Quantification of total genomic DNA and selected repetitive sequences reveals concurrent changes in different DNA families in indica and japonica rice

This paper describes a fluorescence in situ hybridization (FISH) analysis of three different repetitive sequence families, which were mapped to mitotic metaphase chromosomes and extended DNA fibers (EDFs) of the two subspecies of rice (Oryza sativa), indica and japonica (2n=2x=24). The repeat families studied were (1) the tandem repeat sequence A (TrsA), a functionally non-significant repeat; (2) the [TTTAGGG]_n telomere sequence, a non-transcribed, tandemly repeated but functionally significant repeat; and (3) the 5S ribosomal RNA (5S rDNA). FISH of the TrsA repeat to metaphase chromosomes of indica and japonica cultivars revealed clear signals at the distal ends of twelve and four chromosomes, respectively. As shown in a previous report, the 17S ribosomal RNA genes (17S rDNA) are located at the nucleolus organizers (NORs) on chromosomes 9 and 10 of the indica cultivar. However, the japonica rice lacked the rDNA signals on chromosome 10. The size of the 5S rDNA repeat block, which was mapped on the chromosome 11 of both cultivars, was 1.22 times larger in the indica than in the japonica genome. The telomeric repeat arrays at the distal ends of all chromosome arms were on average three times longer in the indica genome than in the japonica genome. Flow cytometric measurements revealed that the nuclear DNA content of indica rice is 9.7% higher than that of japonica rice. Our data suggest that different repetitive sequence families contribute significantly to the variation in genome size between indica and japonica rice, though to different extents. The increase or decrease in the copy number of several repetitive sequences examined here may indicate the existence of a directed change in genome size in rice. Possible reasons for this phenomenon of concurrent evolution of various repeat families are discussed. Received: 9 August 1999 / Accepted: 29 December 1999     

Visual verification of close disposition between a rice A genome-specific DNA sequence (TrsA) and the telomere sequence

A rice A genome-specific tandem repeat sequence (TrsA) and telomeric nucleotide sequences, (TTTAGGG)_n, were simultaneously detected by multicolor fluorescence in situ hybridization (McFISH) using rice prometaphase chromosomes. Six pairs of TrsA sites visualized by fluorescence signals were all localized on the long arms close to the telomeric regions. Differences in the copy number of TrsA at the different sites were visualized both by the size of the telomeric condensation block stained with Giemsa solution and the signal intensity after FISH with TrsA. McFISH analyses using interphase nuclei could resolve close disposition of TrsA and telomere and also gave rough estimation of the distance between them. The functional significance of the close disposition of TrsA and telomere is discussed.     

FISH studies reveal the molecular and chromosomal organization of individual telomere domains in tomato

The molecular and cytological organization of the telomeric repeat (TR) and the subtelomeric repeat (TGR1) of tomato were investigated by fluorescence in situ hybridization (FISH) techniques. Hybridization signals on extended DNA fibres, visualized as linear fluorescent arrays representing individual telomeres, unequivocally demonstrated the molecular co-linear arrangement of both repeats. The majority of the telomeres consisted of a TR and a TGR1 region separated by a spacer. Microscopic measurements of the TR and TGR1 signals revealed high variation in length of both repeats, with maximum sizes of 223 and 1330 kb, respectively. A total of 27 different combinations of TR and TGR1 was detected, suggesting that all chromosome ends have their own unique telomere organization. The fluorescent tracks on the extended DNA fibres were subdivided into four classes: (i) TR–spacer–TGR1; (ii) TR–TGR1; (iii) only TR; (iv) only TGR1. FISH to pachytene chromosomes enabled some of the TR/TGR1 groups to be assigned to specific chromosome ends and to interstitial regions. These signals also provided evidence for a reversed order of the TR and TGR1 sites at the native chromosome ends, suggesting a backfolding telomere structure with the TGR1 repeats occupying the most terminal position of the chromosomes. The FISH signals on diakinesis chromosomes revealed that distal euchromatin areas and flanking telomeric heterochromatin remained highly decondensed around the chiasmata in the euchromatic chromosome areas. The rationale for the occurrence and distribution of the TR and TGR1 repeats on the tomato chromosomes are discussed.     

