Development of canine X-chromosome inactivation pattern analysis for the detection of cell clonality by incorporating the examination of the SLIT and NTRK-like family member 4 (SLITRK4) gene.

X-chromosome inactivation pattern (XCIP) analysis can be used to assess the clonality of cell populations of various origin by distinguishing the methylated X chromosome from the unmethylated X chromosome. In this study, the utility of XCIP analysis was improved by incorporating the examination of AC dinucleotide repeats in SLIT and NTRK-like family member 4 (SLITRK4) gene into the previously reported CAG repeat examination of androgen receptor (AR) gene in dogs. The rate of heterozygosity when both genes were analysed (125/150, 83.3%) was higher than AR gene examination alone (86/150, 57.3%). Blood samples from heterozygous dogs in either AC-1 or AC-2 of SLITRK4 gene were examined for the corrected inactivation allele ratio (CIAR), resulting in the determination of a reference range of CIAR <3.8 in non-neoplastic cell/tissue samples. Using this analytical method, 49% (21/43) of neoplastic tissue samples from dogs showed a CIAR >3.8, indicating the presence of a clonal population. Through the present study, the availability of canine XCIP analysis was improved by incorporating the examination of the SLITRK4 gene, providing a highly useful laboratory examination system for the detection of the clonality of various cell/tissue samples in dogs.

Synaptic configuration of quadrivalents and their association with the XY bivalent in spermatocytes of Robertsonian heterozygotes of Mus domesticus

BACKGROUND: The nuclear architecture of meiotic prophase spermatocytes is based on higher-order patterns of spatial associations among chromosomal domains and consequently is prone to modification by chromosomal rearrangements. We have shown that nuclear architecture is modified in spermatocytes of Robertsonian (Rb) homozygotes of Mus domesticus. In this study we analyse the synaptic configuration of the quadrivalents formed in the meiotic pro- phase of spermatocytes of mice double heterozygotes for the dependent Rb chromosomes: Rbs 11.16 and 16.17. RESULTS: Electron microscope spreads of 60 pachytene spermatocytes from four animals of Mus domesticus 2n = 38 were studied and their respective quadrivalents analysed in detail. Normal synaptonemal complex was found between arms 16 of the Rb metacentric chromosomes, telocentrics 11 and 17 and homologous arms of the Rb metacentric chromosomes. About 43% of the quadrivalents formed a synaptonemal complex between the heterologous short arms of chromosomes 11 and 17. This synaptonemal complex is bound to the nuclear envelope through a fourth synapsed telomere, thus dragging the entire quadrivalent to the nuclear envelope. About 57% of quadrivalents showed unsynapsed single axes in the short arms of the telocentric chromosomes. About 90% of these unsynapsed quadrivalents also showed a telomere-to-telomere association between one of the single axes of the telocentric chromosome 11 or 17 and the X chromosome single axis, which was otherwise normally paired with the Y chromosome. Nucleolar material was associated with two bivalents and with the quadrivalent. CONCLUSIONS: The spermatocytes of heterozygotes for dependent Rb chromosomes formed a quadrivalent where four chromosomes are synapsed together and bound to the nuclear envelope through four telomeres. The nuclear configuration is determined by the fourth shortest telomere, which drags the centromere regions and heterochromatin of all the chromosomes towards the nuclear envelope, favouring the reiterated encounter and eventual rearrangement between the heterologous chromosomes. The unsynapsed regions of quadrivalents are frequently bound to the single axis of the X chromosome, possibly perturbing chromatin condensation and gene expression.

Angiotensin II type 2 receptor- and acetylcholine-mediated relaxation: essential contribution of female sex hormones and chromosomes.

