Development and applications of a set of chromosome-specific cytogenetic DNA markers in potato

Reliable and easy to use techniques for chromosome identification are critical for many aspects of cytogenetic research. Unfortunately, such techniques are not available in many plant species, especially those with a large number of small chromosomes. Here we demonstrate that fluorescence in situ hybridization (FISH) signals derived from bacterial artificial chromosomes (BACs) can be used as chromosome-specific cytogenetic DNA markers for chromosome identification in potato. We screened a potato BAC library using genetically mapped restriction fragment length polymorphism markers as probes. The identified BAC clones were then labeled as probes for FISH analysis. A set of 12 chromosome-specific BAC clones were isolated and the FISH signals derived from these BAC clones serve as convenient and reliable cytological markers for potato chromosome identification. We mapped the 5S rRNA genes, the 45S rRNA genes, and a potato late blight resistance gene to three specific potato chromosomes using the chromosome-specific BAC clones. Received: 19 January 2000 / Accepted: 27 March 2000     

Fingerprinting human chromosomes by polymerase chain reaction-mediated DNA amplification.

We describe here a method for DNA fingerprinting of human chromosomes by Alu-polymerase chain reaction (PCR) amplification of DNA from monochromosomal hybrids, following digestion with restriction endonucleases. DNA digestion with restriction enzymes prior to PCR amplification reduces the total number of amplified fragments. The number and pattern of bands of PCR products observed in an electrophoretic medium are chromosome specific and provide a "fingerprint signature" for individual human chromosomes. Using this approach, we have produced fingerprints for human chromosomes 2, 5, 7, 9, and 12. The applicability of this approach to chromosome identification was assessed by comparing the fingerprints obtained for two different hybrids containing chromosome 7. DNA fragments specific for the long and the short arms of human chromosome 12 have also been identified. In addition, Alu-PCR-generated DNA fragments, specific for different chromosomes, were used to probe Southern blots of a hybrid cell panel to identify human chromosomes present in hybrid cell lines. The chromosomal specificity of these probes permits the identification of intact as well as rearranged chromosomes composed of segments arising from more than one chromosome.     

贵州产蜘蛛抱蛋属植物的细胞分类学研究

报道了6种贵州产蜘蛛抱蛋属植物的染色体数目和核型,并与其相应近缘种对比,联系植物的外部形态特征,探讨核型结构与形态特征的相关性.发现1个种的染色体数目为2n =36,5个种的染色体数目为2n =38,核型公式分别为:平塘蜘蛛抱蛋(Aspidistra pingtangensis),2n =38 =20m +4sm(2sat) +14st;荔波蜘蛛抱蛋(A.liboensis),2n =38 =22m(2sat)+4sm+ 12st;赤水蜘蛛抱蛋(A.chishuiensis),2n =38 =22m(2sat)+8sm +8st;伞柱蜘蛛抱蛋(A.fungilliformis),2n =36=18m (2sat) +4sm+ 14st;四川蜘蛛抱蛋(A.sichuanensis),2n =38 =22m (2sat) +4sm +12st;丛生蜘蛛抱蛋(A caespitosa),2n =38 =20m +6sm(2sat) +12st.核型类型都为2C型.其中平塘蜘蛛抱蛋、荔波蜘蛛抱蛋和赤水蜘蛛抱蛋的染色体数目和核型均为首次报道.研究结果表明,该属植物的核型结构与外部形态特征具有一定的相关性,细胞分类学研究可以为该属植物起源进化研究以及自然分类鉴定提供一定的依据.     

Thermosensitive vector derived from RP1 plasmid for detection of transposable elements

