as a consequence of random X inactivation) but this excess does not increase with age. Further studies of eight of these heterozygotes show that the silent locus does not detectably reactivate spontaneously in culture, but only in response to treatment with 5-aza-2-deoxycytidine, a potent inhibitor of methylation. There is no age difference in the frequency of this reactivation as assayed by HATr clones, and a more sensitive autoradiographic assay shows only a twofold difference between young and old heterozy-gotes. Thus, age-related reactivation is not a feature of all X-linked loci, and may have species, tissue and locus-specific determinants.

Comparison of the structure and cell cycle expression of mRNAs encoded by two histone H3-H4 loci in Saccharomyces cerevisiae.

Abstract: The haploid genome of Saccharomyces cerevisiae contains two nonallelic sets of histone H3 and H4 gene pairs, termed the copy I and copy II loci. The structures of the mRNA transcripts from each of these four genes were examined by nuclease protection and primer extension mapping. For each gene, several species of mRNAs were identified that differed in the lengths of their 5' and 3' untranslated regions. The cell cycle accumulation pattern of the H3 and H4 mRNAs was determined in cells from early-exponential-growth cultures fractionated by centrifugal elutriation. The RNA transcripts from all four genes were regulated with the cell division cycle, and transcripts from the nonallelic gene copies showed tight temporal coordination. Cell cycle regulation did not depend on selection of a particular histone mRNA transcript since the ratio of the multiple species from each gene remained the same across the division cycle. Quantitative measurements showed significant differences in the amounts of mRNA expressed from the two nonallelic gene sets. The mRNAs from the copy II H3 and H4 genes were five to seven times more abundant than the mRNAs from the copy I genes. There was no dosage compensation in the steady-state levels of mRNA when either set of genes was deleted. In particular, there was no increase in the amount of copy I H3 or H4 transcripts in cells in which the high-abundance copy II genes were deleted.

Genomic organization in Caenorhabditis elegans: deficiency mapping on linkage group V (left)

Abstract: In this study we genetically analyse a large autosomal region (23 map units) in Caenorhabditis elegans. The region comprises the left half of linkage group V [LGV(left)] and is recombinationally balanced by the translocation eT1(III; V). We have used rearrangement breakpoints to subdivide the region from the left end of LGV to daf-11 into a set of 23 major zones. Twenty of these zones are balanced by eT1. To establish the zones we examined a total of 110 recessive lethal mutations derived from a variety of screening protocols. The mutations identified 12 deficiencies, 1 duplication, as well as 98 mutations that fell into 59 complementation groups, significantly increasing the number of available genetic sites on LGV. Twenty-six of the latter had more than 1 mutant allele. Significant differences were observed among the alleles of only 6 genes, 3 of which have at least one ‘visible’ allele. Several deficiencies and 3 alleles of let-336 were demonstrated to affect recombination. The duplication identified in this study is sDp30(V;X). Lethal mutations covered by sDp30 were not suppressed uniformly in hermaphrodites. The basis for this non-uniformity may be related to the mechanism of X chromosome dosage compensation in C. elegans.

Cytogenetics of the Hessian Fly: I. Mitotic Karyotype Analysis and Polytene Chromosome Correlations

Abstract: The somatic haploid chromosome set of the Hessian fly, Mayetiola destructor (Say),consists of two autosomes and two sex chromosomes. Female somatic cells have pairs of each of these chromosomes (2n = 8). Male somatic cells have two pairs of autosomes and two monosomic sex chromosomes. C-banding revealed pericentromeric heterochromatin on one arm of each chromosome. Mitotic chromosomes were karyotyped using relative lengths, centromeric positions, presence of secondary constrictions, and position and length of C-positive regions. Silver nitrate stained pericentromeric, heterochromatic regions on all four chromosomes but did not stain the entire C-positive regions. In female larvae, four distinct polytene chromosomes were found in the basal region cells of the salivary glands. In male larvae, two distinct and two diffuse polytene chromosomes were found in basal region cells. Differential polytene chromosome morphology in male and female larvae, polytene chromosome indexes, and the position of the nucleolus were used to establish correlations between mitotic and polytene chromosomes. The diffuse morphology of the polytene sex chromosomes in male larvae was probably related to dosage compensation. The number of sex chromosomes in male and female larvae is discussed in relation to sex determination and the production of monogenous progenies.

Comparative study of DNase I sensitivity at the X-linked human HPRT locus.

Abstract: To examine the association between chromatin structure and gene expression at the human hypoxanthine phosphoribosyltransferase (HPRT) locus, DNase I sensitivity of active and inactive genes was analyzed. In a set of human-hamster hybrid lines containing either an active or an inactive human X chromosome, or a derivative of the latter in which the HPRT gene was reactivated by 5-azacytidine treatment, only the promoter region of the gene was found to contain a hypersensitive domain, and its presence was strictly correlated with gene activity. An S1 nuclease-sensitive site was mapped upstream from the DNase I hypersensitive domain using supercoiled plasmids. The overall level of DNase I sensitivity in the interior of the HPRT gene was also assessed by comparing the degradation of polymorphic restriction fragments on active and inactive alleles in both polyclonal and monoclonal lines of female human cells. In these internally controlled experiments, the active X chromosome was found to be approximately twofold more susceptible to DNase I digestion than the inactive X chromosome.

The relationship between position and expression of genes on the kangaroo X chromosome suggests a tissue-specific spread of inactivation from a single control site.

