DNA hypomethylation causes an increase in DNase-I sensitivity and an advance in the time of replication of the entire inactive X chromosome

Summary We have examined the effect of 5-azacytidine (5-aza-C) induced hypomethylation of DNA on the time of replication and DNase I sensitivity of the X chromosomes of female Gerbillus gerbillus (rodent) lung fibroblast cells. Using in situ nick translation to visualise the potential state of activity of large regions of metaphase chromosomes we show that 5-aza-C causes a dramatic increase in the DNase-I sensitivity of the entire inactive X chromosome of female G. gerbillus cells and this increase in nuclease sensitivity correlates with a large shift in the time of replication of the inactive X chromosome from late S phase to early S phase. These effects of 5-aza-C on the inactive X chromosome are associated with a 15% decrease in DNA methylation. Our results indicate that DNA methylation concomitantly affects both the time of replication and the chromatin conformation of the inactive X chromosome.     

Chondrodysplasia punctata with X;Y translocation

Summary We have studied a family in which the mother and her son were carriers of an X;Y translocation, der(X)t(X;Y) (p22.3;q11). The mother was of slightly short stature and had mildly short upper extremities. The son had epiphyseal punctate calcifications, mildly short extremities, a flattened nasal bridge, and mental retardation (chondrodysplasia punctata). The extra bands on the short arm of the X chromosome were identified as deriving from the long arm of the Y chromosome, using in situ hybridization with a Y-chromosome-specific DNA probe (pHY10). The chondrodysplasia punctata seen in our case may be associated with the abnormality of the distal short arm of the X chromosome caused by X;Y translocation.     

Sequence analysis of the breakpoint regions of an X;5 translocation in a female with Duchenne muscular dystrophy.

X;autosome translocations in females with Duchenne muscular dystrophy (DMD) provide an opportunity to study the mechanisms responsible for chromosomal rearrangements that occur in the germ line. We describe here a detailed molecular analysis of the translocation breakpoints of an X;autosome reciprocal translocation, t(X;5)(p21;q31.1), in a female with DMD. Cosmid clones that contained the X-chromosome breakpoint region were identified, and subclones that hybridized to the translocation junction fragment in restriction digests of the patient's DNA were isolated and sequenced. Primers designed from the X-chromosomal sequence were used to obtain the junction fragments on the der(X) and the der(5) by inverse PCR. The resultant clones were also cloned and sequenced, and this information used to isolate the chromosome 5 breakpoint region. Comparison of the DNA sequences of the junction fragments with those of the breakpoint regions on chromosomes X and 5 revealed that the translocation arose by nonhomologous recombination with an imprecise reciprocal exchange. Four and six base pairs of unknown origin are inserted at the exchange points of the der(X) and der(5), respectively, and three nucleotides are deleted from the X-chromosome sequence. Two features were found that may have played a role in the generation of the translocation. These were (1) a repeat motif with an internal homopyrimidine stretch 10 bp upstream from the X-chromosome breakpoint and (2) a 9-bp sequence of 78% homology located near the breakpoints on chromosomes 5 and X.     

Cytogenetic and molecular characterization of a highly repeated DNA sequence in the peach potato aphid Myzus persicae