Fluorescence in situ hybridization of single copy transgenes in rice chromosomes

Summary Fluorescence in situ hybridization (FISH) is a powerful tool for visualizing the chromosomal location of targeted sequences and has been applied in many areas, including karyotyping, breeding and characterization of genes introduced into the plant genome. A simple, routine and sensitive FISH procedure was developed for localizing single copy genes in rice (Oryza sativa L.) metaphase chromosomes. We used digoxygenin-labeled endogenous or T-DNA sequences as small as 5.6 kb to probe corresponding endogenous sequences or the T-DNA insert in denatured rice metaphase chromosomes prepared from root meristem tissue. The hybridized probe sequence was labeled with cy3-conjugated anti-mouse IgG and visualized using fluorescence microscopy. Single copy and multiple copy introduced T-DNA sequences, as well as endogenous sequences, were localized on the chromosomes. The FISH protocol was effectively used to sereen the chromosomal location of introduced T-DNA and number of integration loci in rice.     

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.     

Involvement of transposition in dispersion of tandem repeat sequences (TrsA) in rice genomes

We describe a method to identify and characterize DNA fragments containing the junction of AA genome-specific tandem repeat sequences (here called TrsA) with adjacent chromosomal sequences of rice by the polymerase chain reaction (PCR) using a pair of primers that hybridize with TrsAs and a flanking non-TrsA sequence. With this method, we obtained results suggesting that TrsA sequences present at two loci (here called trsA1 and trsA2) are flanked by direct repeats of chromosomal sequences of 172 by and about 440 by in length, respectively. These results support the idea that the TrsA sequences have been inserted into each locus by transposition, resulting in duplication of the chromosomal sequence used as target. We also describe a method to identify and characterize TrsA sequences repeated in only a few copies in the rice genome by PCR, using a pair of primers that hybridize with two different portions in the TrsA sequence, and demonstrate that TrsA sequences are present not only in rice strains with the AA genome, but also in those with non-AA genomes. The TrsA sequences were present at the trsA1 locus in all the rice strains examined, indicating that TrsA was inserted and amplified at the locus before the divergence of the various species of rice in the Oryza genus. TrsA sequences were present at the trsA2 locus, however, only in an O. sativa IR36 strain, indicating that TrsA was inserted and amplified at this locus during divergence of rice strains with the AA genome.     

Organization of a Solatium brevidens repetitive sequence related to the TGRI subtelomeric repeats of Lycopersicon esculentum

A species-specific repetitive DNA fragment has been isolated from a genomic library of Solanum brevidens. Sequence analysis revealed a regular organization of three non-homologous subrepeats forming tandemly-arranged composite repetitive units. Interpretation of Southern hybridization patterns based on the known sequence data suggests that the isolated sequence element represents an abundant organization type, although the presence of simple tandem arrays of the subrepeats is also indicated. Seventy-four percent sequence similarity was found between one of the S. brevidens subrepeats (Sb4AX) and a satellite DNA (TGRI) localized as a subtelomeric repeat on almost all Lycopersicon esculentum chromosomes. Insitu hybridization indicated that, similarly to TGRI, the S. brevidens-specific repeats are located at the ends of the arms of several chromosomes. On the basis of the data obtained, a common ancestral sequence can be proposed for the tomato (TGRI) and the S. brevidens (Sb4AX) repeat however, the molecular organization of this element in these two species evolved in a basically different manner.     

