Visualization of the terminal structure of rice chromosomes 6 and 12 with multicolor FISH to chromosomes and extended DNA fibers

High-resolution fluorescence in situ hybridization (FISH) on interphase and pachytene nuclei, and extended DNA fibers enabled microscopic distinction of DNA sequences less than a few thousands of base pairs apart. We applied this technique to reveal the molecular organization of telomere ends in japonica rice (Oryza sativa ssp. japonica), which consist of the Arabidopsis type TTTAGGG heptameric repeats and the rice specific subtelomeric tandem repeat sequence A (TrsA). Southern hybridizations of DNA digested with Bal31 and EcoRI, and FISH on chromosomes and extended DNA fibers demonstrated that (1) all chromosome ends possess the telomere tandem repeat measuring 3–4 kb; (2) the subtelomeric TrsA occurs only at the ends of the long arms of chromosomes 6 and 12, and measure 6 and 10 kb, which corresponds to 231 and 682 copies for these sites, respectively; (3) the telomere and TrsA repeats are separated by at most a few thousands of intervening nucleotide sequences. The molecular organization for a general telomere organization in plant chromosomes is discussed.     

Conserved autonomy of replication of the X-chromosomes in hybrids of Drosophila miranda and Drosophila persimilis

The chromosome complement of hybrid males from the cross between Drosophila miranda female and D. persimilis male provides an interesting chromosomal situation where an autosome, the 3rd chromosome of D. persimilis, coexists with a homologue that developed into a sex chromosome, the X₂ in D. miranda. Except for certain inversions and a few minor translocations, these two chromosomes (X₂ and the 3rd) still look alike as polytene elements. However, in hybrid males pairing of the two chromosomes, the X₂ and 3rd, is rare, while in female hybrids it occurs frequently. — ³H-TdR labeling shows that while the X₂ and 3rd chromosomes replicate synchronously in hybrid female, in the hybrid male the former completes its replication earlier than the 3rd chromosome, as do the two arms of the X₁ (XL and XR). The frequency and relative intensity of ³H-TdR labeling of each site of the X₂ and that of the 3rd chromosome in hybrid males closely agree with those of the corresponding sites in the X₂ of the miranda male and the 3rd chromosome of the persimilis male (or female), respectively. The results suggest that timing and rate of replication of the X₂ are determined autonomously and follow the pattern in the respective parental species.     

Dosage compensation of a retina-specific gene in Drosophila miranda

The X1R chromosome of Drosophila miranda and the 3L autosome of Drosophila melanogaster are thought to have originated from the ancestral D chromosomal element and therefore may contain the same set of genes. It is expected that these genes will be dosage compensated in D. miranda because of their X linkage. To test these possibilities and to study evolution of the dosage compensation mechanism, we used the 3L-linked autosomal head-specific gene 507ml of D. melanogaster to isolate the homologous gene (507 mr) from a D. miranda genomic library. In situ hybridization showed that gene 507 is located at the 12A region of the X1R chromosome of D. miranda, indicating that the chromosomal homology deduced by cytogenetic means is correct. Restriction analysis and cross-specific DNA and RNA blot hybridization revealed the presence of extensive restriction pattern polymorphism and lack of sequence similarity in some areas of the 507 mr and 507 ml DNA, including the 3 portion of the transcribed region. However, the 5 portion of the transcribed region and the DNA sequences, located approximately 0.8 kb upstream and 3 kb downstream from the 507 ml gene showed a high degreee of similarity with the DNA sequences of comparable regions of the 507 mr gene. In both species gene 507 codes for a highly abundant 1.8 kb RNA which is expressed in the retina of the compound eye. Although in D. miranda the males have one and the females have two copies of the 507 gene, the steady-state levels of the 507 mRNA in both sexes were found to be similar, indicating that gene 507 is dosage compensated in D. miranda. Thus, along with the disparate rates of evolution in different areas of the DNA associated with gene 507, in D. miranda this gene has come under the regulation of the X chromosomal dosage compensation mechanism.by M.L. Pardue     

Telomere repeats within the neo-Y-chromosome of Drosophila miranda

The clone Dmir1098, isolated from a genomic lambda library of Drosophila miranda labels exclusively the tips of the giant chromosomes in the highly polytenized nuclei of the female larval salivary glands. However, the in situ hybridizations to male metaphase plates, using the same probe, reveal a massive labeling block within the neo-Y-chromosome in addition to the labeling blocks at both chromosome ends. From the comparison with the Y chromosome labeling pattern of D. pseudoobscura, a sibling species to D. miranda, an end-to-end fusion mechanism involving the telomere repeats would be the most straightforward explanation for the karyotype change in D. miranda.     

