Localization of 5S RNA and rRNA genes in the Norway rat

The genes coding for the two classes of ribosomal RNA molecules, 5S RNA and 18+28S RNA, have been localized in the Norway rat (Rattus norvegicus). The 18+28S RNA cistrons are found on three chromosomes, at secondary constrictions on the short arms of chromosomes 3 and 12 and at the telomere of the short arm of chromosome 11. These sites were confirmed using the silver staining technique for nucleolar organizer regions. Two sites were found for the 5S RNA genes; one is closely linked to the 18+28S gene site on chromosome 12. The second site is at or near the telomere of the long arm of chromosome 19.     

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.     

Ribosomal DNA,tri- and bi-partite pericentromeres in the permanent translocation heterozygote <Emphasis Type="Italic">Rhoeo spathacea</Emphasis>

High- and low-stringency FISH and base-specific fluorescence were performed on the permanent translocation heterozygote Rhoeo spathacea (2n = 12). Our results indicate that 45S rDNA arrays, rDNA-related sequences and other GC-rich DNA fraction(s) are located within the pericentromeric regions of all twelve chromosomes, usually colocalizing with the chromomycin A₃-positive bands. Homogenization of the pericentromeric regions appears to result from the concerted spread of GC-rich sequences, with differential amplification likely. We found new 5S rDNA patterns, which suggest a variability in the breakpoints and in the consequent chromosome reorganizations. It was found that the large 5S rDNA locus residing on each of the 8E and 9E arms consisted of two smaller loci. On each of the two chromosome arms 3b and 4b, in addition to the major subtelomeric 5S rDNA locus, a new minor locus was found interstitially about 40% along the arm length. The arrangement of cytotogenetic landmarks and chromosome arm measurements are discussed with regard to genome repatterning in Rhoeo.     

Detection of 5S and 25S rRNA genes in Sinapis alba, Raphanus sativus and Brassica napus by double fluorescence in situ hybridization

Different ribosomal RNA (5S and 25S) genes were investigated simultaneously by fluorescence in situ hybridization (FISH) in Sinapis alba, Raphanus sativus and Brassica napus. The chromosomes of S. alba carried four 5S and six 25S gene sites, and those of R. sativus four sites of each gene, respectively. These two species have one chromosome pair with both rDNA genes; the two are closely located on a short arm of S. alba, while in R. sativus one is distal on the short arm (5S) and the other more proximal on the long arm (25S). In B. napus we have confirmed 12sites of 25S rDNA. The detection of 5S rDNA genes revealed 14 signals on 12 chromosomes. Of these, six chromosomes had signals for both rDNA genes. The FISH with 5S rDNA probes detected two sites closely adjacent in four chromosomes of B napus. These results are discussed in relation to a probable homoeologous chromosome pair in B. oleracea. Received: 20 July 1999 / Accepted: 8 October 1999     

Chromosome analysis by fluorescence in situ hybridization of callus-derived regenerants in Allium cyaneum R.

Investigations were performed to confirm the optimal in vitro culture condition for callus induction and plant regeneration, to observe if somoclonal variation occurs among regenerated plants at the ploidy level and to analyse the chromosomal location of 5S and 18S-26S rRNA gene families using fluorescence in situ hybridization in callus-derived plants of Allium cyaneum. High-est callus initiation was achieved with bulb explants cultured on MS medium supplemented with 2,4-D and BAP at 1 mg l^–1 each. A total of 195 plants was obtained when using MS medium supplemented with 1 mg l^–1 NAA and 5 mg l^–1 BAP; about 92% were diploid having 2n=16; 8% showed a variation in ploidy level. Using digoxigenin-labelled 5S rRNA and biotin-labelled 18S-26S rRNA gene probes, we compared the fluorescence in situ hybridization patterns of autotetraploid plants with the A. cyaneum wild type. The 5S rRNA gene sites were detected on the interstitial region in the short arm of chromosome 4 and on the interstitial region in both arms of chromosome 7. The 18S-26S rRNA gene sites were detected on the terminal region of the short arm, including the satellite of chromosome 5, as well as on a part of chromosome B. The chromosomal location of both rRNA genes in regenerated autotetraploid plants corresponded to those of the wild species. Received: 20 March 1998 / Revision received: 15 June 1998 / Accepted: 8 July 1998     

Cytogenetics in fruit breeding - localization of ribosomal RNA genes on chromosomes of apple (Malus×domestica Borkh.)

