Isolation and characterization of chromosome-specific DNA sequences from a chromosome arm genomic library of common wheat

Isolation, physical mapping and polymorphism of chromosome-specific DNA sequences in wheat are reported. Following the microdissection of the long arm of chromosome 5B (5BL) of common wheat, its DNA was amplified by degenerate oligonucleotide-primed PCR and directly cloned into plasmid vectors. Characterization of the chromosome arm library showed that ∼55% of the inserts are of low-copy nature. Southern analysis using aneuploid lines of common wheat revealed that five of 11 low-copy inserts analyzed map to chromosome arm 5BL; four of these are 5BL-specific. By deletion mapping, the 5BL-specific sequences were located to sub- chromosome arm regions. Based on the hybridization patterns of three 5BL-specific sequences to DNA from a diverse collection of goat-grass ( Aegilops ) and wheat ( Triticum ) species, it was concluded that these sequences emerged at different times in the course of evolution of this group of plant species.     

Deletion and amplification of the HGPRT locus in Chinese hamster cells.

Somatic cell selective techniques and hybridization analyses with a cloned cDNA probe were used to isolate and identify Chinese hamster cell lines in which the X-linked gene for hypoxanthine-guanine phosphoribosyltransferase (HGPRT) has been altered. Two of 19 HGPRT-deficient mutants selected were found to have major DNA deletions affecting the HGPRT locus. Cytogenetic studies revealed that the X chromosome of each deletion mutant had undergone a translocation event, whereas those from the remaining 17 mutants were normal. Phenotypic revertants of the thermosensitive HGPRT mutant RJK526 were isolated, and amplification of the mutant allele was shown to be the predominant mechanism of reversion. Comparisons of restriction enzyme fragments of DNA from deletion versus amplification strains identified two regions of the Chinese hamster genome that contained homology to the cDNA probe. One was shown to be much larger than the 1,600-nucleotide mRNA for HGPRT and to be comprised of linked fragments that contained the functional HGPRT gene. The second was neither transcribed nor tightly linked to the functional gene. These initial studies of HGPRT alterations at the level of DNA thus identified molecular mechanisms of phenotypic variation.     

Human and mouse amelogenin gene loci are on the sex chromosomes

Enamel is the outermost covering of teeth and is the hardest tissue in the vertebrate body. The enamel matrix is composed of enamelin and amelogenin classes of protein. We have determined the chromosomal locations for the human and mouse amelogenin (AMEL) loci using Southern blot analyses of DNA from human, mouse, or somatic cell hybrids by hybridization to a characterized mouse amelogenin cDNA. We have determined that human AMEL sequences are located on the distal short arm of the X chromosome in the p22.1----p22.3 region and near the centromere on the Y chromosome, possibly at the proximal long arm (Yq11) region. These chromosomal assignments are consistent with the hypothesis that perturbation of the amelogenin gene is involved in X-linked types of amelogenesis imperfecta, as well as with the Y-chromosomal locations for genes that participate in regulating tooth size and shape. Unlike the locus in humans, the mouse AMEL locus appears to be assigned solely to the X chromosome. Finally, together with the data on other X and Y chromosome sequences, these data for AMEL mapping support the notion of a pericentric inversion occurring in the human Y chromosome during primate evolution.     

Molecular detection of a Yp/18 translocation in a 45,X holoprosencephalic male

Summary Prenatal diagnosis in a fetus with holoprosencephaly showed a 45,X karyotype and a suspected 18p abnormality. At birth, the fetus presented with normal male genitalia. Y chromatin was not cytogenetically detectable by Q-, G-, or G11-banding. Mosaicism for a cell line containing a Y chromosome was not observed in amniocytes, lymphocytes, or skin fibroblasts. Southern blot analysis for 11 different Y-DNA loci demonstrated the presence in the patient's genome of sequences derived from the short arm, centromeric region, and proximal long arm of the Y chromosome (intervals 1–5). The distal long arm of the Y (intervals 6 and 7) was absent. In situ hybridization with the Y-derived probe pDP105 showed silver grains over the short arm of the del(18) chromosome, suggesting a Y/18 translocation with loss of 18p and distal Yq material.     