The involvement of the transposable DNA element of E. coli K12 chromosome in integrative recombination of RP1 plasmid was studied. Using temperature sensitive for replication plasmid RP1ts12--the derivative of RP1 which contains mutated transposon Tnl, it was shown that integration of RP1 into host chromosome and Hfr formation may occur according to a mechanism mediated by chromosome IS-elements. Plasmids that are desintegrated from the chromosome of these Hfrs contain discrete DNA segments (IS-elements) and possess elevated frequency of integration into chromosome of rec+ cells. The latter was used for selection of RP1ts12 recombinants carrying chromosome IS. For identification of IS involved in RP1 integration the number of independent RP1ts 12 recombinants was subjected to restriction and heteroduplex analysis. By analysing recombinants integrated into bacterial chromosome with frequency 5 X 10(-3), a new IS-element of E. coli K12 designated IS111 was discovered. IS111-element is about 1500bp of length, contains Smal, Pst1 and BamH1 restriction endonuclease sites and was found in the same position on the plasmid RP1 in two different orientations. IS-elements that have been revealed in a number of other RP1ts12 recombinants were preliminary identified as IS1-like elements. One recombinants plasmid was found to have an IS5-like elements. The activity of IS-elements inserted into RP1ts12 in recA-dependent integrative recombination was estimated. From the data of absolute and relative RP1ts12 integration frequencies mediated by IS111, IS1- and IS5-like elements a conclusion was made about the absence of E. coli K12 chromosome IS-elements in RP1 plasmid. The Hfr-formation and chromosomal gene transfer by recombinant plasmids RP1ts12: IS111 were studied. The possibility to use insertion RP1ts12 derivatives for the estimation of copies number, mapping and definition of orientation of IS-elements in bacterial chromosome and the possibilities for detection of transposable DNA elements using RP1ts12 in a wide range of gram-negative bacteria are discussed.     

Characterization of a novel high-tillering dwarf 3 mutant in rice

Tiller number and culm length are important components of plant architecture and determinate grain production in rice.A line SIL046,derived from an introgression lines population developed by an accession of common wild rice (Oryza rufipogon Griff.) and a high-yielding indica cultivar Guichao 2 (Oryza sativa L.).exhibits a higher tiller number and shorter culm length phenotype than the recipient parent Guichao 2 (GC2).Genetic analysis showed that the high-tillering dwarf phenotype was controlled by a novel single recessive gene,referred to as the high-tillering dwarf3 (htd3),which located within the genetic distance of 13.4 cM between SSR makers RM7003 and RM277 on chromosome 12.By means of fine-mapping strategy,we mapped HTD3 gene within the genetic distance of 2.5 cM and the physical distance of 3100 kb in the centromere of chromosome 12.Further identification of HTD3 gene would provide a new opportunity to uncover the molecular mechanism of the development of culm and tiller,two important components of yields in rice.     

Mapping of DNA markers close to the fragile site on the human X chromosome at Xq27.3.

We report the identification of a new RFLP detected by the DNA probe MN12, which is linked to both the fragile site on the X chromosome at Xq27.3 and the highly polymorphic locus detected by St14 (DXS52). In situ mapping confirms the localisation of MN12 distal to the fragile site. A detailed physical analysis of this region of the X chromosome using pulsed-field gel electrophoresis has shown that MN12, St14 and DX13 (DXS15) are physically linked within a region of 470kb. A long range restriction map around the MN12 locus reveals at least two candidate HTF islands, suggesting the existence of expressed sequences in this region.     

三种小花蝽核型研究(半翅目:花蝽科)

本文利用姬姆萨染色压片法对花蝽科小花蝽属3个种:荷氏小花蝽Orius horvathi(Reuter),微小花蝽Oriusminutus Linnaeus和东亚小花蝽Orius sauteri(Poppius)的核型做了研究,结果表明3种小花蝽单倍体均具有12条染色体,X-Y性别决定机制,但3种核型行为具特定的差异,其核型特征可用于种间分类。     

Cytogenetic characterization of the invasive mussel species Xenostrobus securis Lmk. (Bivalvia: Mytilidae)

The chromosomes of the invasive black-pigmy mussel (Xenostrobus securis (Lmk. 1819)) were analyzed by means of 4',6-diamidino-2-phenylindole (DAPI) / propidium iodide (PI) and chromomycin A3 (CMA) / DAPI fluorescence staining and fluorescent in situ hybridization using major rDNA, 5S rDNA, core histone genes, linker histone genes, and telomeric sequences as probes. The diploid chromosome number in this species is 2n = 30. The karyotype is composed of seven metacentric, one meta/submetacentric, and seven submetacentric chromosome pairs. Telomeric sequences appear at both ends of every single chromosome. Major rDNA clusters appear near the centromeres on chromosome pairs 1 and 3 and are associated with bright CMA fluorescence and dull DAPI fluorescence. This species shows five 5S rDNA clusters close to the centromeres on four chromosome pairs (2, 5, 6, and 8). Three of the four core histone gene clusters map to centromeric positions on chromosome pairs 7, 10, and 13. The fourth core histone gene cluster occupies a terminal position on chromosome pair 8, also bearing a 5S rDNA cluster. The two linker histone gene clusters are close to the centromeres on chromosome pairs 12 and 14. Therefore, the use of these probes allows the unequivocal identification of 11 of the 15 chromosome pairs that compose the karyotype of X. securis.     