Abstract: In marsupials, X chromosome inactivation is paternal and incomplete. The tissue-specific pattern of inactivation of X-linked loci (G6PD, PGK, GLA) has been attributed to a piecemeal inactivation of different regions of the X. We here propose an alternative hypothesis, in which inactivation of the marsupial X is a chromosome-wide event, but is differentially regulated in different tissues. This hypothesis was suggested by the relationship between the positions and activity of genes on the kangaroo paternal X. In the absence of an HPRT polymorphism, we have used somatic cell hybridization to assess the activity of the paternal HPRT allele in lymphocytes and fibroblasts. The absence of the paternal X, and of the paternal forms of G6PD or PGK, from 33 cell hybrids made by fusing HPRT-deficient rodent cells with lymphocytes or fibroblasts of heterozygous females, suggests that the HPRT gene on the paternal X is inactive in both tissues and therefore not selectable. Since HPRT is located medially on the Xq near GLA, which shares the same characteristics of activity, we suggest that the locus-specific and tissue-specific patterns of activity result from a differential spread of inactivation from a single control locus, located near HPRT and GLA, outwards in both directions to G6PD and PGK. The nucleolus organizer region on the short arm does not seem to be part of the inactivated unit.

Genetics of an Unstable White Mutant in Drosophila Simulans: Reversion, Suppression and Somatic Instability

Abstract: A spontaneous white mutation, white-milky (wmky) of Drosophila simulans is moderately unstable and is associated with a 16-kb long DNA insertion into the white gene. wmky, which is an unstable mutation found in D. simulans, has been genetically analyzed. Among nine spontaneous, partial reversions toward wild type, five were white locus mutations. They are phenotypically different from each other and three show eye color sexual dimorphism indicating a failure of the dosage compensation mechanism. Two w locus mutations whose eye color appeared identical between males and females were also isolated. Of the other back-mutants, three were associated with a recessive suppressor of wmky and one was a semidominant suppressor. These suppressor loci are located on the third chromosome at map positions about 90 and 120, respectively. The suppressor mutations demonstrate specific effects on w locus mutations derived from wmky which lack in the gene dosage compensation. Somatic instability was detected at the frequency of 5.6 X 10(-4) in wmky flies heterozygous for the recessive suppressor and the frequency was increased 10-fold when the suppressor mutation was placed in a different genetic background.

X Chromosome Duplications Affect a Region of the Chromosome They Do Not Duplicate in Caenorhabditis elegans

Abstract: X chromosome duplications have been used previously to vary the dose of specific regions of the X chromosome to study dosage compensation and sex determination in Caenorhabditis elegans. We show here that duplications suppress and X-linked hypomorphic mutation and elevate the level of activity of an X-linked enzyme, although these two genes are located in a region of the X chromosome that is not duplicated. The effects do not depend on the region of the X chromosome duplicated and is stronger in strains with two doses of a duplication than in strains with one dose. This is evidence for a general elevation of X-linked gene expression in strains carrying X-chromosome duplications, consistent with the hypothesis that the duplications titrate a repressor acting on many X-linked genes.

Developmental regulation of the rosy locus in Drosophila melanogaster.

Abstract: The rosy locus of Drosophila melanogaster encodes the enzyme xanthine dehydrogenase (XDH, xanthine: NAD oxidoreductase). The most thoroughly studied gene-enzyme system in higher eukaryotes (Sang, 1984), and long a paradigm for eukaryotic gene organization, the rosy locus has recently become a fertile system for analysis of eukaryotic gene regulation. The major reasons for this are: 1 The locus encodes an enzyme (XDH) that has been isolated and characterized and is easily assayed in extracts of small numbers of whole organisms or isolated tissues (see Sections 3 and 6). 2 The locus has been subject to intensive fine-structure genetic analyses that have defined its limits and organization (see Sections 4 and 7). 3 Effective phenotypic selection and screening protocols are available (see Section 2), taking advantage of conditional lethality at the locus, and resulting in the isolation of a wealth of mutants, including regulatory variants (see Section 7). 4 It is clear that expression of rosy is regulated at both the stage and tissue levels during development (see Section 6); mutants that affect particular stages and tissues have been identified (see Section 7). 5 The locus has been cloned and sequenced. Thus, molecular probes are available for analyses at the DNA and RNA levels (see Sections 4 and 5). 6 The technique of P element-mediated transformation was initiated using the rosy system, and genetic and molecular analyses using transposons are now quite advanced (see Sections 4 and 7). Manipulation of rosy transposons also permits analysis of a range of regulatory phenomena not exclusive to XDH, including euchromatic position effects, heterochromatic position effects, and dosage compensation (see Section 7).

Ag-NOR staining in the Bennett wallaby, Macropus rufogriseus: evidence for dosage compensation

Abstract: Silver staining of cells in metaphase and interphase nuclei of both sexes of the Bennett wallaby, Macropus rufogriseus, has shown that (1) the nucleolus organizer region (NOR) is located only on the X chromosome (single Ag-NOR); (2) both X chromosomes in the female cells stain with silver; (3) the amounts of silver staining of metaphase chromosomes and interphase nuclei of both sexes are very similar; (4) the single X chromosome is hyperactive in male cells to equalize the expression of rRNA genes in the female cells with two X chromosomes; and (5) the mechanism of dosage compensation for rRNA genes in this species is similar to that reported for Drosophila salivary gland cells.