Electrophoresis following digestion of Myzus persicae genomic DNA with HindIII showed the presence of a prominent band of approximately 200 bp whereas a faint electrophoretic band corresponding to DNA fragments of about 3000 bp was observed after digestion with ApaI. In situ digestion with restriction enzymes, followed by in situ nick translation, showed that ApaI targets are localized at the nucleolus organizer-bearing X telomeric region, whereas HindIII restriction sites are clustered in intercalary C-positive areas on the same X chromosome. Fluorescent in situ hybridization (FISH) carried out by using digoxygenin-labeled HindIII repeats as probe fully confirmed overlapping between the hybridization sites of this probe and the AT-rich intercalary heterochromatic bands on the X chromosome. These findings, together with published data, allow us to conclude that the M. persicae genome possesses three classes of C-positive heterochromatin: (i) a GC-rich argentophilic band located on one telomere of the X chromosome that contains ApaI targets; (ii) AT-rich intercalary bands located on the X chromosome containing clustered HindIII fragments; (iii) AT-rich telomeric bands, located on autosomes, consisting of HaeIII repeats. Molecular analysis has shown that the length of the HindIII repeat consensus sequence is 189 bp with an AT content of 67%. Southern blotting with HindIII monomers revealed a regular ladder of bands composed of multimers of basic length that are characteristic of satellite DNAs. The HindIII repeat displays other features typical of eukaryotic satellite arrays such as overlapping with heterochromatic bands and a high degree of sequence similarity among monomers (84%–94%). A similarity plot showed that sequences were particularly variable in the 50–100 bp region whereas they proved to be highly conservative in the first 50 bp, thus suggesting that this portion of the repeat might be functionally important. Received: 23 February 1999; in revised form: 21 July 1999 / Accepted: 28 July 1999     

Cytogenetic and molecular investigations of an abnormal Y chromosome: evidence for a pseudo-dicentric (Yq) isochromosome.

A derivative Y chromosome was found in a 55-year-old man with Lambert-Eaton paraneoplasic pseudomyastheniform disease. Small testicles, azoospermia were noticed and hormonal level values were as in the Klinefelter syndrome. A 45,X/46,XYp+ mosa?cism was described on peripheral blood lymphocytes. Cytogenetic investigations with R-G-C- and Q-banding have been performed. In situ hybridization with the GMGY 10 DNA probe showed two copies of proximal Yp sequences. Southern blot analyses were performed using the Y DNA probes 27a, 47z, 64a7, 50f2 disclosing specific Yp and Yq sequences from the pseudoautosomal boundary to the Yq proximal portion. The der(Y) has been defined as a dicentric isochromosome for the long arm with one active and one apparently suppressed centromere. The breakpoint leading to the der(Y), has been located in the pairing segment of the Y short arm (i.e. Yp11.32). So the der(Y) was interpreted as a psu dic(Y) (qter-->cen-->p11.32 ::p11.32-->qter). There was thus an almost complete duplication of the Y chromosome.     

Pattern of late DNA replication of the allocyclic X chromosome in Cebus apella and Leontopithecus rosalia chrysomelas fibroblasts

The pattern of late DNA replication in the allocyclic X chromosome has been studied in the primary fibroblasts of two neotropical primates (Cebus apella and Leontopithecus rosalia chrysomelas). A comparison with previous reports showed a pattern identical with that of (1) the allocyclic X chromosome of human fibroblasts, and (2) the allocyclic X chromosome of rhesus and Cebus lymphocytes. Our results show that at least in one species (C. apella), and probably in rhesus and Leontopithecus, there is no tissue-specific difference between the late DNA replication patterns of the allocyclic X chromosome as there is between human lymphocytes and fibroblasts.     

Molecular and cytological characterization of repetitive DNA sequences from the centromeric heterochromatin of <Emphasis Type="Italic">Sciara coprophila</Emphasis>

Sciara coprophila (Diptera, Nematocera) constitutes a classic model to analyze unusual chromosome behavior such as the somatic elimination of paternal X chromosomes, the elimination of the whole paternal, plus non-disjunction of the maternal X chromosome at male meiosis. The molecular organization of the heterochromatin in S. coprophila is mostly unknown except for the ribosomal DNA located in the X chromosome pericentromeric heterochromatin. The characterization of the centromeric regions, thus, is an essential and required step for the establishment of S. coprophila as a model system to study fundamental mechanisms of chromosome segregation. To accomplish such a study, heterochromatic sections of the X chromosome centromeric region from salivary glands polytene chromosomes were microdissected and microcloned. Here, we report the identification and characterization of two tandem repeated DNA sequences from the pericentromeric region of the X chromosome, a pericentromeric RTE element and an AT-rich centromeric satellite. These sequences will be important tools for the cloning of S. coprophila centromeric heterochromatin using libraries of large genomic clones.     

