Genes controlling chromosome activity

Normal propagation of Y chromosome lampbrush loops was used as a screening tool in order to recover X-linked mutations controling Y chromosome activation. The nature of the most extreme mutationthus recovered, sterile (1) XL2, is described. It is a recessive gene mutation, readily mapped 2 cross over units distally to white. The mutation exerts its sterilizing effect by blocking normal unfolding of all Y lampbrush loops, but does not affect the unique shape of each diminutive loop. The degree to which a loop forming site is developed is partially temperature sensitive. It is independent however, on its map location or the dose of homologous as well as heterologous sites. It was provisionally concluded therefore that site response to the XL2 effect is a stage specific and not a quantitative one. The possible ways by which non homologous genes control Y chromosome activity are discussed.     

HGPRT mutation induction by N-ethyl-N-nitrosourea as measured by 6-thioguanine resistance is higher in male than in female Syrian hamster fetuses

BACKGROUND: The consequences of mutations in embryonic and fetal cells are serious and contribute to high prenatal sensitivity to mutagenic agents. An understanding of the factors that influence the yield of such mutations is important for management of adverse effects of perinatal exposures. Resistance to 6-thioguanine (6-TG) can be utilized to study mutational events at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus. HGPRT is X-linked and recessive. According to the Lyon hypothesis, male cells have only one X-chromosome and female cells randomly inactivate the second X-chromosome. This leads to the prediction that X-linked genes should be equally sensitive to the mutagenic effects of toxicants in male and female fetuses. METHODS: We tested this supposition by in utero exposure of Syrian hamster fetuses to N-ethyl-N-nitrosourea (ENU) at day 12 of gestation. ENU is a strong carcinogen and mutagen. HGPRT mutations were detected by selection with 6-TG. RESULTS: Surprisingly. the male cells had 4 to 5 times more 6-TG mutants than female cells, in two separate experiments (p<0.001). Ouabain resistance, reflecting a co-dominant autosomal locus, was used as a control, and we found that there was no significant difference between male and female cells (p=0.549). CONCLUSIONS: Possible reasons for the sex difference in mutations include escape of the second X-chromosome from inactivation in some of the female cells, or higher mutability in male cells. In any event, there is a gender difference in vulnerability to mutation of an X-linked gene that has previously not been appreciated, and that may be relevant to toxicological studies of such genes. HGPRT is frequently used to monitor mutagenic events in human fetuses.     

Dynamic Elements at Both Cytoplasmically and Extracellularly Facing Sides of the UapA Transporter Selectively Control the Accessibility of Substrates to Their Translocation Pathway

In the UapA uric acid-xanthine permease of Aspergillusnidulans, subtle interactions between key residues of the putative substrate binding pocket, located in the TMS8-TMS9 loop (where TMS is transmembrane segment), and a specificity filter, implicating residues in TMS12 and the TMS1-TMS2 loop, are critical for function and specificity. By using a strain lacking all transporters involved in adenine uptake (ΔazgA ΔfcyB ΔuapC) and carrying a mutation that partially inactivates the UapA specificity filter (F528S), we obtained 28 mutants capable of UapA-mediated growth on adenine. Seventy-two percent of mutants concern replacements of a single residue, R481, in the putative cytoplasmic loop TMS10-TMS11. Five missense mutations are located in TMS9, in TMS10 or in loops TMS1-TMS2 and TMS8-TMS9. Mutations in the latter loops concern residues previously shown to enlarge UapA specificity (Q113L) or to be part of a motif involved in substrate binding (F406Y). In all mutants, the ability of UapA to transport its physiological substrates remains intact, whereas the increased capacity for transport of adenine and other purines seems to be due to the elimination of elements that hinder the translocation of non-physiological substrates through UapA, rather than to an increase in relevant binding affinities. The additive effects of most novel mutations with F528S and allele-specific interactions of mutation R481G (TMS10-TMS11 loop) with Q113L (TMS1-TMS2 loop) or T526M (TMS12) establish specific interdomain synergy as a critical determinant for substrate selection. Our results strongly suggest that distinct domains at both sides of UapA act as selective dynamic gates controlling substrate access to their translocation pathway.     