Frequency of telomere repeat units in the Drosophila miranda genome

Cloned DNA fragments of Drosophila miranda which label all chromosome ends show a basic tandem repeat unit of 4.4 kb. The D. miranda telomere specific tandem repeats do not cross-hybridize with genomic D. melanogaster DNA which itself contains telomere repeat units of 3 kb. For a more detailed analysis of the functional criteria of telomere specific sequences we determined the repetition frequency of the tandem repeat units. As a low estimate we found a repetition frequency of 20 for female D. miranda DNA. This is on average equivalent to 2 telomere repeat units per chromosome end in the female D. miranda karyotype. However, a variable number of tandem repeat units per chromosome end would describe more closely the obtained differences in the labeling intensity between the individual chromosomes (X¹L-5). For the D. miranda male DNA we determined a repetition frequency of 90. The frequency difference of 70 copies between male and female DNA must be due to the Y-chromosome.     

利用粗线期染色体和DNA纤维的FISH分析水稻端粒序列

利用粗线期染色体和DNA纤维的荧光原位杂交(FISH)技术分析了水稻广陆矮四号(Oryzasativassp.indicacv.GuangluaiNo.4)的端粒序列。粗线期染色体荧光原位杂交结果表明,大多数染色体的末端都有端粒串联重复,但信号的强度在不同染色体上是不同的。伸展DNA纤维荧光原位杂交结果显示,端粒最长的线状信号长度为6.55μm,最短的为1.82μm,依据2.51kb/μm的标准,它们分别相当于16.44kb和4.56kb。端粒的平均信号长度为3.62±1.32μm,相当于9.09±3.31kb。由此可以估计,最长的、最短的和平均长度的端粒拷贝数约为2349、651和1298±473。     

Pneumocystis carinii telomere repeats are composed of TTAGGG and the subtelomeric sequence contains a gene encoding the major surface glycoprotein

We have cloned a telomere and adjacent sequences from rat-derived Pneumocystis carinii using the ability of foreign telomeres to complement a yeast artificial chromosome (YAC) deficient by one telomere in Saccharomyces cerevisiae . Characterization of the cloned DNA in the recombinant YAC demonstrated that it was a chimera of two P. carinii sequences, namely a 13.5 kb fragment of mitochondrial DNA and an 8.3 kb distal portion consisting of subtelomeric DNA. The P. carinii telomere repeat was demonstrated to be TTAGGG, the most common telomere repeat found in organisms from the animal and fungal kingdoms. Karyotype analysis confirmed that this sequence was present on all the P. carinii chromosomes. Sequence adjacent to the telomere repeats was shown by Bal 31 exonuclease digestion to be located at the chromosome ends. Analysis of the subtelomeric fragment revealed homology to the gene encoding the major surface glycoprotein of P. carinii     

The large-scale genomic organization of repetitive DNA families at the telomeres of rye chromosomes.

Repetitive DNA sequences in the terminal heterochromatin of rye (Secale cereale) chromosomes have consequences for the structural and functional organization of chromosomes. The large-scale genomic organization of these regions was studied using the telomeric repeat from Arabidopsis and clones of three nonhomologous, tandemly repeated, subtelomeric DNA families with complex but contrasting higher order structural organizations. Polymerase chain reaction analysis with a single primer showed a fraction of the repeat units of one family organized in a "head-to-head" orientation. Such structures suggest evolution of chromosomes by chromatid-type breakage-fusion-bridge cycles. In situ hybridization and pulse field gel electrophoresis showed the order of the repeats and the heterogeneity in the lengths of individual arrays. After Xbal digestion and pulse field gel electrophoresis, the telomeric and two subtelomeric clones showed strong hybridization signals from 40 to 100 kb, with a maximum at 50 to 60 kb. We suggest that these fragments define a basic higher order structure and DNA loop domains of regions of rye chromosomes consisting of arrays of tandemly organized sequences.     