The location of highly repetitious DNA in the somatic chromosomes of Drosophila melanogaster

In situ hybridization of Drosophila melanogaster somatic chromosomes has been used to demonstrate the near exact correspondence between the location of highly repetitious DNA and classically defined constitutive heterochromatin. The Y chromosome, in particular, is heavily labeled even by cRNA transcribed from female (XX) DNA templates (i.e., DNA from female Drosophila with 2 Xs and 2 sets of autosomes). This observation confirms earlier reports that the Y chromosome contains repeated DNA sequences that are shared by other chromosomes. In grain counting experiments the Y chromosome shows significantly heavier label than any other chromosome when hybridized with cRNA from XY DNA templates (i.e., DNA from male Drosophila with 1 X and 1 Y plus 2 sets of autosomes). However, the preferential labeling of the Y is abolished if the cRNA is derived from XX DNA. We interpret these results as indicating the presence of a class of Y chromosome specific repeated DNA in D. melanogaster. The relative inefficiency of the X chromosome in binding cRNA from XY and XYY DNA templates, coupled with its ability to bind XX derived cRNA, may also indicate the presence of an X chromosome specific repeated DNA.     

Chimaerism detection in bovine twins,triplets and quadruplets using sex chromosome‐linked markers

The phenomenon of chimaerism occurs in the majority of cattle twin pregnancies. The objectives of this study were to develop a powerful diagnostic test for chimaerism in bovine male and female co‐twins using X and Y chromosome‐linked markers and to determine the extent of chimaerism in twins, triplets and quadruplets. We developed a multiplex PCR set of three polymorphic markers on chromosome X (DIK2865, DIK2283, AGLA257), where the presence of >1 and >2 alleles per marker is sufficient to prove chimaerism in males and females, respectively. In addition, a specific segment on chromosome Y (BOV97M) is included in the set to indicate chimaerism in females. Visualization of chimaeric alleles was best for DNA extracted from blood, fair for DNA from vaginal smears and failed for DNA extracted from hair. The power of chimaerism identification using this set of markers for DNA extracted from blood was calculated as 99% in males and virtually 100% in females. All females and males in heterosexual twins, triplets and quadruplets displayed evidence of a chimaeric allele in at least one and maximum of three of three X chromosome markers analysed. In addition, all females showed the presence of the BOV97M segment and were validated as chimaeric by the standard clinical diagnosis of impaired vaginal length. Quantitative PCR analysis of BOV97M copies in all twins vs. their sires showed a mean ratio of 45–68% in females and 39–49% in males, indicating a substantial symmetrical exchange of cells among all co‐twins. The proposed analysis of X and Y chromosome‐linked markers is advantageous to previous methods based on Y chromosome sequences only, because it detects chimaerism in both male and female co‐twins.     

A t(Y;15) translocation with a deletion of the proximal Yq in a boy with mixed gonadal dysgenesis

Summary A Japanese boy with genital malformation and mixed gonadal dysgenesis is described. The karyotype appeared to be 46,X t(15;Y)(p13;q11). A comparison of the Q-positive segment on der(15) with that of the paternal Y chromosome revealed, however, the loss of over half of the Q-positive segment from the paternal Y during t(15;Y) translocation. The father had an unusually long Y chromosome that corresponded to a chromosome 18. DNA analysis further revealed a deletion of the non-fluorescent part of the long arm of the Y chromosome spanning interval 5–6.     

Meiosis in males of the brown planthopper, Nilaparvata lugens

Meiosis in spermatocytes was studied among the three biotypes of the brown planthopper, Nilaparvata lugens (Stål). The chromosome number, morphology, and behavior of the biotypes were similar. The autosomes divided normally, reductional for the first division and equational for the second. The X chromosome had an unstable segregation behavior; it may segregate equationally or reductionally during the first division depending on its chromatid's orientation. Metaphase I plates contained 14 autosomal bivalents and an X chromosome (14_II + X). The co-oriented sister chromatids of the X chromosome moved together to either one of the poles during anaphase I, while auto-oriented sister chromatids segregated to opposite poles. Because of the anomalous segregation behaviour of the X chromosome, metaphase II plates may both contain 14_I + X chromatid, or one with only 14_I and the other with 14_I + X chromosome. Male N. lugens has a diploid chromosome number of 29.