Repeated DNA sequences in the distal long arm of the human X chromosome

Summary Two DNA probes from within a single large insert from a recombinant phage-DNA library that was constructed from flow-sorted chromosomes enriched for the human X chromosome were shown to hybridize with repeated X-specific and autosomal DNA sequences. The X-chromosomal repeated sequences were assigned to the distal long arm of the X chromosome by both hybrid mapping and in situ hybridization. Fine mapping places these repeats in a region of Xq28 between DX13 (DXS15, in distal Xq28) and factor VIII (F8C, in proximal Xq28). The location of the X-specific repeats makes them potentially useful for future investigations of discases mapping to the distal long arm of the X chromosome, such as the fragile X syndrome.     

Three cases of 45,X/46,XYnf mosaicism

Summary Three patients with 45,X/46,XYnf mosaicism were investigated by Southern hybridization using both X- and Y-specific DNa probes. Our patients seem to be hemizygous for the X chromosomal loci tested. Single-copy and low-copy repeated Y chromosomal sequences assigned to the short arm, centromere, and euchromatin of the long arm have been detected in our patients, suggesting the Y chromosomal origin of the marker chromosome both in male and female cases studied. Densitometry of autoradiographs revealed a double dose of Yp-specific fragments of the DXYS1 locus. None of the patients tested showed either the 3.4- or the 2.1-kb Hae III malespecific repeated DNa sequences. It seems likely that the Ynf is a pseudodicentric chromosome with duplication of Yp and euchromatic Yq sequences, the Yq heterochromatin being lost. Our findings indicate structural heterogeneity of the marker chromosome and in addition provide further information on the relative position of DNa sequences detectected by DNA probes 50f2, M1A, and pDP105.     

Comparative genetic mapping of cellular rel sequences in man, mouse, and the domestic cat.

We used in situ hybridization techniques to assign the human c-rel locus to the centromere-proximal portion of the short arm of chromosome 2 (2cent-2p13). We also determined the chromosomal location of c-rel sequences in the domestic cat and the laboratory mouse by using a human c-rel fragment to screen panels of rodent X cat and hamster X mouse somatic cell hybrid DNAs. The c-rel locus apparently maintains similar syntenic relationships with other known genetic markers in the human and cat, but displays different linkage relationships in the mouse.     

Assignment of Three Gene Loci (Pgk, Hgprt, G6pd) to the Long Arm of the Human X Chromosome by Somatic Cell Genetics

The intrachromosomal localization of three X-linked gene loci (PGK, HGPRT and G6PD) has been determined using a somatic cell genetic approach. A human cell line possessing an X/14 translocation was used as one parent in the formation of human/mouse hybrids. The translocation separates the human X into two parts: Xp and t(Xq14q). The data indicate that all three X-linked loci segregate with the t(Xq14q) rearrangement product thus permitting their assignment to the X chromosome's long arm. Secondary rearrangements and data from other laboratories suggest that the order of the the three markers from the centromere to the distal end of the X long arm is PGK, HGPRT, G6PD. It was also observed that NP, an autosomal locus, segregated with the t(Xq14q) chromosome. This provides strong support for the assignment of NP to 14.     

A PCR product derived from female DNA with regional localization on the Y chromosome.

A 154-bp PCR product amplified from human female DNA mapped onto the Y chromosome under high-stringency in situ hybridization conditions. The female DNA sequence revealed an 89% homology with the HSDYZ1 sequence. When the same primers were used to amplify male DNA, a 154-bp DNA fragment was also obtained, showing a 98% homology with HSDYZ1. However, although the HSDYZ1 sequence is widely distributed along the long arm of the Y chromosome, both of these particular PCR products are di-regionally localized within this distal block of constitutive heterochromatin. In situ hybridization under lower stringency showed that these 154-bp sequences map both onto the autosomes and the Y chromosome. Overall, this paper shows (i) a new class of DNA sequences shared by the autosomes and the Y chromosome; and (ii) a substructured organization of some DNA repeats within the DYZ1 family that forms a large part of the constitutive heterochromatin of the Y chromosome.     