AT-rich sequences containing Arabidopsis-type telomere sequence and their chromosomal distribution in Pinus densiflora

Japanese red pine Pinus densiflora has 2 n=24 chromosomes and after FISH-detection of Arabidopsis-type (A-type) telomere sequences, many telomere signals were observed on these chromosomes at interstitial and proximal regions in addition to the chromosome ends. These interstitial and proximal signal sites were observed as DAPI-positive bands, suggesting that the interstitial and proximal telomere signal sites are composed of AT-rich highly repetitive sequences. Four DNA clones (PAL810, PAL1114, PAL1539, PAL1742) localized at the interstitial telomere signals were selected from AluI-digested genomic DNA library using colony blot hybridization probed with A-type telomere sequences and characterized using FISH and Southern blot hybridization. The AT-contents of these selected four clones were 60.8–76.3%, and repeat units of the telomere sequence and degenerated telomere sequences were found in their nucleotide sequences. Except for two sites of PAL1114, FISH signals of the four clones co-localized with interstitial and proximal A-type telomere sequence signals. FISH signals a showed similar distribution pattern, but the patterns of signal intensity were different among the four clones. PAL810, PAL1539 and PAL 1742 showed similar FISH signal patterns, and the differences were only with respect to the signal intensity of some signal sites. PAL1114 had unique signals that appeared on chromosomes 7 and 10. Based on results of the Southern blot hybridization these four sequences are not arranged tandemly. Our results suggest that the interstitial A-type telomere sequence signal sites were composed of a mixture of several AT-rich repetitive sequences and that these repetitive sequences contained A-type telomere sequences or degenerated A-type telomere sequence repeats.     

Telomeric DNA in chromosomes of five opisthorchid species

The analysis of telomere repeat distribution in chromosomes of five opisthorchid species (Opisthorchis felineus (Rivolta, 1884), Opisthorchis viverrini (Poirier, 1886), Metorchis xanthosomus (Creplin, 1846), Metorchis bilis (Braun, 1890), Clonorchis sinensis (Cobbold, 1875)) was performed with fluorescent in situ hybridization (FISH) of labeled (TTAGGG)n DNA-probe and PNA telomere probe on mitotic and meiotic chromosomes of these species. It was shown that chromosome telomeres of all studied species contain large clusters of (TTAGGG)n telomeric repeats. Interstitial clusters of the (TTAGGG)n repeats have not been revealed in the chromosomes of any studied species even when FISH of PNA telomere probe on pachytene chromosomes was performed. Furthermore interstitial clusters of the (TTAGGG)n repeats have not been detected in the chromosomes of O. viverrini, one of chromosomes of this species is the result of a fusion of two ancestral opisthorchid chromosomes.     

Analysis and location of a rice BAG clone containing telomeric DNA sequences

BAC2, a rice BAC clone containing (TTTAGGG)_n homologous sequences, was analyzed by Southern hybridization and DNA sequencing of its subclones. It was disclosed that there were many tandem repeated satellite DNA sequences, called TA352, as well as simple tandem repeats consisting of TTTAGGG or its variant within the BAC2 insert. A 0. 8 kb (TTTAGGG)_n-containing fragment in BAC2 was mapped in the telomere regions of at least 5 pairs of rice chromosomes by using fluorescencein situ hybridization (FISH). By RFLP analysis of low copy sequences the BAC2 clone was localized in one terminal region of chromosome 6. All the results strongly suggest that the telomeric DNA sequences of rice are TTTAGGG or its variant, and the linked satellite DNA TA352 sequences belong to telomere-associated sequences.     

Analysis and location of a rice BAC clone containing telomeric DNA sequences

BAC2, a rice BAC clone containing (TTTAGGG)_n homologous sequences, was analyzed by Southern hybridization and DNA sequencing of its subclones. It was disclosed that there were many tandem repeated satellite DNA sequences, called TA352, as well as simple tandem repeats consisting of TTTAGGG or its variant within the BAC2 insert. A 0. 8 kb (TTTAGGG)_n-containing fragment in BAC2 was mapped in the telomere regions of at least 5 pairs of rice chromosomes by using fluorescencein situ hybridization (FISH). By RFLP analysis of low copy sequences the BAC2 clone was localized in one terminal region of chromosome 6. All the results strongly suggest that the telomeric DNA sequences of rice are TTTAGGG or its variant, and the linked satellite DNA TA352 sequences belong to telomere-associated sequences.     