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Zusammenfassung Die Meiose in Spermatozyten wurde in drei Biotypen von Nilaparvata lugens (Stål) studiert. Die Anzahl der Chromosomen, die Morphologie und das Verhalten der Biotypen waren ähnlich. Die Autosomen teilten in einer normalen Weise, reduzierend für die erste und äquatorial für die zweite Teilung. Das X-Chromosom hatte ein unstabiles Teilungsverhalten; es kann sich äquatorial oder aber auch reduktional trennen, je nachdem wie sich sein Chromatid orientiert hat. Die Ebenen der Metaphase I enthalten 14 autosome Bivalente und ein X-Chromosom (14_II + X). Die co-orientierten Schwester-chromatiden des X-Chromosoms bewegen sich während der Anaphase I zusammen zu einem der beiden Pole, während auto-orientierte Schwester-chromatiden sich nach entgegengesetzten Polen trennen. Wegen des ungewöhnlichen Teilungsverhaltens des X-Chromosoms, können beide Metaphase II Ebenen 14_I und X Chromatid enthalten, oder aber eine Ebene enthält 14_I und die andere 14_I + X.

     

Common sequence motifs at the rearrangement sites of a constitutional X/autosome translocation and associated deletion.

Reciprocal chromosome translocations are common de novo rearrangements that occur randomly throughout the human genome. To learn about causative mechanisms, we have cloned and sequenced the breakpoints of a cytologically balanced constitutional reciprocal translocation, t(X;4)(p21.2;q31.22), present in a girl with Duchenne muscular dystrophy (DMD). Physical mapping of the derivative chromosomes, after their separation in somatic cell hybrids, reveals that the translocation disrupts the DMD gene in Xp21 within the 18-kb intron 16. Restriction mapping and sequencing of clones that span both translocation breakpoints as well as the corresponding normal regions indicate the loss of approximately 5 kb in the formation of the derivative X chromosome, with 4-6 bp deleted from chromosome 4. RFLP and Southern analyses indicate that the de novo translocation is a paternal origin and that the father's X chromosome contains the DNA that is deleted in the derivative X. Most likely, deletion and translation arose simultaneously from a complex rearrangement event that involves three chromosomal breakpoints. Short regions of sequence homology were present at the three sites. A 5-bp sequence, GGAAT, found exactly at the translocation breakpoints on both normal chromosomes X and 4, has been preserved only on the der(4) chromosome. It is likely that the X-derived sequence GGAATCA has been lost in the formation of the der(X) chromosome, as it matches an inverted GAATCA sequence present on the opposite strand exactly at the other end of the deleted 5-kb fragment. These findings suggest a possible mechanism which may have juxtaposed the three sites and mediated sequence-specific breakage and recombination between nonhomologous chromosomes in male meiosis.     

Linkage assignment of a DNA sequence (ERCC2L1) homologous to a human DNA repair gene in Xiphophorus fishes: Implications for the evolutionary derivation of human chromosome 19