Toward closing rice telomere gaps: mapping and sequence characterization of rice subtelomere regions

Despite the collective efforts of the international community to sequence the complete rice genome, telomeric regions of most chromosome arms remain uncharacterized. In this report we present sequence data from subtelomere regions obtained by analyzing telomeric clones from two 8.8 × genome equivalent 10-kb libraries derived from partial restriction digestion with HaeIII or Sau3AI (OSJNPb HaeIII and OSJNPc Sau3AI). Seven telomere clones were identified and contain 25–100 copies of the telomere repeat (CCCTAAA)_n on one end and unique sequences on the opposite end. Polymorphic sequence-tagged site markers from five clones and one additional PCR product were genetically mapped on the ends of chromosome arms 2S, 5L, 10S, 10L, 7L, and 7S. We found distinct chromosome-specific telomere-associated tandem repeats (TATR) on chromosome 7 (TATR7) and on the short arm of chromosome 10 (TATR10s) that showed no significant homology to any International Rice Genome Sequencing Project (IRGSP) genomic sequence. The TATR7, a degenerate tandem repeat which is interrupted by transposable elements, appeared on both ends of chromosome 7. The TATR10s was found to contain an inverted array of three tandem repeats displaying an interesting secondary folding pattern that resembles a telomere loop (t-loop) and which may be involved in a protective function against chromosomal end degradation.Electronic Supplementary Material Supplementary material is available for this article at     

Defining the orientation of the tandem fusions that occurred during the evolution of Indian muntjac chromosomes by BAC mapping

The Indian muntjac (Muntiacus muntjak vaginalis) has a karyotype of 2n=6 in the female and 7 in the male, the karyotypic evolution of which through extensive tandem fusions and several centric fusions has been well-documented by recent molecular cytogenetic studies. In an attempt to define the fusion orientations of conserved chromosomal segments and the molecular mechanisms underlying the tandem fusions, we have constructed a highly redundant (more than six times of whole genome coverage) bacterial artificial chromosome (BAC) library of Indian muntjac. The BAC library contains 124,800 clones with no chromosome bias and has an average insert DNA size of 120 kb. A total of 223 clones have been mapped by fluorescent in situ hybridization onto the chromosomes of both Indian muntjac and Chinese muntjac and a high-resolution comparative map has been established. Our mapping results demonstrate that all tandem fusions that occurred during the evolution of Indian muntjac karyotype from the acrocentric 2n=70 hypothetical ancestral karyotype are centromere–telomere (head–tail) fusions.     

The Saccharomyces cerevisiae chromosome III left telomere has a type X, but not a type Y'', ARS region.

A yeast Saccharomyces cerevisiae telomeric region was isolated by chromosome walking from HML alpha, the most distal known gene on the chromosome III left (IIIL) end. The terminal heterodisperse 3.3-kilobase (kb) SalI fragment on chromosome IIIL, 8.6 kb distal to HML alpha, was cloned in a circular vector to generate a telomeric probe. Southern hybridization and DNA sequencing analyses indicated that 0.6 kb (+/- 200 base pairs) of 5'-C1-3A-3' simple tandem repeat sequence, adjacent to a 1.2-kb type X ARS region, constitutes the telomere on the chromosome IIIL end, and no type Y' ARS region homologies exist between HML alpha and the IIIL terminus.     

Multiple sex chromosomes in Drosophila miranda: A system to study the degeneration of a chromosome

Drosophila miranda possesses an intriguing sex chromosome constitution. While female metaphase plates have 10 chromosomes (diploid set), in males only 9 chromosomes can be identified. The missing homologue has been translocated to the Y, forming a neo-Y chromosome which is polytenized in the salivary gland cells. This report presents a detailed characterization of DNA, isolated from D. miranda flies. In situ hybridizations, using cRNA transcribed from unfractionated D. miranda DNA, reveal hybridization to the neo-Y with label distributed over the entire chromosome. The original partner of the translocated chromosome, X², is essentially unlabelled. These results suggest that repetitive DNA sequences invade the translocated chromosome. This result is discussed with reference to the hypothesis of degeneration of the Y chromosome, formulated by Muller (1918, 1932a).     

DNA amplifications and deletions in Streptomyces lividans 66 and the loss of one end of the linear chromosome

Thirty-two 2-deoxygalactose-resistant mutants with DNA amplifications were isolated from Streptomyces lividans 66 strains carrying plasmid pMT664, which carries an agarase gene (dagA) and IS466. Thirty-one of the mutants carried amplified DNA sequences from a 70 kb region about 300 kb from one end of the linear chromosome in this species. In 28 of the mutants, all the wild-type sequences between the amplified region and the start of the 30 kb inverted repeat that forms the chromosome end were deleted. Thus, there appeared to be loss of one chromosome end and its replacement by the DNA amplification. In some mutants there amplification of a previously characterised 5.7 kb sequence that lies about 600 kb from the other chromosome end was also noted.     