Assignment of genes encoding metallothioneins I and II to Chinese hamster chromosome 3: evidence for the role of chromosome rearrangement in gene amplification.

Cadmium resistant (Cdr) variants with coordinately amplified metallothionein I and II (MTI and MTII) genes have been derived from both Chinese hamster ovary and near-euploid Chinese hamster cell lines. Cytogenetic analyses of Cdr variants consistently revealed breakage and rearrangement involving chromosome 3p. In situ hybridization with a Chinese hamster MT-encoding cDNA probe localized amplified MT gene sequences near the translocation breakpoint involving chromosome 3p. These observations suggested that both functionally related, isometallothionein loci are linked on Chinese hamster chromosome 3. Southern blot analyses of DNAs isolated from a panel of Chinese hamster X mouse somatic cell hybrids which segregate hamster chromosomes confirmed that both MTI and MTII are located on chromosome 3. We speculate that rearrangement of chromosome 3p could be causally involved with the amplification of MT genes in Cdr hamster cell lines.     

Rye chromosome translocations in hexaploid wheat: a re-evaluation of the loss of heterochromatin from rye chromosomes

Summary Using in situ hybridization techniques, we have been able to identify the translocated chromosomes resulting from whole arm interchanges between homoeologous chromosomes of wheat and rye. This was possible because radioactive probes are available which recognize specific sites of highly repeated sequence DNA in either rye or wheat chromosomes. The translocated chromosomes analysed in detail were found in plants from a breeding programme designed to substitute chromosome 2R of rye into commercial wheat cultivars. The distribution of rye highly repeated DNA sequences showed modified chromosomes in which (a) most of the telomeric heterochromatin of the short arm and (b) all of the telomeric heterochromatin of the long arm, had disappeared. Subsequent analyses of these chromosomes assaying for wheat highly repeated DNA sequences showed that in type (a), the entire short arm of 2R had been replaced by the short arm of wheat chromosome 2B and in (b), the long arm of 2R had been replaced by the long arm of 2B. The use of these probes has also allowed us to show that rye heterochromatin has little effect on the pairing of the translocated wheat arm to its wheat homologue during meiosis. We have also characterized the chromosomes resulting from a 1B-1R translocation event.From these results, we suggest that the observed loss of telomeric heterochromatin from rye chromosomes in wheat is commonly due to wheat-rye chromosome translocations.     

Semi-automatic laser beam microdissection of the Y chromosome and analysis of Y chromosome DNA in a dioecious plant, Silene latifolia

Silene latifolia has heteromorphic sex chromosomes, the X and Y chromosomes. The Y chromosome, which is thought to carry the male determining gene, was isolated by UV laser microdissection and amplified by degenerate oligonucleotide-primed PCR. In situ chromosome suppression of the amplified Y chromosome DNA in the presence of female genomic DNA as a competitor showed that the microdissected Y chromosome DNA did not specifically hybridize to the Y chromosome, but hybridized to all chromosomes. This result suggests that the Y chromosome does not contain Y chromosome-enriched repetitive sequences. A repetitive sequence in the microdissected Y chromosome, RMY1, was isolated while screening repetitive sequences in the amplified Y chromosome. Part of the nucleotide sequence shared a similarity to that of X-43.1, which was isolated from microdissected X chromosomes. Since fluorescence in situ hybridization analysis with RMY1 demonstrated that RMY1 was localized at the ends of the chromosome, RMY1 may be a subtelomeric repetitive sequence. Regarding the sex chromosomes, RMY1 was detected at both ends of the X chromosome and at one end near the pseudoautosomal region of the Y chromosome. The different localization of RMY1 on the sex chromosomes provides a clue to the problem of how the sex chromosomes arose from autosomes.     