X-linked gene expression and sex determination in Caenorhabditis elegans

The signal for sex determination in the nematode Caenorhabditis elegans is the ratio between the number of X chromosomes and the number of sets of autosomes (the X/A ratio). Animals with an X/A ratio of 0.67 (a triploid with two X chromosomes) or less are males. Animals with an X/A ratio of 0.75 or more are hermaphrodites. Thus, diploid males have one X chromosome and diploid hermaphrodites have two X chromosomes. However, the difference in X-chromosome number between the sexes is not reflected in general levels of X-linked gene expression because of the phenomenon of dosage compensation. In dosage compensation, X-linked gene expression appears to be 'turned down' in 2X animals to the 1X level of expression. An intriguing and unexplained finding is that mutations and X-chromosome duplications that elevate X-linked gene expression also feminize triploid males. One way that this relationship between sex determination and X-linked gene expression may be operating is discussed.     

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

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.     

Nonsyndromic X-linked mental retardation: where are the missing mutations?

Analysis of linkage intervals from 125 unrelated families with nonsyndromic X-linked mental retardation (NS-XLMR) has revealed that the respective gene defects are conspicuously clustered in defined regions of the human X-chromosome, with approximately 30% of all mutations being located on the proximal Xp. In 83% of these families, underlying gene defects are not yet known. Our observations should speed up the search for mutations that are still missing and pave the way for the molecular diagnosis of this common disorder.     

Genetic Analysis of X-Chromosome Dosage Compensation in Caenorhabditis elegans

We have shown that the phenotypes resulting from hypomorphic mutations (causing reduction but not complete loss of function) in two X-linked genes can be used as a genetic assay for X-chromosome dosage compensation in Caenorhabditis elegans between males (XO) and hermaphrodites (XX). In addition we show that recessive mutations in two autosomal genes, dpy-21 V and dpy-26 IV, suppress the phenotypes resulting from the X-linked hypomorphic mutations, but not the phenotypes resulting from comparable autosomal hypomorphic mutations. This result strongly suggests that the dpy-21 and dpy-26 mutations cause increased X expression, implying that the normal function of these genes may be to lower the expression of X-linked genes. Recessive mutations in two other dpy genes, dpy-22 X and dpy-23 X, increase the severity of phenotypes resulting from some X-linked hypomorphic mutations, although dpy-23 may affect the phenotypes resulting from the autosomal hypomorphs as well. The mutations in all four of the dpy genes show their effects in both XO and XX animals, although to different degrees. Mutations in 18 other dpy genes do not show these effects.     

The interaction between daughterless and sex-lethal in triploids: a lethal sex-transforming maternal effect linking sex determination and dosage compensation in Drosophila melanogaster

Regulation of Drosophila sex determination and X-chromosome dosage compensation in response to the X-chromosome/autosome (X/A) balance of the zygote is shown to require proper functioning of both the da+ gene in the mother and the Sxl+ gene in the zygote. Previous studies led to the hypothesis that zygotic Sxl+ alleles are differentially active in females (XXAA) vs males (XYAA) in response to the X/A balance, and that maternal da+ gene product acts as a positive regulator in this connection. Sxl+ activity was proposed to impose the female developmental sequence on cells which would follow the male sequence in its absence. Important predictions of this proposal are verified. This study focuses primarily on the phenotype of triploid intersexes (XXAAA, X/A = 0.67). They are shown here to survive effects of da and Sxl mutations that would be lethal to diploids. The ambiguous X/A signal of intersexes normally causes them to develop as phenotypic mosaics of male and female tissue. Loss of maternal da+ or zygotic Sxl+ gene function shifts their somatic sexual phenotype to the male alternative. A gain-of-function mutation at Sxl has the opposite effect, imposing female development regardless of the maternal genotype with respect to da. It also reduces their rate of X-linked gene expression. The effects of a duplication of Sxl+ resemble those of the constitutive Sxl allele, but are less extreme. The role of these genes in the process of X-chromosome dosage compensation is inferred indirectly from the strict dependence of the mutations' lethal effects on the X/A balance in haploids, diploids, and triploids, and more directly from the effects of the mutations on the phenotypes of the X-linked neomorphic mutations, Bar and Hairy-wing. The relationship of da+ and Sxl+ gene functions to those of other sex-specific lethal loci in D. melanogaster, and to sex determination mechanisms in other species, is discussed.     

