High-resolution organization of mouse telomeric and pericentromeric DNA

We studied the organization of telomeric, major and minor satellite DNA sequences located in the pericentromeric regions of mouse telocentric and Robertsonian metacentric chromosomes by high-resolution fluorescence in situ hybridization. Molecular data have already proved that in telocentrics, from the physical chromosome end, telomeric sequences are followed by minor and then by major satellite DNA. We showed that the three families of repetitive DNA are organized as uninterrupted long-range cluster repeats and that there is no intermingling between telomeric and minor satellite DNA or between the major and the minor tandem repeats or with non-satellite DNA. The pericentromeric region of metacentric chromosomes consists of a small block of minor satellite DNA sandwiched between two blocks of major satellite DNA.     

Robertsonian metacentrics of the house musk shrew (Suncus murinus, Insectivora, Soricidae) lose the telomeric sequences in the centromeric area

The house musk shrew, Suncus murinus, is polymorphic for five Robertsonian translocations (Rb8.17, 9.13, 10.12, 11.16, 14.15). Fluorescence in situ hybridisation with a biotin-labelled oligonucleotide, (TTAGGG)7, was performed to localise the telomeric DNA sequences at Rb chromosomes of heterozygous shrews. Hybridisation signals were observed at both ends of all chromosomes, but not at the pericentromeric areas of any of the Robertsonian metacentrics. Our results indicate a complete loss of the telomeric sequences at the fusion points of the Rb metacentrics in S. murinus.     

Non-telomeric sites as evidence of chromosomal rearrangement and repetitive (TTAGGG)n arrays in heterochromatic and euchromatic regions in four species of Akodon (Rodentia, Muridae)

Comparative studies among four species--Akodonazarae (2n = 38), A. lindberghi (2n = 42), A. paranaensis (2n = 44) and A. serrensis (2n = 46)--employing classic cytogenetics (C- and G-bands) and fluorescence in situ hybridization with telomeric (TTAGGG)n sequencesare reported here. Non-telomeric signals in addition to the regular telomeric sites were detected in three species:A. azarae, A. lindberghi and A. serrensis. One interstitial telomeric site (ITS) was observed proximally at the long arm of chromosome 1 of A. azarae. The comparison of G-banding patterns among the species indicated that the ITS was due to a tandem fusion/fission rearrangement. Non-telomeric signals of A. lindberghi and A. serrensis were not related to chromosomal rearrangements; instead, the sequences co-localized with (i) heterochromatic regions of all chromosomes in A. serrensis; (ii) some heterochromatic regions in A. lindberghi, and (iii) both euchromatic and heterochromatic regions in the metacentric pair of A. lindberghi. These exceptional findings revealed that ITS in Akodon can be related to chromosomal rearrangements and repetitive sequences in the constitutive heterochromatin and that the richness of TTAGGG-like sequences in the euchromatin could be hypothesized to be a result of amplification of the referred sequence along the chromosome arms.     

Cytogenetic characterization and mapping of rDNAs,core histone genes and telomeric sequences in <Emphasis Type="Italic">Venerupis aurea</Emphasis> and <Emphasis Type="Italic">Tapes rhomboides</Emphasis> (Bivalvia: Veneridae)

We describe the chromosomal location of GC-rich regions, 28S and 5S rDNA, core histone genes, and telomeric sequences in the veneroid bivalve species Venerupis aurea and Tapes (Venerupis) rhomboides, using fluorochrome staining with propidium iodide, DAPI and chromomycin A3 (CMA) and fluorescent in situ hybridization (FISH). DAPI dull/CMA bright bands were coincident with the chromosomal location of 28S rDNA in both species. The major rDNA was interstitially clustered at a single locus on the short arms of the metacentric chromosome pair 5 in V. aurea, whereas in T. rhomboides it was subtelomerically clustered on the long arms of the subtelocentric chromosome pair 17. 5S rDNA also was a single subtelomeric cluster on the long arms of subtelocentric pair 17 in V. aurea and on the short arms of the metacentric pair 9 in T. rhomboides. Furthermore, V. aurea showed four telomeric histone gene clusters on three metacentric pairs, at both ends of chromosome 2 and on the long arms of chromosomes 3 and 8, whereas histone genes in T. rhomboides clustered interstitially on the long arms of the metacentric pair 5 and proximally on the long arms of the subtelocentric pair 12. Double and triple FISH experiments demonstrated that rDNA and H3 histone genes localized on different chromosome pairs in the two clam species. Telomeric signals were found at both ends of every single chromosome in both species. Chromosomal location of these three gene families in two species of Veneridae provides a clue to karyotype evolution in this commercially important bivalve family.     