Acquisition of telomere repeat sequences by transfected DNA integrated at the site of a chromosome break.

Previous analysis of plasmid DNA transfected into 108 cell clones demonstrated extensive polymorphism near the integration site in one clone. This polymorphism was apparent by Southern blot analysis as diffuse bands that extended over 30 kb. In the present study, nucleotide sequence analysis of cloned DNA from the integration site revealed telomere repeat sequences at the ends of the integrated plasmid DNA. The telomere repeat sequences at one end were located at the junction between the plasmid and cell DNA. The telomere repeat sequences at the other end were located in the opposite orientation in the polymorphic region and were shown by digestion with BAL 31 to be at the end of the chromosome. Telomere repeat sequences were not found at this location in the plasmid or parent cell DNA. Although the repeat sequences may have been acquired by recombination, a more likely explanation is that they were added to the ends of the plasmid by telomerase before integration. Comparison of the cell DNA before and after integration revealed that a chromosome break had occurred at the integration site, which was shown by fluorescent in situ hybridization to be located near the telomere of chromosome 13. These results demonstrate that chromosome breakage and rearrangement can result in interstitial telomere repeat sequences within the human genome. These sequences could promote genomic instability, because short repeat sequences can be recombinational hotspots. The results also show that DNA rearrangements involving telomere repeat sequences can be associated with chromosome breaks. The introduction of telomere repeat sequences at spontaneous or ionizing radiation-induced DNA strand breaks may therefore also be a mechanism of chromosome fragmentation.     

A Novel MS7 Repeat Specific for Heterochromatin of Sex Chromosomes in Voles of Genus Microtus, Group arvalis: Genomic Organization and Chromosomal Localization

The tandemly arranged MS4 repeat with monomeric units of 4.1 kb is species-specifically distributed in heterochromatin of sex chromosomes of four common vole species of genus Microtus, group arvalis. In this work, we studied the genomic organization of the MS4 homolog in euchromatin of the X chromosome of M. arvalis. It has been shown by analyzing the phage genomic clones that one MS4 copy makes a part of a monomeric unit exceeding 8.5 kb that also includes a new MS7 repeat and, possibly, LINE fragments. MS7 is located together with MS4 in heterochromatin of common vole sex chromosomes, but in a substantially lesser amount. Probably, as a result of an evolutionary transition of an original repeat from euchromatin of the X chromosome to heterochromatin of the Y chromosome, MS4 underwent multiple amplification, and MS7 spread throughout heterochromatin, being surrounded by the MS4 tandem arrays.     

Telomere and 45S rDNA sequences are structurally linked on the chromosomes in <Emphasis Type="Italic">Chrysanthemum segetum</Emphasis> L.

Some reports have shown that nucleolar organizer regions are located at the telomeric region and have a structural connection with telomeres at the cellular level in many organisms. In this study, we found that all 45S ribosomal DNA (rDNA) signals were located at telomeric regions on the chromosomes in Chrysanthemum segetum L., and the 45S rDNA showed distinct signal patterns on different metaphase chromosome spreads. The bicolor fluorescence in situ hybridization experiment on the extended fibers revealed that telomere repeats were structurally connected with or interspersed into rDNA sequences. The close cytological structure relation between rDNA and telomere sequences led us to use PCR with combinations of the telomere primer and the rDNA primer to obtain some fragments, which were flanked by different rDNA and telomere primer sequences. One representative clone CHS2 contains closely connected rDNA and telomere sequences, suggesting that the telomere sequence invaded into the conserved rDNA sequence. In addition, the sequences of some PCR clones were flanked by the single telomeric primer sequence or the rDNA primer sequence. These results suggested that homologous recombination occurred between tandem repeat units of rDNA sequences or telomere repeats at the chromosome terminus.     

Evolutionary changes in the organization of the major LCP gene cluster during sex chromosomal differentiation in the sibling speciesDrosophila persimilis,D. pseudoobscura andD. miranda

The major larval cuticle protein (LCP) genes I–IV ofDrosophila melanogaster are clustered on the right arm of the second chromosome. By cross-hybridization we cloned the corresponding genes from three different members of theobscura group:D. persimilis, D. pseudoobscura andD. miranda. InD. pseudoobscura andD. persimilis the gene cluster maps to autosome3. In contrast, inD. miranda it was found on theX2 andY sex chromosome. Hence, this exceptional karyotypic situation offers a unique opportunity to analyse the molecular processes underlying the phenomenon of chromosome degeneration. Comparison of LCP genes I–IV in theX2 andY chromosomal region inD. miranda revealed extensive DNA rearrangements at the latter. TheY chromosomal LCP cluster is characterized by DNA insertions which are absent in the correspondingX2 chromosomal DNA, suggesting that these DNA sequences must have invaded this area. In addition, part of the analysedY chromosomal region is duplicated.     