 The localization of rRNA genes was studied by fluorescent in situ hybridization (FISH) on chromosomes of the cultivated apple, M.×domestica ‘Pinova’ (2n=34). The 18S/25S rRNA loci were detected in terminal positions of the short arms of two submetacentric and two metacentric chromosome pairs. One 5S rRNA gene locus was found in the proximal region of the short arm of a small metacentric chromosome pair. Received : 21 June 1996 / Accepted : 28 June 1996     

Dual-color FISH karyotype analyses using rDNAs in three Cucurbitaceae species

Dual-color fluorescence in situ hybridization (FISH) analysis of three Cucurbitaceae species from different genera was conducted using 5S and 45S rDNA probes. In Benincasa hispida (Thunb.) Cogn. (2n=24), the 45S rDNA probe hybridized on two chromosomes, one in the short arm of a medium-sized metacentric chromosome and another at the satellite of a chromosome. The 5S rDNA hybridized at a site proximal to the centromere of the same short arm of the 45S rRNA gene locus that occupied almost the entire short arm. For Citrullus lanatus (Thunb.) Matsum & Nakai (2n=22), the 45S rDNA probe hybridized at sites in the short arms of two chromosomes and the 5S rDNA probe was co-localized with the 45S rRNA locus at the region proximal to the centromere in one chromosome. The 45S rRNA loci occupied almost all of the short arms in both chromosomes. In Cucurbita moschata Duch. (2n=40), the 45S rDNA probe hybridized in five chromosomes in which the 45S rRNA genes occupied almost two-thirds of the chromosomes in two large chromosomes and the entire short arm of a medium-sized chromosome. Two other loci were present in two medium-sized chromosomes, one in the proximal region in the short arm of a chromosome and another at the tip of the long arm of a chromosome. Chromosomes of B. hispida were relatively larger than those of the other two species. The karyotype of B. hispida is composed of two metacentrics and 10 submetacentrics, while that of C. lanatus is composed of seven metacentrics and four submetacentrics and that of C. moschata is composed of 18 metacentrics and two submetacentrics. Comparative chromosome evolution among the three Cucurbitaceae species was attempted using the karyotypes and the chromosomal distribution patterns of the 5S and 45S rDNAs. The results presented herein will be useful in elucidating the phylogenetic relationships among Cucurbitaceae species, and will provide basic data for their breeding programs.     

Molecular hybridization of iodinated 4S, 5S, and 18S + 28S RNA to salamander chromosomes

4S, 5S, AND 18S + 28S RNA from the newt Taricha granulosa granulosa were iodinated in vitro with carrier-free 125I and hybridized to the denatured chromosomes of Taricha granulosa and Batrachoseps weighti. Iodinated 18S + 28S RNA hybridizes to the telomeric region on the shorter arm of chromosome 2 and close to the centromere on the shorter arm of chromosome 9 from T. granulosa. On this same salamander the label produced by the 5S RNA is located close to or on the centromere of chromosome 7 and the iodinated 4S RNA labels the distal end of the longer arm of chromosome 5. On the chromosomes of B. wrighti, 18S + 28S RNA hybridizes close to the centromeric region on the longer arm of the largest chromosome. Two centromeric sites are hybridized by the iodinated 5S RNA. After hybridization with iodinated 4S RNA, label is found near the end of the shorter arm of chromosome 3. It is concluded that both ribosomal and transfer RNA genes are clustered in the genome of these two salamanders.     

Physical mapping of the 5S ribosomal RNA genes in Arabidopsis thaliana by multi-color fluorescence in situ hybridization with cosmid clones