Characterization and comparative analysis of DNA from the pericentric heterochromatin of chromosome 2 of Anopheles atroparvus V. Tiel (Culicidae, Diptera)

The minilibrary containing DNA sequences from the diffuse pericentric heterochromatin from the right arm of Anopheles atroparvus V. Tiel (Culicidae, Diptera) chromosome 2 (2R) was generated by use of chromosome microdissection technique. Southern-blot hybridization of the minilibrary fragments with the labeled genomic DNA of A. atroparvus and analysis of their primary structure showed that this heterochromatin region contained repeated DNA sequences differed by their primary structure and the number of copies. These were mostly AT-rich sequences harboring the features characteristic of the S/MAR regions. Based on the clones homology to the sequences from the An. gambiae and Drosophila melanogaster genomes, it was demonstrated that the pericentric heterochromatin from the right arm of An. atroparvus chromosome 2 contained gypsy-like transposable elements, as well as the sequences homologous to the structural genes. In situ hybridization with the chromosomes of A. atroparvus and of the two representatives of the Anopheles maculipennis species complex, A. messeae and A. beklemishevi, showed that pericentric regions of all these chromosomes contained DNA sequences homologous to the sequences from the region-specific minilibrary. Cloned fragments of conserved repetitive DNA revealed upon interspecific Southern-blot hybridization of the clones with the labeled genomic DNA of A. messeae can be utilized in further investigations of evolutionary rearrangements of the pericentric heterochromatin within the Anopheles maculipennis species complex.     

人14p＋标记染色体的分子细胞遗传学研究

一例23岁女性患者因近五、六年来出现胡须、四肢多毛及偶有月经不规则而就诊。细胞遗传学检查发现一个短臂明显增大的亚中着丝粒的14号标记染色体14p ·p 区域GTG显带呈浅染,C-带暗染,都呈均匀的染色区。硝酸银染色在p 远侧端显现一个Ag-NOR,其大小与正常近端着丝粒染色体的无明显差异。应用~3H标记的7.3 kb长的rRNA基因探针进行染色体原位杂交,自显影银颗粒沿整个p 区域分布,p 上的银颗粒数是正常近端着丝粒染色体短臂上银颗粒平均数的5倍。这些结果排除了Y或其他染色体参加的重排形成p 的可能性,并表明Ag-NOR的大小或NOR的数目并不一定与rRNA基因的数量成正比。研究Dp 或Gp 类型的染色体变异,对了解人二倍体细胞内rRNA基因表达的调控有重要意义。     

Characteristics of the distribution of DNA repetitive sequences in the sex chromosomes of 4 species of rodent

Four rodent species with very large heterochromatic regions on the sex chromosomes have been studied using in situ DNA/DNA hybridization techniques. Repetitious DNA fractions were obtained at C0t 0-0.01. Heterochromatic regions of X and X chromosomes of Cricetulus barabensis and Phodopus sungorus, and the heterochromatic long arm of the Y chromosome of Mesocricetus auratus do not contain disproportionately high amounts of repeated DNA sequences. Heterochromatic regions on sex chromosomes of Microtus subarvalis contain high amounts of repeated DNA sequences. Additional heterochromatic autosomal arms, a heterochromatic arm of the X chromosome, and a short arm of the Y chromosome of Mesocricetus auratus contain high amounts of repeated DNA sequences too.     

Characterization of a low copy repetitive element S232 involved in the generation of frequent deletions of the distal short arm of the human X chromosome.