A novel avian W chromosome DNA repeat sequence in the lesser black-backed gull (Larus fuscus)

The phenol emulsion reassociation technique was used to isolate and clone a female specific, repetitive DNA sequence fromLarus fuscus. The repeat, designated P2000-17, is restricted to the W chromosome, although related sequences occur elsewhere in the genome ofL. fuscus. Similar sequences were detected in the genome of six other bird species from outside the genus Laridae, but the sequence occurs less frequently and to a similar extent in both sexes. The 298 bp DNA sequence of P2000-17 was determined and found to have extensive sequence identity to the rabbit dihydropyridine (DHP) receptor calcium channel. P2000-17 is represented once within a larger 8.6 kb tandem repeat (LfW-1), which has a complex internal DNA sequence. LfW-1 is highly conserved between repeat motifs and may comprise 3% of the female genome. The possible evolutionary origin of LfW-1 is discussed in relation to the repeat types found on the W and Y chromosomes of other species.     

Demonstration of W chromosome-specific repetitive DNA sequences in the domestic fowl,Gallus g. domesticus

Evidence is presented to demonstrate the presence of W chromosome-specific repetitive DNA sequences in the female White Leghorn chicken, Gallus g. domesticus, based on two different experimental approaches. First, ³H-labelled, female chicken DNA was hybridized with excess, unlabelled, mercurated, male DNA, and unhybridized single-stranded ³H-DNA (³H-SHU-DNA) was recovered by SH-Sepharose and hydroxyapatite column chromatography. Approximately 24% of the hybridizable ³H-SHU-DNA was female-specific and localized on the W chromosome. The second approach was to examine female-specific DNA fragments among the digests of chicken DNA with various restriction endonucleases. Among them, we found that digestion with XhoI produced two prominent female-specific bands of 0.60 kb (= kilobase pairs) and 1.1 kb. The 0.60 kb fragment was isolated and ³H-labelled by nick-translation. Female-specificity of the ³H-XhoI—0.60 kb DNA was judged to be at least 95% under the conditions of hybridization with membrane filter-bound DNA. Presence of amplified XhoI—0.60 kb DNA on the W chromosome seems to be limited to different lines of G. g. domesticus and no such repeat was detected in three species belonging to other genera in the order Galliformes and in three species belonging to other avian orders.     

Male-specific repeats in wild Bovidae

In this study, we investigated repetitive sequences localized on Y chromosomes. Repetitive DNA sequences represent a substantial part of the eukaryotic genome and, among them, a large portion comprises sequences repeated in tandem. Efficient and rapid isolation of repeat units is possible due to a laser microdissection technique used for Y chromosome separation, followed by polymerase chain reaction (PCR), cloning, and sequence analysis. We applied the derived repeat units to members of nine tribes within the Bovidae. Apart from the Y chromosomes of Bos taurus and Bubalus bubalis, where we used known sequences of repetition, the derived sequences were used as probes for fluorescent in situ cross-hybridization to members of the nine tribes of the Bovidae. We investigated the distribution of repeat units within the tribes and their localization on the Y chromosome. Sharing of sequence variants would indicate common descent, while the rapid horizontal evolution should allow discrimination between closely related species or subspecies.     

Structure and variability of human chromosome ends.

Mammalian telomeres are thought to be composed of a tandem array of TTAGGG repeats. To further define the type and arrangement of sequences at the ends of human chromosomes, we developed a direct cloning strategy for telomere-associated DNA. The method involves a telomere enrichment procedure based on the relative lack of restriction endonuclease cutting sites near the ends of human chromosomes. Nineteen (TTAGGG)n-bearing plasmids were isolated, two of which contain additional human sequences proximal to the telomeric repeats. These telomere-flanking sequences detect BAL 31-sensitive loci and thus are located close to chromosome ends. One of the flanking regions is part of a subtelomeric repeat that is present at 10 to 25% of the chromosome ends in the human genome. This sequence is not conserved in rodent DNA and therefore should be a helpful tool for physical characterization of human chromosomes in human-rodent hybrid cell lines; some of the chromosomes that may be analyzed in this manner have been identified, i.e., 7, 16, 17, and 21. The minimal size of the subtelomeric repeat is 4 kilobases (kb); it shows a high frequency of restriction fragment length polymorphisms and undergoes extensive de novo methylation in somatic cells. Distal to the subtelomeric repeat, the chromosomes terminate in a long region (up to 14 kb) that may be entirely composed of TTAGGG repeats. This terminal segment is unusually variable. Although sperm telomeres are 10 to 14 kb long, telomeres in somatic cells are several kilobase pairs shorter and very heterogeneous in length. Additional telomere reduction occurs in primary tumors, indicating that somatic telomeres are unstable and may continuously lose sequences from their termini.