Fish gene mapping studies have identified several syntenic groups showing conservation over more than 400 million years of vertebrate evolution. In particular, Xiphophorus linkage group IV has been identified as a homolog of human chromosomes 15 and 19. During mammalian evolution, loci coding for glucosephosphate isomerase, peptidase D, muscle creatine kinase, and several DNA repair genes (ERCC1, ERCC2, and XRCC1) appear as a conserved syntenic group on human chromosome 19. When X. clemenciae and X. milleri PstI endonuclease-digested genomic DNA was used in Southern analysis with a human ERCC2 DNA repair gene probe, a strongly cross-hybridizing restriction fragment length polymorphism was observed. Backcrosses to X. clemenciae from X. milleri × X. clemenciae F₁ hybrids allowed tests for linkage of the ERCC2-like polymorphism to markers covering a large proportion of the genome. Statistically significant evidence for linkage was found only for ERCC2L1 and CKM (muscle creatine kinase), with a total of 41 parents and 2 recombinants (4.7% recombination, χ² = 35.37, P < 0.001); no evidence for linkage to GPI and PEPD in linkage group IV was detected. The human chromosome 19 synteny of ERCC2 and CKM thus appears to be conserved in Xiphophorus, while other genes located nearby on human chromosome 19 are in a separate linkage group in this fish. If Xiphophorus gene arrangements prove to be primitive, human chromosome 19 may have arisen from chromosome fusion or translocation events at some point since divergence of mammals and fishes from a common ancestor.     

Organization and chromosomal distribution of a novel repetitive DNA component from Muntiacus muntjak vaginalis with a repeat length of more than 40 kb

The organization and chromosomal distribution of the repetitive DNA component IB from Muntiacus muntjak vaginalis (MMV) was investigated. DNA fragments of component IB were cloned in cosmids and their structure analysed using restriction nucleases and blot-hybridization experiments. Two cosmids were found to be practically identical by restriction enzyme mapping. The repeat unit of component IB DNA is more than 40 kb and contains the 11 and 18 kb Bam HI fragments, which have previously been shown to cross-hybridize with MMV satellite IA. In addition, the repeat unit contains long stretches of DNA sequences which are unique to component IB. In situ hybridization experiments showed that component IB has the properties characteristic of long interspersed repetitive DNA rather than tandemly repeated satellite DNA. Consistent with this conclusion, only a minor fraction of component IB is located on the X chromosome as demonstrated by the analysis of somatic cell hybrids. This is in marked contrast to satellite IA that is specific for the X chromosome. These results have interesting implications for the evolution of the component I DNA family of the MMV genome.     

Karyotype analysis of the Russian wheat aphid, <Emphasis Type="Italic">Diuraphis noxia</Emphasis> (Kurdjumov) (Hemiptera: Aphididae) reveals a large X chromosome with rRNA and histone gene families

The Russsian wheat aphid (RWA), Diuraphis noxia (Kurdjumov), is a worldwide pest of cereals. Despite its economic importance, little is known about its genome. Here we investigated physical genomic features in RWA by karyotype analysis using differential staining with AgNO₃, CMA₃, and DAPI, by chromosomal localization of ribosomal DNA (rDNA), H3 and H4 histone genes, and the “arthropod” telomeric sequence (TTAGG) _n using fluorescence in situ hybridization (FISH), and by measuring the RWA genome size using flow cytometry. The female karyotype, 2n = 10, is composed of four autosome pairs and a pair of X chromosomes, whereas the male karyotype, 2n = 9, has a single X. The X chromosome is the largest element in the karyotype. All three molecular markers used, i.e., 18S rRNA and both H3 and H4 probes are co-localized at one end of the X chromosome. The FISH probes revealed that the AgNO₃-positive bridge between two prometaphase X chromosomes of females, which is believed to be responsible for the elimination of one X chromosome in aphid oocytes determined to undergo male development, contains clusters of both histone genes, in addition to an rDNA cluster. Interestingly, RWA lacks the (TTAGG) _n telomeric sequence in its genome, in contrast to several previously investigated aphid species. Additionally, we compared female and male genome sizes. The female genome size is 2C = 0.86 pg, whereas the male genome size is 2C = 0.70 pg. The difference between the DNA content in the two genders suggests that the RWA X chromosome occupies about 35% of the female haploid genome (1C = 0.43 pg), which makes it one of the largest sex chromosomes in the animal kingdom.     