Defining the cause of skewed X-chromosome inactivation in X-linked mental retardation by use of a mouse model

Extreme skewing of X-chromosome inactivation (XCI) is rare in the normal female population but is observed frequently in carriers of some X-linked mutations. Recently, it has been shown that various forms of X-linked mental retardation (XLMR) have a strong association with skewed XCI in female carriers, but the mechanisms underlying this skewing are unknown. ATR-X syndrome, caused by mutations in a ubiquitously expressed, chromatin-associated protein, provides a clear example of XLMR in which phenotypically normal female carriers virtually all have highly skewed XCI biased against the X chromosome that harbors the mutant allele. Here, we have used a mouse model to understand the processes causing skewed XCI. In female mice heterozygous for a null Atrx allele, we found that XCI is balanced early in embryogenesis but becomes skewed over the course of development, because of selection favoring cells expressing the wild-type Atrx allele. Unexpectedly, selection does not appear to be the result of general cellular-viability defects in Atrx-deficient cells, since it is restricted to specific stages of development and is not ongoing throughout the life of the animal. Instead, there is evidence that selection results from independent tissue-specific effects. This illustrates an important mechanism by which skewed XCI may occur in carriers of XLMR and provides insight into the normal role of ATRX in regulating cell fate.     

Association of CHRDL1 Mutations and Variants with X-linked Megalocornea,Neuh?user Syndrome and Central Corneal Thickness

We describe novel CHRDL1 mutations in ten families with X-linked megalocornea (MGC1). Our mutation-positive cohort enabled us to establish ultrasonography as a reliable clinical diagnostic tool to distinguish between MGC1 and primary congenital glaucoma (PCG). Megalocornea is also a feature of Neuhäuser or megalocornea-mental retardation (MMR) syndrome, a rare condition of unknown etiology. In a male patient diagnosed with MMR, we performed targeted and whole exome sequencing (WES) and identified a novel missense mutation in CHRDL1 that accounts for his MGC1 phenotype but not his non-ocular features. This finding suggests that MMR syndrome, in some cases, may be di- or multigenic. MGC1 patients have reduced central corneal thickness (CCT); however no X-linked loci have been associated with CCT, possibly because the majority of genome-wide association studies (GWAS) overlook the X-chromosome. We therefore explored whether variants on the X-chromosome are associated with CCT. We found rs149956316, in intron 6 of CHRDL1, to be the most significantly associated single nucleotide polymorphism (SNP) (p = 6.81×10⁻⁶) on the X-chromosome. However, this association was not replicated in a smaller subset of whole genome sequenced samples. This study highlights the importance of including X-chromosome SNP data in GWAS to identify potential loci associated with quantitative traits or disease risk.     

sdc-1: a link between sex determination and dosage compensation in C. elegans

Mutations in the X-linked gene sdc-1 affect both sex determination and X-chromosome dosage compensation in C. elegans, providing evidence that these two pathways share a common step. In XX animals (normally hermaphrodites), sdc-1 mutations cause partial masculinization and elevated levels of X-linked gene expression, an apparent shift of both pathways toward their XO modes of expression. The masculinization occurs through effects on the major sex determination pathway, upstream of all previously identified sex-determining genes. XO animals are apparently unaffected by the sdc-1 mutations. We propose a model in which the wild-type sdc-1 activity is either a component of the primary sex-determining signal (the X/Autosome ratio) or involved in transmitting information about this signal to both the sex determination and dosage compensation pathways.     