There are several copies of related sequences on the distal short arm of the human X chromosome and the proximal long arm of the Y chromosome which were originally detected by cross hybridization with a genomic DNA clone, CRI-S232. Recombination between two S232-like sequences flanking the steroid sulfatase locus has been shown to cause frequent deletions in the X chromosome short arm, resulting in steroid sulfatase deficiency. We now report the characterization of several S232-like sequences. Restriction mapping and sequence analysis show that each S232 unit contains 5 kb of unique sequence in addition to two elements, RU1 and RU2, composed of a variable number of tandem repeats. RU1 consists of 30 bp repeating units and its length shows minimal variation between individuals. The RU2 elements in the hypervariable S232 loci on the X chromosome consist of repeating sequences which are highly asymmetric, with about 90% purines and no C's on one strand. The X-derived RU2 elements range from 0.6 kb to over 23 kb among different individuals, accounting entirely for the observed polymorphism at the S232 loci. Although the repeating units of the RU2 elements in the nonpolymorphic S232 loci on the Y chromosome share high sequence homology with those on the X chromosome, they exhibit much higher intrarepeat sequence variation. S232 homologous sequences are found in great apes, old world and new world monkeys. In chimpanzees and gorillas the S232-like sequences are polymorphic in length.     

A 45,X male with Y-specific DNA translocated onto chromosome 15.

A 20-year-old male patient with chromosomal constitution 45,X, testes and normal external genitalia was examined. Neither mosaicism nor a structurally aberrant Y chromosome was observed when routine cytogenetic analysis was performed on both lymphocytes and skin fibroblasts. Y chromosome-specific single-copy and repeated DNA sequences were detected in the patient's genome by means of 11 different recombinant-DNA probes of known regional assignment on the human Y chromosome. Data indicated that the short arm, the centromere, and part of the long-arm euchromatin of the Y chromosome have been retained and that the patient lacks deletion intervals 6 and 7 of Yq. High-resolution analysis of prometaphase chromosomes revealed additional euchromatic material on the short arm of one of the patient's chromosomes 15. After in situ hybridization with the Y chromosome-specific probe pDP105, a significant grain accumulation was observed distal to 15p11.2, suggesting a Y/15 chromosomal translocation. We conclude that some 45,X males originate from Y-chromosome/autosome translocations following a break in the proximal long arm of the Y chromosome.     

Molecular cloning of zebra finch W chromosome repetitive sequences: evolution of the avian W chromosome

The zebra finch (Taeniopygia guttata) has a large Z chromosome and highly condensed W chromosome. We used the random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) technique to isolate female-specific sequences ZBM1 and ZBM2. Southern blot hybridization to male and female zebra finch genomic DNA suggested that these sequences were located on the W chromosome, although homologous sequences appeared to be autosomal or Z-linked. Fluorescent in situ hybridization (FISH) using bacterial artificial chromosome (BAC) clones corresponding to ZBM sequences showed hybridization to the whole W chromosome, suggesting that the BACs encode sequences that are repeated across the entire W chromosome. Based on the sequencing of a ZBM repetitive sequence and Z chromosome derived BAC clones, we demonstrate a random distribution of repeat sequences that are specific to the W chromosome or encoded by both Z and W. The positions of ZW-common repeat sequences mapped to a noncoding region of a Z chromosome BAC clone containing the CHD1Z gene. The apparent lineage-specificity of W chromosome repeat sequences in passerines and galliform birds suggest that the W chromosome had not differentiated well from the Z at the time of divergence of these lineages. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A 45,X male with a Yp/18 translocation

Summary A patient described as a 45,X male (Forabosco et al. 1977) was examined for the presence of Y-specific DNA by using various probes detecting restriction fragments from different regions of the Y chromosome. Positive hybridization signals were obtained for Yp fragments only. In situ hybridization with two different probes, pDP31 and the pseudoautosomal probe 113F, led to a clear assignment of the Yp sequences to the short arm of one chromosome 18. Cytogenetically, the presence of all of Yp including the Y centromere on 18p could be demonstrated replacing a segment of similar size of 18p. Thus, the Y/18 translocation chromosome is dicentric structurally, but it was shown to be monocentric functionally with the no. 18 centromere active. Gene dosage studies with the probe B74 defining a sequence at 18p11.3 demonstrated a single dose of this sequence in the patient. In agreement with these observations, the patient shows clinical signs of the 18p-syndrome. It is concluded that in XO males in general, the X is of maternal origin while the maleness is due to a de novo Y/autosome translocation derived from the father. Depending on the nature of the autosomal deficiency caused by the Y/autosome translocation, the patient may have congenital malformations.