The 5S ribosomal RNA genes were mapped to mitotic chromosomes of Arabidopsis thaliana by fluorescence in situ hybridization (FISH). In the ecotype Landsberg erecta, hybridization signals appeared on three pairs of chromosomes, two of which were metacentric and the other acrocentric. Hybridization signals on one pair of metacentric chromosomes were much stronger than those on the acrocentric and the other pair of metacentric chromosomes, probably reflecting the number of copies of the genes on the chromosomes. Other ecotypes, Columbia and Wassilewskija, had similar chromosomal distribution of the genes, but the hybridization signals on one pair of metacentric chromosomes were very weak, and detectable only in chromosomes prepared from young flower buds. The chromosomes and arms carrying the 5S rDNA were identified by multi-color FISH with cosmid clones and a centromeric 180 bp repeat as co-probes. The metacentric chromosome 5 and its L arm carries the largest cluster of the genes, and the short arm of acrocentric chromosome 4 carries a small cluster in all three ecotypes. Chromosome 3 had another small cluster of 5S rRNA genes on its L arm. Chromosomes 1 and 2 had no 5S rDNA cluster, but they are morphologically distinguishable; chromosome 1 is metacentric and 2 acrocentric. Using the 5S rDNA as a probe, therefore, all chromosomes of A. thaliana could be identified by FISH. Chromosome 1 is large and metacentric; chromosome 2 is acrocentric carrying 18S-5.8S-25S rDNA clusters on its short arm; chromosome 3 is metacentric carrying a small cluster of 5S rDNA genes on its L arm; chromosome 4 is acrocentric carrying both 18S-5.8S-25S and 5S rDNAs on its short (L) arm; and chromosome 5 is metacentric carrying a large cluster of 5S rDNA on its L arm.     

The effect of heterochromatin on synapsis of the sex chromosomes of Peromyscus (Rodentia, Cricetidae)

The pairing behavior of the sex chromosomes in male and female individuals representing seven species of Peromyscus was analyzed by electron microscopy of silver-stained zygotene and pachytene configurations. Six species possess submetacentric or metacentric X chromosomes with heterochromatic short arms. Sex-chromosome pairing in these species is initiated during early pachynema at an interstitial position on the X and Y axes. Homologous synapsis then progresses in a unidirectional fashion towards the telomeres of the X short arm and the corresponding arm of the heterochromatic Y chromosome. The distinctive pattern of synaptic initiation allowed a late-synapsing bivalent in fetal oocytes to be tentatively identified as that of the X chromosomes. In contrast to the other species, Peromyscus megalops possesses an acrocentric X chromosome and a very small Y chromosome. Sex-chromosome pairing in this species is initiated at the proximal telomeric region during late zygonema, and then proceeds interstitially towards the distal end of the Y chromosome. These observations suggest that the presence of X short-arm heterochromatin and corresponding Y heterochromatin interferes with late-zygotene alignment of the pairing initiation sites, thereby delaying XY synaptic initiation until early pachynema. The pairing initiation sites are conserved in the vicinity of the X and Y centromeres in Peromyscus, and consequently the addition of heterochromatin during sex-chromosome evolution essentially displaces these sites to an interstitial position.     

18-26S rDNA在4种重楼属植物中的定位

为探讨rDNA在重楼属Paris L.中的分布规律,利用荧光原位杂交（FISH）对4种重楼属植物 的18-26S rDNA进行了定位。所有植物均为二倍体,基因组由A、B、C、D和 E5条染色体构成。（1）滇重楼P.polyphylla var.yunnanensis:2n=10=6m+4t,C和D染色体的 短臂上各有1个18-26S rDNA位点;（2）长柱重楼P.forrestii:2n=10=6m+4t,B染色体的长臂 、C和D染色体的短臂上各有1个位点;（3）五指莲P.axialis:2n=10=6m（2sat）+4t（2sat） +1-2B,C和D染色体的短臂上各有1个位点;在有1个B染色体的细胞中,B染色体没有信号点, 而有2个B染色体的细胞中,只有1个B染色体上有信号点,表明B染色体上有基因存在且其分裂 不均等;（4）大理重楼P.daliensis:2n=10=4m+2sm+2st+2t,C染色体的短臂上有1个位点。1 8-26S rDNA位点不仅出现在染色体的次缢痕上,也出现在非次缢痕位点。另外,4个种中C染 色体短臂末端均有18-26S rDNA。     

Physical mapping of rDNA sequences in four karyotypes of Ranunculus silerifolius (Ranunculaceae)

The chromosomal locations of the 18S-5.8S-26S rDNA and 5S rDNA sequences were examined in four cytotypes of Ranunculus silerifolius (the Matsuyama, Mugi, Otaru, and Karatsu types) using fluorescence in situ hybridization (FISH). Using the 18S-5.8S-26S rDNA probe, one pair of probe hybridization sites was detected by FISH in the interstitial region corresponding to the secondary constriction on the short arm of a satellite chromosome (chromosome pair 6) in all four karyotypes. FISH using 5S rDNA identified one pair of sites. The 5S rDNA locus was on different chromosomes in the four karyotypes: in the interstitial region of the short arm of the largest metacentric chromosome (chromosome pair 1) in the Matsuyama type, in the interstitial region of the short arm of the subtelocentric chromosome (pair 2) in the Mugi and Otaru types, and in the interstitial region of the short arm of the metacentric chromosome (pair 2) in the Karatsu type. This physical mapping of the 5S rDNA provides valuable information about karyotype evolution in R. silerifolius. Possible mechanisms of chromosome evolution are discussed.     