Karyotypic characterization and genomic organization of the 5S rDNA in <Emphasis Type="Italic">Polypterus senegalus</Emphasis> (Osteichthyes,Polypteridae)

Polypteridae (Cladistia) is a family of archaic fishes, confined to African freshwaters. On account of their primitiveness in anatomical and morphological characters and mosaic relationships among lower Osteichthyans fishes, they constitute an important subject for the study of evolution in vertebrates. Very little is known about the karyological structure of these species. In this article, a cytogenetic analysis on twenty specimens of Polypterus senegalus (Cuvier, 1829) was performed using both classical and molecular techniques. Karyotype (2n = 36; FN = 72), chromosome location of telomeric sequences (TTAGGG) _n , (GATA)₇ repeats and ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA₃ staining and FISH. Staining with Ag-NOR showed the presence of two GC rich NORs on the p arm of the chromosome pair no. 1. CMA₃ marked all centromerical and some (no. 1 and no. 14) telomeric regions. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair no. 14. FISH with 18S rDNA marked the telomeric region of the p arm of the chromosome pair no. 1, previously marked by Ag-NOR. (GATA)₇ repeats marked the subtelomeric regions of all chromosome pairs, with the exclusion of the no. 1, 3 and 14. Hybridization with telomeric probes (TTAGGG) _n showed bright signals at the end of all chromosomes. After cloning, the 5SrDNA alignment revealed an organization of sequences made up of two different classes of tandem arrays (5S type I and 5S type II) of different lengths.     

Complex and Tandem Repeat Structure of Subtelomeric Regions in the Taiwan Cricket, <Emphasis Type="BoldItalic">Teleogryllus taiwanemma</Emphasis>

Telomeres of most insects are composed of simple (TTAGG) _n repeats that are synthesized by telomerase. However, in some dipteran insects such as Drosophila melanogaster, (TTAGG) _n repeats or telomerase activity has not been detected. Although telomere structure is well documented in Diptera and Lepidoptera, very limited information is available on lower insect groups. To understand general aspects of telomere function and evolution in insects, we endeavored to characterize structures of the telomeric and subtelomeric regions in a lower insect, the Taiwan cricket, Teleogryllus taiwanemma. FISH analysis of this insect's chromosomes demonstrated (TTAGG) _n repeat elements in all distal ends. Just proximal to the telomeric repeats, the highly conserved 9-kb long terminal unit (LTU) sequences are tandemly repeated. These were observed in four of six chromosomes, three autosomal ends, and one X-chromosomal end. LTU sequences represent about 0.2% of the T. taiwanemma genome. Each LTU contains a core (TTAGG)₈-like sequence (TRLS) and five types of conserved sequences—ST (short telomere associated), J (joint), X, SR (satellite sequence rich), and Y—which vary in length from about 150 bp to 2.7 kb. The LTU sequence is defined as ST–J–TRLS–SR–X–Y–X–Y–X. Most LTU regions may be derived from the ancestral common sequence, which is observed in ST regions six times and at many other LTU sites. We could not find the LTU-like sequence in three other crickets including the closest species, T. emma, suggesting that the LTU in T. taiwanemma has been rapidly amplified in subtelomeric regions through recent evolutional events. It is also suggested that the highly conserved structure of the LTU is maintained by recombination and may contribute to telomere elongation, as seen in dipteran insects. Received: 6 August 2001/Accepted: 10 October 2001     

Sequencing and characterization of telomere and subtelomere regions on rice chromosomes 1S, 2S, 2L, 6L, 7S, 7L and 8S