Selective staining of X chromosome segments in Schistocerca gregaria after denaturation and reassociation procedures

The DNA of fixed mitotic and meiotic chromosomes and of spermatides of Schistocerca gregaria males was heat denaturated and then differentially reassociated in a Giemsa buffer or in acridine orange buffer solution. After this procedure, two to three large, selectively stained regions are seen in the X chromosome of spermatocytes and spermatides. Denaturation and reassociation experiments have shown that after differential reassociation such a selective stainability of chromosome regions is characteristic for the presence of fast-reassociating, i.e., repetitive DNA (Stockert and Lisanti, 1972). The possible presence of repetitive DNA in the X chromosome regions concerned can not be the only reason for the occurrence of the heavily stained segments after reassociation because (1) these segments are obtained in positively heteropycnotic X chromosomes, but not in negatively heteropycnotic Xs and (2) they do not occur in positively heteropycnotic X chromosomes when the histones have been extracted before the denaturation and reassociation processes. Contrary to the latter statement, the heavily stained X chromosomal regions are preserved when the histones are removed after the denaturation and reassociation steps. — It is assumed that the heavily stained X chromosome segments represent DNA reassociation complexes which are only formed if histones are present. It is discussed whether the formation of the X chromosome complexes depends on a specific chromatin configuration within positively heteropycnotic X chromosomes.     

Characterization of a set of X-linked sequences and of a panel of somatic cell hybrids useful for the regional mapping of the human X chromosome

Summary We have characterized 19 DNA fragments originating from the human X chromosome. Most of them have been isolated from an X chromosome genomic library (Davies et al. 1981) using a systematic screening procedure. These DNA probes have been used to search for restriction fragment length polymorphisms (RFLP). The frequency of restriction polymorphisms (1 per 350 bp analysed) was lower than expected from data obtained with autosomal fragments. The various probes have been mapped within 12 subchromosomal regions using a panel of human-rodent hybrid cell lines. The validity of the panel was established by hybridization experiments performed with 27 X-specific DNA probes, which yielded information on the relative position of translocation break-points on the X chromosome. The DNAs from the various hybrid lines are blotted onto a reusable support which allows one to quickly map any new X-specific DNA fragment. The probes already isolated should be of use to map unbalanced X chromosome aberrations or to characterize new somatic cell hybrid lines. The probes which detect RFLPs define new genetic markers which will help to construct a detailed linkage map of the human X chromosome, and might also serve for the diagnosis of carriers or prenatal diagnosis.     

正常中国人和DMD患者X染色体Xp~(21)区的RFLP研究

本文应用从人类X柒色体Xp~(21)区不同部位分离得到的9种DNA探针,分析了100名正常中国人,38名DMD患者及其母亲X柒色体Xp~(21)区的14个限制性位点多态性(RSP;又称限制性片段长度多态性,RFLP)。发现正常的X染色体与携带DMD基因的X染色体Xp~(21)区的RFLP频率没有明显差别;在38例DMD患者中有7例的X染色体有DNA片段缺失;在本文分析的24例患者母杀中有17例是DMD基因携带者,她们在Xp~(21)区的RFLP均存在杂合的多态性,因此可以应用RFLP连锁分析对这些家系进行DMD的产前诊断。     

Late replication patterns in adult and embryonic mice carrying Searle's X-autosome translocation

Bromodeoxyuridine-dye technique analysis of X chromosome DNA synthesis in female adult and fetal mice carrying the balanced form of the T(X; 16) 16H translocation demonstrated that the structurally normal X chromosome was late replicating (and hence presumably inactive) in 93% of the adult cells and 99% of the 9-day embryo cells, with the X¹⁶ chromosome late replicating in the remaining cells. We conclude from these results that in T16H/+ females either there is preferential inactivation of the normal X chromosome or that, if inactivation is random, cell selection takes place before 9 days of development. Two 9-day female embryos with an unbalanced karyotype were also studied; both had two late-replicating chromosomes in most of their cells, one being the chromosome 16^X, the other a normal X chromosome. These results, together with the presence of a late-replicating X¹⁶ chromosome in T16H/+ adult and fetal mice, support the concept that more than one inactivation center is present on the X chromosome of the mouse because the X¹⁶ and the 16^x chromosomes can be late replicating.