Structural variability of human chromosome 9 in relation to its evolution

Summary Human chromosome 9 shows a high susceptibility for structural rearrangements, particularly pericentric inversions, which often are transmitted. Three types of pericentric inversions can be observed on No. 9: 1) Type I, showing the total constitutive heterochromatin in the short arm. 2) Type II with part of the C heterochromatin on the short arm, the rest located on the long arm proximal to the centromere. 3) Type III: a subtelocentric chromosome with part of the C heterochromatin in the very short arm and the rest located interstitially on the long arm. With these inversions as well as with other structural rearrangements, e.g. translocations, the break-points are located preferentially within the C heterochromatin or close to the heterochromatic-euchromatic junctions. These findings are in contrast to the findings in lymphocytes from 5 patients with Fanconi's anemia and after irradiation in vitro, reported in the literature. In lymphocytes break-points seem to be distributed more or less by chance. These observations together led us to speculate that human chromosome 9 primarily was an acrocentric chromosome; in morphology and at least in some functions similar to D-and G-group chromosomes. During evolution this acrocentric chromosome changed to a submetacentric one due to a pericentric inversion.The author is sponsored by the Deutsche Forschungsgemeinschaft.     

Physical mapping of 5S and 18S-26S ribosomal RNA gene families inAllium victorialis var.platyphyllum

A physical map of the 5S and 18S–26S rRNA genes was determined using bi-color fluorescencein situ hybridization technique inA. victorialis var.platyphyllum. 5S rRNA genes were positioned in the intercalary regions of the short arms in homologous chromosomes 6. Two major loci of the 18S-26S rRNA genes were detected in the secondary constrictions flanking with a pair of satellite and terminal region of short arm in chromosome 4. And two additional minor loci were heterotype, representing one signal on the terminal region of the short arm in one homolog of chromsome 2, and other on one homolog of chromosome 6 with linked 5S rRNA loci. In addition chromomycin A₃ (CMA,) fluorescent banding method was used to identify the relation between Nucleolus Organizer Region (NOR) sites and CMA, positive heterochromatin sites. In homologous chromosome 4 showing 18S–26S rDNA hybridization signals revealed also distinct CMA, positive band.     

New 18S·26S ribosomal RNA gene loci: chromosomal landmarks for the evolution of polyploid wheats

Three new 18S·26S rRNA gene loci were identified in common wheat by sequential N-banding and in situ hybridization (ISH) analysis. Locus Nor-A7 is located at the terminal area of the long arm of 5A in both diploid and polyploid wheats. Locus Nor-B6 is located in N-band 1BL2.5 of the long arm of chromosome 1B in Triticum turgidum and Triticum aestivum. ISH sites, similar to Nor-B6, were also detected on the long arms of chromosomes 1G in Triticum timopheevii and 1S in Aegilops speltoides, but their locations on the chromosomes were different from that of Nor-B6, indicating possible chromosome rearrangements in 1GL and 1BL during evolution. The third new locus, Nor-D8, was only found on the short arm of chromosome 3D in the common wheat Wichita. The loss of rRNA gene locus Nor-A3 and gain of repetitive DNA sequence pSc119 on the terminal part of 5AS suggest a structural modification of 5AS. Comparative studies of the location of the 18S·26S rRNA gene loci in polyploid wheats and putative A and B (G) genome progenitor species support the idea that: (1) Triticum monococcum subsp. urartu is the donor of both the A and A^t genome of polyploid wheats. (2) Ae. speltoides is closer to the B and G genome of polyploid wheats than Aegilops longissima and is the most probable progenitor of these two genomes.     

Identification of the Y chromosome in chinook salmon (Oncorhynchus tshawytscha)

The Y chromosome in chinook salmon, Oncorhynchus tshawytscha, was identified using fluorescence in situ hybridization (FISH) with a probe to a male-specific repetitive sequence isolated from this species. The probe highlights the distal end of the short arm of an acrocentric chromosome with a DAPI-bright interstitial band of variable size. The proximal portion of the short arm of the Y chromosome contains 5S rDNA sequences, which are also found on the short arms of six other acrocentric chromosomes in this species.     