Angiotensin-induced vasodilation, involving type 2 receptor (AT2R)-induced generation of nitric oxide (NO; by endothelial NO synthase) and endothelium-derived hyperpolarizing factors, may be limited to women. To distinguish the contribution of female sex hormones and chromosomes to AT2R function and endothelium-derived hyperpolarizing factor-mediated vasodilation, we made use of the four-core genotype model, where the testis-determining Sry gene has been deleted (Y(-)) from the Y chromosome, allowing XY(-) mice to develop a female gonadal phenotype. Simultaneously, by incorporating the Sry gene onto an autosome, XY(-)Sry and XXSry transgenic mice develop into gonadal male mice. Four-core genotype mice underwent a sham or gonadectomy (GDX) operation, and after 8 weeks, iliac arteries were collected to assess vascular function. XY(-)Sry male mice responded more strongly to angiotensin than XX female mice, and the AT2R antagonist PD123319 revealed that this was because of a dilator AT2R-mediated effect occurring exclusively in XX female mice. The latter could not be demonstrated in XXSry male and XY(-) female mice nor in XX female mice after GDX, suggesting that it depends on both sex hormones and chromosomes. Indeed, treating C57bl/6 GDX male mice with estrogen could not restore angiotensin-mediated, AT2R-dependent relaxation. To block acetylcholine-induced relaxation of iliac arteries obtained from four-core genotype XX mice, both endothelial NO synthase and endothelium-derived hyperpolarizing factor inhibition were required, whereas in four-core genotype XY animals, endothelial NO synthase inhibition alone was sufficient. These findings were independent of gonadal sex and unaltered after GDX. In conclusion, AT2R-induced relaxation requires both estrogen and the XX chromosome sex complement, whereas only the latter is required for endothelium-derived hyperpolarizing factors.

X-chromosome dosage affects male sexual behavior.

Sex differences in the brain and behavior are primarily attributed to dichotomous androgen exposure between males and females during neonatal development, as well as adult responses to gonadal hormones. Here we tested an alternative hypothesis and asked if sex chromosome complement influences male copulatory behavior, a standard behavior for studies of sexual differentiation. We used two mouse models with non-canonical associations between chromosomal and gonadal sex. In both models, we found evidence for sex chromosome complement as an important factor regulating sex differences in the expression of masculine sexual behavior. Counter intuitively, males with two X-chromosomes were faster to ejaculate and display more ejaculations than males with a single X. Moreover, mice of both sexes with two X-chromosomes displayed increased frequencies of mounts and thrusts. We speculate that expression levels of a yet to be discovered gene(s) on the X-chromosome may affect sexual behavior in mice and perhaps in other mammals.

Species-dependent expression patterns of DNA methyltransferase genes in mammalian oocytes and preimplantation embryos.

DNA methyltransferases (DNMTs) comprise a family of proteins involved in the establishment and maintenance of DNA methylation patterns in the mammalian genome. DNA methylation involves the transfer of the methyl group of the coenzyme S-adenosyl-L-methionine to the 5 position of cytosine residues within CpG dinucleotides. DNA methylation is implicated in the control of imprinted genes expression, X chromosome silencing, development of certain types of cancer, and embryonic development. DNA methylation is also believed to protect the genome from parasitic elements such as transposons, retrotransposons, and viruses. The aim of this study was to analyze the expression patterns of DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L genes in rhesus macaque (Macaca mulatta) oocytes and preimplantation stage embryos from fertilization to the hatched blastocyst stage, and to compare these results with the expression profiles in the mouse and other mammalian species. We describe species-dependent differences as well as similarities in expression patterns of DNMT genes among mammals.

Expression analysis of the steroidogenic acute regulatory protein (STAR) gene in developing porcine conceptuses.