Tandem insertions of Alu elements

The technique of chromosomal orientation and direction fluorescence in situ hybridization (COD-FISH) was adapted for plant chromosomes in order to study long-range organization of two families of satellite repeats, VicTR-B of Vicia sativa and PisTR-B of Pisum sativum. The technique allowed FISH to be performed on mitotic chromosomes in a strand-specific manner, resulting in visualization of the repeat orientation along the chromosomes and with respect to the direction of telomeric repeats. The VicTR-B probe applied to V. sativa chromosomes produced signals on a single chromatid at most regions containing corresponding sequences, thus confirming a presence of long arrays of head-to-tail arranged repeat monomers which is typical for satellite DNA. However, hybridization signals of different or equal intensities on both chromatids were also detected at some loci, suggesting a more complex arrangement of the repeats. Similar observations were made for PisTR-B repeats on P. sativum chromosomes, although the proportion of loci displaying signals on both chromatids was lower. In contrast to VicTR-B, orientation of the PisTR-B clusters with respect to telomeric sequences appeared to be conserved among subtelomeric regions of metacentric chromosomes and of the short arms of acrocentric chromosomes.     

Distribution of telomeric (TTAGGG)<Subscript>n</Subscript> sequences in avian chromosomes

The physical ends of mammalian and other vertebrate chromosomes consist of tandemly repeated (TTAGGG)(n) hexamers, nucleating a specialized telomeric structure. However, (TTAGGG)(n) sequences can also occur at non-telomeric sites, providing important insights into karyotypic evolution. By fluorescence in situ hybridization (FISH) we studied the chromosomal distribution of (TTAGGG)(n) sequences in 16 bird species, representing seven different orders. Many species, in particular the ratites, display (TTAGGG)(n) hybridization signals in interstitial and centromeric regions of their macrochromosomes in addition to the typical telomeric signals. In some but not all species these non-telomeric sites coincide with C-band-positive heterochromatin. The retention and/or amplification of telomeric (TTAGGG)(n) repeats at interstitial and centromeric sites may indicate the fusion of ancestral chromosomes. Compared with the macrochromosomes, the microchromosomes of most species are enriched with (TTAGGG)(n) sequences, displaying heterogeneous hybridization patterns. We propose that this high density of (TTAGGG)(n) repeats contributes to the exceptionally high meiotic recombination rate of avian microchromosomes.     

Ribosomal DNA distribution and a genus-wide phylogeny reveal patterns of chromosomal evolution in Alstroemeria (Alstroemeriaceae)

? Premise of the study: Understanding the flexibility of monocot genomes requires a phylogenetic framework, which so far is available for few of the ca. 2800 genera. Here we use a molecular tree for the South American genus Alstroemeria to place karyological information, including fluorescent in situ hybridization (FISH) signals, in an explicit evolutionary context. ? Methods: From a phylogeny based on plastid, nuclear, and mitochondrial sequences for most species of Alstroemeria, we selected early-branching (Chilean) and derived (Brazilian) species for which we obtained 18S-25S and 5S rDNA FISH signals; we also analyzed chromosome numbers, 1C-values, and telomere FISH signals (in two species). ? Key results: Chromosome counts for Alstroemeria cf. rupestris and A. pulchella confirm 2n = 16 as typical of the genus, which now has chromosomes counted for 29 of its 78 species. The rDNA sites are polymorphic both among and within species, and interstitial telomeric sites in Alstroemeria cf. rupestris suggest chromosome fusion. ? Conclusions: In spite of a constant chromosome number, closely related species of Alstroemeria differ drastically in their rDNA, indicating rapid increase, decrease, or translocations of these genes. Previously proposed Brazilian and Chilean karyotype groups are not natural, and the n = 8 chromosomes in Alstroemeria compared to n = 9 in its sister genus Bomarea may result from a Robertsonian fusion.     