Mapping of18-26S rDNA loci in four species of the genus Paris by fluorescence in situ hybridization（FISH）

18-26S rDNA loci were mapped on chromosomes in four species of Par is,and the num-ber and position of rDNA sites in these species were compared f or analysis of the distribution of the sites. All the plants were diploids,and t he genome consisted of five chromosomes,A,B,C,D and E. （1）P. polyphylla var. yunnanensis,2n=10=6m+4t. Two18-26S rDNA loci were de-tected on the short arms o f C and D chromosomes;（2）P. forrestii,2n=10=6m+4t. One locus was detected on th e long arm of B chromosome,and also two loci on the short arms of C and D chromosomes;（3）P. axialis. 2n=10=6m（2sat）+4t（2sat）+1-2B. Two loci were detected o n the short arms of C and D chromosomes. One locus was detected in the cell with t wo B-chromosomes（B）,but none was detected in that with only one B chromosome, indicating that rRNA gene existed on B chromsome,and an unequal division occurr ed during mitotic cycle of B-chromosomes. （4）P. daliensis,2n=10=4m+2sm+2st+2t. O ne locus was detected on the short arm of D chromo-some. The signals of18-26S rD NA appeared not only in the second constriction but also in the other regions of chromosome. It is noteworthy that one locus was detected in the terminal region o n the short arm of C chromosome in all the four species studied.     

Physical mapping of rRNA genes by fluorescent in-situ hybridization and structural analysis of 5S rRNA genes and intergenic spacer sequences in sugar beet (Beta vulgaris)

A digoxigenin-labelled 5S rDNA probe (pTa-794) and a rhodamine-labelled 18S-5.8S-25S rDNA probe (pTa71) were used for double-target in-situ hybridization to root-tip metaphase, prophase and interphase chromosomes of cultivated beet,Beta vulgaris L. After in-situ hybridization with the 18S-5.8S-25S rDNA probe, one major pair of sites was detected which corresponded to the secondary constriction at the end of the short arm of chromosome 1. The two rDNA chromosomes were often associated and the loci only contracted in late metaphase. In the majority of the metaphase plates analyzed, we found a single additional minor hybridization site with pTa71. One pair of 5S rRNA gene clusters was localized near the centromere on the short arm of one of the three largest chromosomes which does not carry the 18S-5.8S-25S genes. Because of the difficulties in distinguishing the very similarly-sizedB. vulgaris chromosomes in metaphase preparations, the 5S and the 18S-5.8S-25S rRNA genes can be used as markers for chromosome identification. TwoXbaI fragments (pXV1 and pXV2), comprising the 5S ribosomal RNA gene and the adjacent intergenic spacer, were isolated. The two 5S rDNA repeats were 349 bp and 351 bp long, showing considerable sequence variation in the intergenic spacer. The use of fluorescent in-situ hybridization, complemented by molecular data, for gene mapping and for integrating genetic and physical maps of beet species is discussed.     

MAMMALIAN CHROMOSOMES IN VITRO : XVIII. DNA Replication Sequence in the Chinese Hamster

The complete DNA replication sequence of the entire complement of chromosomes in the Chinese hamster may be studied by using the method of continuous H³-thymidine labeling and the method of 5-fluorodeoxyuridine block with H³-thymidine pulse labeling as relief. Many chromosomes start DNA synthesis simultaneously at multiple sites, but the sex chromosomes (the Y and the long arm of the X) begin DNA replication approximately 4.5 hours later and are the last members of the complement to finish replication. Generally, chromosomes or segments of chromosomes that begin replication early complete it early, and those which begin late, complete it late. Many chromosomes bear characteristically late replicating regions. During the last hour of the S phase, the entire Y, the long arm of the X, and chromosomes 10 and 11 are heavily labeled. The short arm of chromosome 1, long arm of chromosome 2, distal portion of chromosome 6, and short arms of chromosomes 7, 8, and 9 are moderately labeled. The long arm of chromosome 1 and the short arm of chromosome 2 also have late replicating zones or bands. The centromeres of chromosomes 4 and 5, and occasionally a band on the short arm of the X are lightly labeled.     

Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.