Steroidogenesis in porcine non-conceptus tissue is regulated by the STAR-dependent transport of cholesterol from the outer to inner mitochondrial membrane. Previous serial analysis of gene expression (SAGE) identified a STAR mRNA transcript in the porcine peri-implantation conceptus during trophectoderm elongation and increased conceptus estrogen synthesis between gestational day 11 and 12. To assess a potential role for STAR in the modulation of conceptus steroidogenesis via cholesterol transport, the conceptus STAR transcript was PCR cloned and temporal expression of mRNA and protein were examined. Northern analysis of day 12 corpora lutea and pig conceptus RNA detected multiple STAR transcripts in both tissues and identified the cloned transcript as the longest variant. The transcript had a 99% similarity to a truncated ovarian STAR transcript. The conceptus STAR transcript was temporally regulated during elongation but trace expression was present in day 6 blastocysts and day 25 conceptuses. Differential regulation of STAR mRNA was concomitant with the presence of the stimulatory transcription factor steroidogenic factor 1, and absence of the inhibitory transcription factor dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1, transcripts. In contrast to peak STAR mRNA expression at the filamentous stage, Western blot analyses revealed STAR protein levels were highest in tubular conceptuses. These data confirm the presence of STAR mRNA and protein during porcine conceptus elongation and suggest regulation of STAR at two levels, transcriptionally, in part, through differential regulation of transcription factors, and post-transcriptionally, evidenced by the disparity of protein to RNA in filamentous conceptuses.

XY chromosomal bivalent: nucleolar attraction.

Nucleolar organization by autosomal bivalents occurs during male meiotic prophase in mammalian species. During late leptotene-early zygotene stages, several autosomal bivalents are engaged in ribosomal RNA synthesis. At pachytene stage, nucleolar masses detach from the sites of primary autosomal origin, relocate close to the XY chromosomal pair, and nucleolar components become segregated. In early pachytene, an extensive synaptonemal complex at the pseudoautosomal region, links X and Y chromosomes in close juxtaposition along most of the length of the Y chromosome, except for a terminal region of the Y that diverges from the pairing region. As meiotic prophase advances, X and Y chromosomes progressively desynapse and, at diplotene, the XY pair is associated end-to-end. Xmr (Xlr-related, meiosis regulated) is a protein component of the nucleolus associated to the XY pair and of the asynapsed portions of the X and Y axial cores. Xmr, like SCP3, is a component of the lateral element of the synaptonemal complex. Both share structural homology in their C-terminal region. This region contains several putative coiled-coil domains known to mediate heterodimeric protein-protein interactions and to provide binding sites to regulatory proteins. Like Xmr, the tumor repressor protein BRCA1 is present along the unsynapsed cores of the XY bivalent. Both Xmr and BRCA1 have been implicated in a mechanism leading to chromatin condensation and transcription inactivation of the XY bivalent. The BRCA1-ATR kinase complex, as recent research suggests, triggers the phosphorylation of histone H2AX, which predominates in the condensed chromatin of the XY chromosomal pair. Xmr is not present in the XY bivalent when the expression of histone H2AX is deficient. The role of Xmr in chromatin condensation of the XY bivalent has not been determined. The partial structural homology of SCP3 and Xmr, their distribution along the unsynapsed axial cores of the X and Y chromosomes, and the presence of Xmr in the XY pair-associated nucleolus raises the possibility that Xmr, and other proteins including protein kinases, may be recruited to the nucleolus to perform functions related to chromosomal synapsis, chromatin condensation and recombination processes, as well as cell cycle progression.

Modulation of ISWI function by site-specific histone acetylation.

Mutations in Drosophila ISWI, a member of the SWI2/SNF2 family of chromatin remodeling ATPases, alter the global architecture of the male X chromosome. The transcription of genes on this chromosome is increased 2-fold relative to females due to dosage compensation, a process involving the acetylation of histone H4 at lysine 16 (H4K16). Here we show that blocking H4K16 acetylation suppresses the X chromosome defects resulting from loss of ISWI function in males. In contrast, the forced acetylation of H4K16 in ISWI mutant females causes X chromosome defects indistinguishable from those seen in ISWI mutant males. Increased expression of MOF, the histone acetyltransferase that acetylates H4K16, strongly enhances phenotypes resulting from the partial loss of ISWI function. Peptide competition assays revealed that H4K16 acetylation reduces the ability of ISWI to interact productively with its substrate. These findings suggest that H4K16 acetylation directly counteracts chromatin compaction mediated by the ISWI ATPase.

Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells.

The resetting of a somatic epigenotype to a totipotential state has been demonstrated by successful animal cloning, via transplantation of somatic nuclei into enucleated oocytes. We have established an experimental system, which reproduces the nuclear reprogramming of somatic cells in vitro by fusing adult thymocytes with embryonic stem (ES) cells. Analysis of the lymphoid-cell-specific V-(D)-J DNA rearrangement of the T cell receptor and immunoglobin genes shows that the ES cells have hybridized with differentiated cells. In these ES cell hybrids, the inactivated X chromosome derived from a female thymocyte adopts some characteristics of an active X chromosome, including early replication timing and unstable Xist transcription. We also found that an Oct4-GFP transgene, which is normally repressed in thymocytes, is reactivated 48 hr after cell fusion. The pluripotency of the ES-thymocyte hybrid cells is shown in vivo, since they contribute to all three primary germ layers of chimeric embryos. The somatic DNA methylation pattern of the imprinted H19 and Igf2r genes is maintained in these hybrids, unlike hybrids between ES and EG (embryonic germ) cells in which the differential methylation is erased. Thus, ES cells have the capacity to reset certain aspects of the epigenotype of somatic cells to those of ES cells.

Unusual electrophysiological findings in X-linked dominant Charcot-Marie-Tooth disease.

X-linked Charcot-Marie-Tooth disease (CMTX) is the second most common form of Charcot-Marie-Tooth disease. Variable histopathological and nerve conduction velocity (NCV) results have suggested either a primary demyelinating or axonal polyneuropathy. We identified five individuals across three generations in a family with CMTX associated with a mutation in the gene coding for connexin 32. All individuals were studied by clinical neurological examination, DNA analysis, and nerve conduction studies. The proband (1174/KD) also underwent a sural nerve biopsy. As expected, all the affected males were more clinically affected than the females. All affected males and obligate female carriers exhibited some electrophysiological characteristics of demyelination. However, striking heterogeneity of nerve conduction velocities was seen. This family shows that CMTX is a heterogeneous and distinctly nonuniform demyelinating polyneuropathy, the severity of which varies with sex and age. Such electrophysiological variability is unique among hereditary neuropathies.

Clonal analysis of a case of multiple meningiomas using multiple molecular genetic approaches: pathology case report.

OBJECTIVE: Multiple meningiomas are uncommon brain tumors occurring concurrently in several intracranial locations in the same patient. In the present study, we determined the clonality, methylation status of deoxyribonucleic acid, and relationship of genetic alterations in eight meningiomas from one female patient. METHODS: Six molecular genetic techniques, including two methylation-based clonality assays and one transcription-based clonality assay, methylation analysis of CpG islands by methylation-specific polymerase chain reaction, loss of heterozygosity, microsatellite instability, and mutational analysis of the NF2 gene on chromosome 22, were used in comparative investigations on clonality and genetic alterations. RESULTS: The presence of clonal tumor cells was demonstrated by 1) loss of the same copy of chromosome 22 in all eight tumors; 2) transcription of the human AR gene from the same allele in six of eight tumors; 3) a common unmethylated allele at the AR locus in all eight tumors; and 4) the identical single-basepair insertion mutation in exon 9 of the NF2 gene in six of eight tumors. In addition, loss of a copy of the X chromosome in one tumor nodule and microsatellite instability in another nodule were observed. CONCLUSION: Taken together, this case of multiple meningiomas was most likely monoclonal in origin. Loss of chromosome 22 was an early event during the development of multiple meningiomas and was followed by mutations at the NF2 locus. Later events, including loss of the X chromosome, variation of AR gene expression, or microsatellite instability, may also have played a role in the development of multiple meningiomas in this patient.

Chromosomes exhibit preferential positioning in nuclei of quiescent human cells.