Robertsonian metacentrics of the house mouse lose telomeric sequences but retain some minor satellite DNA in the pericentromeric area

A combination of cytogenetic and molecular biology techniques were used to study the molecular composition and organisation of the pericentromeric regions of house mouse metacentric chromosomes, the products of Robertsonian (Rb) translocations between telocentrics. Regardless of whether mitotic or meiotic preparations were used, in situ hybridisation failed to reveal pericentromeric telomeric sequences on any of the Rb chromosomes, while all metacentrics retained detectable, although reduced (average 50 kb), amounts of minor satellite DNA in the vicinity of their centromeres. These results were supported by slot blot hybridisation which indicated that mice with 2n=22 Rb chromosomes have 65% of telomeric sequences (which are allocated to the distal telomeres of both Rb and telocentric chromosomes and to the proximal telomeres of telocentrics) and 15% the amount of minor satellite, compared with mice with 2n=40 all-telocentric chromosomes. Pulsed field gel electrophoresis and Southern analysis of DNA from Rb mice showed that the size of the telomeric arrays is similar to that of mice with all-telocentric chromosomes and that the minor satellite sequences were hybridising to larger fragments incorporating major satellite DNA. Since the telomeric sequences are closer to the physical end of the chromosome than the minor satellite sequences, the absence of telomeric sequences and the reduced amount of minor satellite sequences at the pericentromeric region of the Rb metacentrics suggest that the breakpoints for the Rb translocation occur very close to the minor satellite-major satellite border. Moreover, it is likely that the minor satellite is required for centromeric function, 50–67 kb being enough DNA to organise one centromere with a functionally active kinetochore.     

Chromosomal localization of telomeric sequences in three species of Akodon (Rodentia, Sigmodontinae)

The distribution of the vertebrate telomeric sequence T2AG3 in three species of the rodent genus Akodon was examined by FISH with a peptide nucleic acid probe. In addition to the expected telomeric hybridization, non-telomeric signals were observed in the three species. In A. dolores, centromeric signals were visible in two of the four biarmed autosome pairs featuring Robertsonian polymorphism, indicating the retention of at least part of the telomeric sequences during the fusion process, and an interstitial signal of lower intensity was observed in the short arm of another. In A. boliviensis, a strong signal was observed near the centromeric end of the first chromosome pair. The first pair of A. azarae (homologous to the first pair of A. boliviensis) showed a similar but markedly amplified signal, and a subcentromeric signal in the X chromosome corresponding to a heterochromatic region; additionally, interstitial signals of lower intensity were present in one to four chromosomes in the majority of cells examined.     

拟南芥DNA探针的比较基因组原位杂交揭示的拟南芥与远缘植物基因组间的同源性

采用生物素标记的拟南芥基因组DNA探针在75%杂交严谨度下对双子叶植物番茄、蚕豆和单子叶植物水稻、玉米、大麦的染色体进行了比较基因组荧光原位杂交（comparative genomic in situ hybridization,cGISH）分析,以揭示拟南芥与远缘植物基因组间的同源性.cGISH信号代表了拟南芥基因组DNA中的重复DNA与靶物种染色体上同源序列的杂交.探针DNA在所有靶物种的全部染色体上都产生了杂交信号.杂交信号为散在分布,并呈现随基因组增大,杂交信号增多,且分布更加分散的趋势.所有靶物种的核仁组织区（NOR）都显示了明显强于其他区域的杂交信号,表明拟南芥基因组DNA探针可用于植物NOR的物理定位.在所有的靶物种中,信号主要分布在染色体的臂中间区和末端,着丝粒或近着丝粒区有少数信号分布.大麦染色体显示了与C-和N-带不同的独特的cGISH信号带型,表明此探针可用于不同植物染色体的识别.这些结果表明,拟南芥基因组与远缘植物基因组之间,除rDNA和端粒重复序列外,还存在其它同源的重复DNA;一些重复DNA序列在被子植物分歧进化为单子叶和双子叶植物之前就已存在,虽经历了长期的进化过程,至今在远缘物种之间仍保持了较高的同源性.结果还提示,大基因组中古老而保守的重复DNA在进化过程中发生了明显的扩增.     

Molecular-cytogenetic characterization of a higher plant centromere/kinetochore complex

The centromeric region of a telocentric field bean chromosome that resulted from centric fission of the metacentric satellite chromosome was microdissected. The DNA of this region was amplified and biotinylated by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR)/linker-adapter PCR. After fluorescence in situ hybridization (FISH) the entire chromosome complement of Vicia faba was labelled by these probes except for the nucleolus organizing region (NOR) and the interstitial heterochromatin, the chromosomes of V. sativa and V. narbonensis were only slightly labelled by the same probes. Dense uniform labelling was also observed when a probe amplified from a clearly delimited microdissected centromeric region of a mutant of Tradescantia paludosa was hybridized to T. paludosa chromosomes. Even after six cycles of subtractive hybridization between DNA fragments amplified from centromeric and acentric regions no sequences specifically located at the field bean centromeres were found among the remaining DNA. A mouse antiserum was produced which detected nuclear proteins of 33 kDa and 68 kDa; these were predominantly located at V. faba kinetochores during mitotic metaphase. DNA amplified from the chromatin fraction adsorbed by this serum out of the sonicated total mitotic chromatin also did not cause specific labelling of primary constrictions. From these results we conclude: (1) either centromere-specific DNA sequences are not very conserved among higher plants and are — at least in species with large genomes — intermingled with complex dispersed repetitive sequences that prevent the purification of the former, or (2) (some of) the dispersed repeats themselves specify the primary constrictions by stereophysical parameters rather than by their base sequence.     