Mutational analysis of hemophilia B in Russia: Molecular-genetic study

Hemophilia B is a hereditary X-linked coagulation disorder. This pathology is caused by various defects in the factor IX gene, which is, being about 34 kb long and consisting of eight exons, localized in the Xq27 locus of the X-chromosome long arm. Mutations were revealed in 56 unrelated patients with hemophilia B in this study by using direct sequencing of factor IX gene functionally important fragments. Forty-six mutations were found with prevailing missense mutations (n = 30). The rest of the mutations were nonsense (n = 4) and splicing (n = 4) mutations, large deletions (n = 3), microdeletions (n = 2), microinsertions (n = 2), and promoter mutations (n = 1). Eleven of 46 mutations were previously unknown for human populations.     

The Prevention of Repeat-Associated Deletions in Saccharomyces Cerevisiae by Mismatch Repair Depends on Size and Origin of Deletions

We have investigated the effects of mismatch repair on 1- to 61-bp deletions in the yeast Saccharomyces cerevisiae. The deletions are likely to involve unpaired loop intermediates resulting from DNA polymerase slippage. The mutator effects of mutations in the DNA polymerase δ (POL3) gene and the recombinational repair RAD52 gene were studied in combination with mismatch repair defects. The pol3-t mutation increased up to 1000-fold the rate of extended (7-61 bp) but not of 1-bp deletions. In a rad52 null mutant only the 1-bp deletions were increased (12-fold). The mismatch repair mutations pms1, msh2 and msh3 did not affect 31- and 61-bp deletions in the pol3-t but increased the rates of 7- and 1-bp deletions. We propose that loops less than or equal to seven bases generated during replication are subject to mismatch repair by the PMS1, MSH2, MSH3 system and that it cannot act on loops >=31 bases. In contrast to the pol3-t, the enhancement of 1-bp deletions in a rad52 mutant is not altered by a pms1 mutation. Thus, mismatch repair appears to be specific to errors of DNA synthesis generated during semiconservative replication.     

Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein.     

Isolation of a homozygous X-linked translocation stock with two additional sex-chromosomes in the onion fly Hylemya antiqua Meigen

Summary The onion fly, Hylemya antiqua Meigen, was subjected to irradiation and selection based on observations of fertility and cytogenetics, in order to isolate structural chromosome mutations which might be used for genetic control of this species. To the present time, only a simple X-linked translocation could be obtained as a homozygous stock. Sibcrossing was carried out using translocation trisomics (TN + X) obtained from test-crossed translocation heterozygous females (TN) showing numerical nondisjunction. A homozygous stock was obtained with two additional sex-chromosomes. This is a unique case because normally an X-linked translocation can not be made homozygous in the male sex, which normally only carries one X-chromosome.     

The synaptonemal complexes of Caenorhabditis elegans: Pachytene karyotype analysis of the rad-4 radiation-sensitive mutant

In the rad-4 mutant of C. elegans there is a specific increase in the number of ‘Disjunction Regulator Regions’ (DDR) present on the synaptonemal complexes (SC) in pachytene nuclei. These DDRs either promote disjunction or inhibit nondisjunction of the X-chromosome as evidenced by the 10-fold decrease in the rate of X-chromosome nondisjunction as compared to the wild-type. The structure of the tripartite SC is normal, thus, the decrease in the rate of X-chromosome nondisjunction in the rad-4 mutant is not related to the structure of the SC but may be related to the number of DDRs. Other changes are also associated with the sensitivity to irradiation, i.e. the pachytene nuclear morphology is altered such that nuclei and nucleoli are 50% the size of wild-type. In addition, the autosomal: X-chromosome size ratio is reduced in the rad-4 mutant. That there are six SCs confirms n = 6 in this mutant and of these six SCs three can be identified: (1) the XX bivalent, SC No. 1, is the shortest and pairs synchronously with the autosomes; (2) the longest bivalent, SC No. 6, carries the nucleolar organizer region at one extreme end; and (3) SC No. 5 has two DDRs located approximately on μm apart from each other.