The relative spatial positioning of chromosomes 7, 8, 16, X and Y was examined in nuclei of quiescent (noncycling) diploid and triploid human fibroblasts using fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes and digital imaging. In quiescent diploid cells, interhomolog distances and chromosome homolog position maps revealed a nonrandom, preferential topology for chromosomes 7, 8 and 16, whereas chromosome X approximated a more random distribution. Variations in the orientation of nuclei on the culture substratum tended to hinder detection of an ordered chromosome topology at interphase by biasing homolog position maps towards random distributions. Using two chromosome X homologs as reference points in triploid cells (karyotype = 69, XXY), the intranuclear location of chromosome Y was found to be predictable within remarkably narrow spatial limits. Dual-FISH with various combinations of chromosome-specific DNA probes and contrasting fluorochromes was used to identify adjacent chromosomes in mitotic rosettes and test whether they are similarly positioned in interphase nuclei. From among the combinations tested, chromosomes 8 and 11 were found to be closely apposed in most mitotic rosettes and interphase nuclei. Overall, results suggest the existence of an ordered interphase chromosome topology in quiescent human cells in which at least some chromosome homologs exhibit a preferred relative intranuclear location that may correspond to the observed spatial order of chromosomes in rosettes of mitotic cells.

Analysis of clonality in human endometriotic cysts based on evaluation of X chromosome inactivation in archival formalin-fixed, paraffin-embedded tissue.

The clonality of epithelial cells in ovarian endometriotic cysts was evaluated on the basis of a polymorphism of the X-linked phosphoglycerate kinase gene (PGK). The problems associated with clonality analysis of DNA samples extracted from archival formalin-fixed, paraffin-embedded tissue were addressed with the use of newly designed primers and a modified stepdown program for PCR amplification. The method relies on digestion of DNA with the methylation-sensitive restriction enzyme SnaBI, PCR amplification of PGK, and detection of a BstXI polymorphism. With this approach, only the inactive (methylated) PGK allele is selectively amplified by PCR. A total of 25 epithelial cells in endometriotic cysts and 25 matched normal ovarian stromal tissue specimens were analyzed. All of the control tissue samples yielded PCR products, with 11 of the 25 samples found to be heterozygous for the BstXI polymorphism. Ten of the 25 epithelial cells in endometriosis specimens were informative at this locus, and all of these 10 were shown to be monoclonal on the basis of PGK methylation. The results indicate that this new method of clonal analysis with archival formalin-fixed tissue is reliable and confirm that endometriotic cysts are monoclonal in origin.

Mutational analysis and genetic cloning of the agnostic locus, which regulates learning ability in Drosophila.

P-insertion mutations were obtained and localized by in situ methods at the agnostic gene (agn: 1-38.9; 11AB) in Drosophila. All agn mutants showed a wide spectrum of pleiotropic effects: an EMS-induced mutation of the agn-ts398 improved the ability to develop a conditioned defensive response and increased the activity of cAMP metabolic enzymes; spontaneous mutation of agnX1 showed morphological defects of the brain. P-insertion mutations were used to clone the gene; a restriction map of 80 kb in length was determined, and the insertion was localized to a fragment of 9 kb.

Potential role of the inactivated X chromosome in ovarian epithelial tumor development.