Chromosomal distribution of telomeric and telomeric-associated sequences in Hordeum chilense by in situ hybridization

The chromosomal distribution of telomeric repeat pAtT4 from Arabidopsis thaliana and telomeric associated repetitive sequence HvT01 from Hordeum vulgare have been studied by FISH (fluorescence in situ hybridization) in two accessions (H1 and H7) of Hordeum chilense. The telomeric sequence pAtT4 is present at the end of all chromosome arms in H1 and H7 accessions. In contrast, the telomeric associated sequence homologous to HvT01 showed variability for size, intensity and position of the signals for each line. In H1, HvT01 was present in every chromosome whereas only four chromosomes were labeled in H7 accession. Physical distribution of GAA-satellite sequence on both H1 and H7 metaphase chromosomes was also studied. Polymorphism for hybridization signals between the two accessions for GAA-banding pattern was also found. Based on differences in position and intensity of the hybridization signals found for both GAA and HvT01-homologous sequences, karyotypes for the in situ hybridization patterns are presented for H1 and H7 accessions of H. chilense.     

The presence of interstitial telomeric sequences in constitutional chromosome abnormalities.

We describe a novel chromosome structure in which telomeric sequences are present interstitially, at the apparent breakpoint junctions of structurally abnormal chromosomes. In the linear chromosomes with interstitial telomeric sequences, there were three sites of hybridization of the telomere consensus sequence within each derived chromosome: one at each terminus and one at the breakpoint junction. Telomeric sequences also were observed within a ring chromosome. The rearrangements examined were constitutional chromosome abnormalities with a breakpoint assigned to a terminal band. In each case (with the exception of the ring chromosome), an acentric segment of one chromosome was joined to the terminus of an apparently intact recipient chromosome. One case exhibited apparent instability of the chromosome rearrangement, resulting in somatic mosaicism. The rearrangements described here differ from the telomeric associations observed in certain tumors, which appear to represent end-to-end fusion of two or more intact chromosomes. The observed interstitial telomeric sequences appear to represent nonfunctional chromosomal elements, analogous to the inactivated centromeres observed in dicentric chromosomes.     

Characterization of TTAGG telomeric repeats,their interstitial occurrence and constitutively active telomerase in the mealybug <Emphasis Type="Italic">Planococcus lilacinus</Emphasis> (Homoptera; Coccoidea)

We confirmed the occurrence of the insect TTAGG telomeric repeats in the mealybug Planococcus lilacinus, a radiation-resistant coccid, by single primer polymerase chain reaction (PCR) and Southern hybridization. Analysis of Bal31 nuclease-digested DNA by Southern hybridization and chromosomes by FISH suggests that these repeats occur mainly at the ends of the chromosomes. However, sequence analysis of the PCR products of TTAGG-associated sequences from genomic DNA showed their interstitial occurrence and association with certain unrelated low-copy repeats. Because of their shorter length, the interstitial TTAGG sequences were detectable by primed in situ hybridizations but not by FISH. Analysis of chromosomes recovered after irradiation by fluorescent in situ hybridization suggested acquisition of TTAGG repeats at a majority of the healed ends. We also observed mild telomerase activity in unirradiated insects which was further enhanced after irradiation. Taken together, these results suggest that the mealybug has an efficient mechanism of formation of TTAGG repeats at radiation-induced chromosome ends and constitutively active telomerase may be a feature associated with rapid recovery of chromosome ends damaged by ionizing radiation.     

Chromosome fission associated with growth of ribosomal DNA in Neodiprion abietis (Hymenoptera: Diprionidae)

The haploid complement consists of seven metacentric chromosomes in most diprionid species but has evolved to n = 8 by fission in Neodiprion abietis. This fission generated a small telocentric chromosome and a large pseudoacrocentric chromosome with a short arm carrying a satellite. In situ hybridization indicated that the location of the rRNA gene cluster corresponds to the whole short arm. This suggests that (i) the breaking point was located close to an rRNA gene cluster, and (ii) fission was associated with growth of rDNA. These results suggest rDNA as a preferential breaking point but with a role in the healing of naked chromosome ends.     