Telomeres, which are important for chromosome maintenance, are composed of long, repetitive DNA sequences associated with a variety of telomere-binding proteins. We characterized the organization and structure of rice telomeres and adjacent subtelomere regions on the basis of cytogenetic and sequence analyses. The length of the rice telomeres ranged from 5.1 to 10.8 kb, as revealed by both fibre-fluorescent in situ hybridization and terminal restriction-fragment assay. Physical maps of the chromosomal ends were constructed from a fosmid library. This facilitated sequencing of the telomere regions of chromosomes 1S, 2S, 2L, 6L, 7S, 7L and 8S. The resulting sequences contained conserved TTTAGGG telomere repeats, which indicates that the physical maps partly covered the telomere regions of the respective chromosome arms. These repeats were organized in the order of 5'-TTTAGGG-3' from the chromosome-specific region, except in chromosome 7S, in which seven inverted copies also existed in tandem array. Analysis of the telomere-flanking regions revealed the occurrence of deletions, insertions, or chromosome-specific substitutions of single nucleotides within the repeat sequences at the junction between the telomere and subtelomere. The sequences of the 500-kb regions of the seven chromosome ends were analysed in detail. A total of 598 genes were predicted in the telomeric regions. In addition, repetitive sequences derived from various kinds of retrotransposon were identified. No significant evidence for segmental duplication could be detected within or among the subtelomere regions. These results indicate that the rice chromosome ends are heterogeneous in both sequence and characterization.     

Identification,characterisation and clinical applications of cosmids from the telomeric and centromeric regions of the long arm of chromosome 22

Using human telomeric repeats and centromeric alpha repeats, we have identified adjacent single copy cosmid clones from human chromosome 22 cosmid libraries. These single copy cosmids were mapped to chromosome 22 by fluorescence in situ hybridisation (FISH). Based on these cosmids, we established contigs that included part of the telomeric and subtelomeric regions, and part of the centromeric and pericentromeric regions of the long arm of human chromosome 22. Each of the two cosmid contigs consisted of five consecutive steps and spanned approximately 100–150 kb at both extreme ends of 22q. Moreover, highly informative polymorphic markers were identified in the telomeric region. Our results suggest that the telomere specific repeat (TTAGGG) _n encompasses a region that is larger than 40 kb. The cosmid contigs and restriction fragment length polymorphism markers described here are useful tools for physical and genetic mapping of chromosome 22, and constitute the basis of further studies of the structure of the subtelomeric and pericentromeric regions of 22q. We also demonstrate the use of these clones in clinical diagnosis of different chromosome 22 aberrations by FISH.     

BAC-FISH in wheat identifies chromosome landmarks consisting of different types of transposable elements

Fluorescence in situ hybridization (FISH) has been widely used in the physical mapping of genes and chromosome landmarks in plants and animals. Bacterial artificial chromosomes (BACs) contain large inserts making them amenable for FISH mapping. We used BAC-FISH to study genome organization and evolution in hexaploid wheat and its relatives. We selected 56 restriction fragment length polymorphism (RFLP) locus-specific BAC clones from libraries of Aegilops tauschii (the D-genome donor of hexaploid wheat) and A-genome diploid Triticum monococcum. Different types of repetitive sequences were identified using BAC-FISH. Two BAC clones gave FISH patterns similar to the repetitive DNA family pSc119; one BAC clone gave a FISH pattern similar to the repetitive DNA family pAs1. In addition, we identified several novel classes of repetitive sequences: one BAC clone hybridized to the centromeric regions of wheat and other cereal species, except rice; one BAC clone hybridized to all subtelomeric chromosome regions in wheat, rye, barley and oat; one BAC clone contained a localized tandem repeat and hybridized to five D-genome chromosome pairs in wheat; and four BAC clones hybridized only to a proximal region in the long arm of chromosome 4A of hexaploid wheat. These repeats are valuable markers for defined chromosome regions and can also be used for chromosome identification. Sequencing results revealed that all these repeats are transposable elements (TEs), indicating the important role of TEs, especially retrotransposons, in genome evolution of wheat.Communicated by P.B. Moens