BACKGROUND: Ovarian epithelial tumors can be divided into subcategories often regarded as different stages of neoplastic transformation. Cystadenomas belong to the least aggressive subgroup and are noninvasive and nonmetastatic. Ovarian tumors of low malignant potential (LMP) are intermediate between cystadenomas and carcinomas and show markedly reduced invasive and metastatic abilities. Invasion and metastasis are the hallmarks of carcinomas, which constitute the most aggressive subgroup and can be further subdivided into different grades. PURPOSE: We performed comparative allelotype analyses of ovarian cystadenomas, LMP tumors, and carcinomas, reasoning that such analyses could provide clues about the molecular determinants of their phenotypic differences. Because we realized that allelic losses involving the X chromosome might be associated with LMP tumor development, we determined whether such losses were interstitial and whether they involved the active or the inactive X chromosome. METHODS: Frequencies of loss of heterozygosity (LOH) at specific loci in every chromosomal arm were determined in 16 ovarian cystadenomas, 23 ovarian LMP tumors, 15 low-grade ovarian carcinomas, and 35 high-grade ovarian carcinomas by use of either the polymerase chain reaction (PCR) or Southern blot analyses. We took advantage of the fact that DNA methylation is an important mechanism of X-chromosome inactivation to determine whether losses involving the X chromosome were in the active or the inactive copy. We analyzed the methylation status of retained alleles on the X chromosome by determining whether they could be amplified by PCR after digestion with the methylation-sensitive restriction endonuclease Hpa II. RESULTS: High-grade carcinomas contained frequent(>50%) LOH in four autosomal chromosome arms, i.e., 6q, 13q, 17p, and 17q. Except for 13q, these same chromosomal arms showed frequent LOH in low-grade carcinomas. LOH in autosomal chromosomes was comparatively rare in LMP tumors and was absent in cystadenomas. In contrast, half (eight of 16) of LMP tumors informative for a locus in the proximal portion of chromosome Xq showed LOH at that locus. These losses were the result of interstitial deletions in six of the eight cases and involved the inactive copy of the X chromosome exclusively. Similar losses in the X chromosome were not seen in either cystadenomas or low-grade carcinomas. CONCLUSIONS AND IMPLICATIONS: LOH at multiple loci is associated with the development of ovarian carcinomas but not with the development of cystadenomas and LMP tumors. However, the integrity of a locus in chromosome Xq that possibly escapes X-chromosome inactivation is important for the control of LMP tumor development. The fact that this locus does not appear to be involved in the genesis of low-grade carcinomas suggests that LMP tumors are not precursors of such carcinomas.

[Mutational analysis and genetic cloning of the agnostic locus controlling learning ability in Drosophila]. / Mutatsionnyi analiz i geneticheskoe klonirovanie lokusa agnostic, kontroliruiuschego sposobnost'k obucheniiu, u drozofily.

The ts-mutations of the agnostic gene either increased or decreased the activity of the AC and PDE. A chromosomal deficiency in the 11B region failed to complement with the agnostic behavioral defect like agn P29 and P40, led to an increased proportion of the PDE-1 in total PDE and to the structural defects in the central complex thus indicating that the P insertion is responsible for the mutant agnostic phenotype.

XY chromosome behaviour in the germ-line of the human male: a FISH analysis of spatial orientation, chromatin condensation and pairing.

We have used multicolour fluorescence in situ hybridization to study the behaviour of the X and Y chromosomes in relation to a representative autosome, chromosome 1, on air-dried testicular preparations from normal fertile human males. In a proportion of Sertoli cells at interphase as well as spermatogonial metaphases there is an apparent selective undercondensation of the heterochromatic block of the long arm of the Y, which may be of functional significance with respect to Y-specific gene activity, initiating and maintaining spermatogenesis; we suggest that this may involve a mechanism similar to heterochromatin position-effect variegation in Drosophila. In the supporting Sertoli as well as pre-meiotic and leptotene cells the X and Y occupy relatively restricted domains at opposite poles of the nuclear membrane, while the chromosome 1 centromere regions are located interstitially and appear prealigned. The XY pairing and 'sex vesicle' formation comprises a complex series of spatial movement and differential condensation patterns. On the basis of these observations we propose that: the XIST/Xist gene, known to be involved in somatic X inactivation, imposes a chromatin reorganization leading to bending at the X-inactivation centre both at first meiotic prophase in males and in the soma in females; and the differential X and Y segments are protected from potentially deleterious meiotic exchanges by their separate spatial orientation. In addition, there is an indication that the timing of pairing and first meiotic segregation of the sex chromosomes is different, and precocious in comparison to the pairing and segregation of the autosomes, which may explain the high incidence of sex chromosome aneuploidy in sperm.