Analysis of radiation-induced micronuclei by fluorescence in situ hybridization (FISH) simultaneously using telomeric and centromeric DNA probes.

Fluorescence in situ hybridization using simultaneously a combination of DNA probes for the telomeric hexamer repeat (TTAGGG) and the centromerically repeated murine gamma-satellite DNA was applied to analyze the nature of radiation-induced micronuclei in mouse NIH 3T3 fibroblasts. After subtraction of spontaneously occurring micronuclei independent from the dose and time after irradiation, approximately 22% of the radiation-induced micronuclei did not reveal any hybridization signal. Approximately 17% showed one centromeric hybridization signal and about four telomeric signals, suggesting their origin from whole chromosomes. Almost 60% of radiation-induced micronuclei had telomeric signals only, suggesting their origin from acentric fragments. A fraction of micronuclei were found to contain two or more acentric fragments. Micronuclei derived from whole chromosomes or from multiple acentric fragments might, together with DNA synthesis in micronuclei, explain the occurrence of radiation-induced micronuclei with DNA contents greater than the largest chromosome arm.     

Telomeres and mechanisms of Robertsonian fusion

The Robertsonian (Rb) fusion, a chromosome rearrangement involving centric fusion of two acro-(telo)centric chromosomes to form a single metacentric, is one of the most frequent events in mammalian karyotype evolution. Since one of the functions of telomeres is to preserve chromosome integrity, a prerequisite for the formation of Rb fusions should be either telomere loss or telomere inactivation. Possible mechanisms underlying the formation of various types of Rb fusion are discussed here. For example, Rb fusion in wild mice involves complete loss of p-arm telomeres by chromosome breakage within minor satellite sequences. By contrast, interstitial telomeric sites are found in the pericentromeric regions of chromosomes originating from a number of vertebrate species, suggesting the occurrence of Rb-like fusion without loss of telomeres, a possibility consistent with some form of telomere inactivation. Finally, a recent study suggests that telomere shortening induced by the deletion of the telomerase RNA gene in the mouse germ-line leads to telomere loss and high frequencies of Rb fusion in mouse somatic cells. Thus, at least three mechanisms in mammalian cells lead to the formation of Rb fusions. Received: 11 November 1997 / Accepted: 21 December 1997     

Molecular cytological evidence for gradual telomere synthesis at the broken chromosome ends in wheat

Telomere formation of the normal and broken chromosomes of common wheat,Triticum aestivum, was investigated byin situ hybridization using the biotin-labeled probe of telomere repetitive sequences (pAtT4) ofArabidopsis thaliana with subsequent amplification by an antibody. After double and triple amplification, prominent signals appeared at all the telomeric regions of the normal chromosomes. Prominent signals also emerged at the broken ends of the telocentric and deletion chromosomes that had passed through more than one generation since the appearance. However, broken ends that had passed through only the stages of gametogenesis, fertilization, embryogenesis and root development did not show complete signals such as found in normal telomeres. These findings indicate that a certain time or stage is required for synthesis of the telomeric repetitive sequences with a complete length. Nevertheless, because the broken ends without complete telomere sequences were also healed, restoration of the normal complement of telomere sequences is not necessary for healing of broken ends.     

Chromosomal studies in four species of genus Chaunus (Bufonidae, Anura): localization of telomeric and ribosomal sequences after fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) using telomeric and ribosomal sequences was performed in four species of toad genus Chaunus: C. ictericus, C. jimi, C. rubescens and C. schneideri. Analyses based on conventional, C-banding and Ag-NOR staining were also carried out. The four species present a 2n = 22 karyotype, composed by metacentric and submetacentric chromosomes, which were indistinguishable either after conventional staining or banding techniques. Constitutive heterochromatin was predominantly located at pericentromeric regions, and telomeric sequences (TTAGGG)(n) were restricted to the end of all chromosomes. Silver staining revealed Ag-NORs located at the short arm of pair 7, and heteromorphism in size of NOR signals was also observed. By contrast, FISH with ribosomal probes clearly demonstrated absence of any heteromorphism in size of rDNA sequences, suggesting that the difference observed after Ag-staining should be attributed to differences in chromosomal condensation and/or gene activity rather than to the number of ribosomal cistrons.