Human chromosomes 9, 12, and 15 contain the nucleation sites of stress-induced nuclear bodies

We previously reported the identification of a novel nuclear compartment detectable in heat-shocked HeLa cells that we termed stress-induced Src-activated during mitosis nuclear body (SNB). This structure is the recruitment center for heat shock factor 1 and for a number of RNA processing factors, among a subset of Serine-Arginine splicing factors. In this article, we show that stress-induced SNBs are detectable in human but not in hamster cells. By means of hamster>human cell hybrids, we have identified three human chromosomes (9, 12, and 15) that are individually able to direct the formation of stress bodies in hamster cells. Similarly to stress-induced SNB, these bodies are sites of accumulation of hnRNP A1-interacting protein and heat shock factor 1, are usually associated to nucleoli, and consist of clusters of perichromatin granules. We show that the p13-q13 region of human chromosome 9 is sufficient to direct the formation of stress bodies in hamster>human cell hybrids. Fluorescence in situ hybridization experiments demonstrate that the pericentromeric heterochromatic q12 band of chromosome 9 and the centromeric regions of chromosomes 12 and 15 colocalize with stress-induced SNBs in human cells. Our data indicate that human chromosomes 9, 12, and 15 contain the nucleation sites of stress bodies in heat-shocked HeLa cells.     

不同地域乌拉尔甘草基因组的FISH分析与染色体识别

在核型分析与染色体识别基础上,分别以番茄45S和5S rDNA为探针,对3种不同地域的乌拉尔甘草进行FISH分析.结果表明:内蒙古鄂托克前旗的乌拉尔甘草核型公式为2n=2x=16=6m+10sm (2SAT),新疆阿勒泰地区的乌拉尔甘草核型公式为2n=2x=16=4m+12sm(2SAT),内蒙古喀喇沁旗乌拉尔甘草核型公式为2n=2x=16=4m+12sm(2SAT);其第8染色体均带有随体.3种乌拉尔甘草基因组内均有1对5S rDNA和1对45S rDNA杂交位点.核型分析显示,5S rDNA杂交位点均位于第2染色体的短臂部位,45S rDNA杂交位点均位于第8染色体的次缢痕和随体部位.45S与5S rDNA在3种乌拉尔甘草中期分裂相上的位点数和分布情况高度一致,表明来自3种不同地域的乌拉尔甘草在染色体结构水平上没有较大的分化.     

Sister chromatid differentiation and chromosomal in situ suppression hybridization: a combined methodology for analyzing cell proliferation and SCEs in individual chromosomes.

A technique combining sister chromatid differentiation (SCD) with chromosomal in situ suppression (CISS) hybridization is described. This combined methodology allows simultaneous analysis of cell-proliferation kinetics and sister chromatid exchanges (SCEs) in chromosomes identified by probes. To demonstrate the usefulness of this approach, cultured fibroblasts from a patient with Pallister-Killian syndrome, mos46/47,+i(12p), a chromosome mosaicism disorder, were studied. The fibroblasts were cultured in the presence of 5-bromodeoxyuridine (BrdU) for 72 h. Chromosome preparations were stained by a modified fluorescence-plus-Giemsa method to obtain SCD. For identification of the normal chromosome 12 and the i(12p), CISS hybridization with a biotin-labeled chromosome 12-specific library probe (LA 12NS01) was carried out after SCD. The hybridization was detected by an indirect immunofluorescence technique. For the analysis of cell kinetics and SCEs, the technique allows rapid, reliable identification of abnormal and normal cell populations. It also allows analysis of SCEs in individual chromosomes.     

Construction and characterization of band-specific DNA libraries

Summary A universally primed polymerase chain reaction was developed to amplify DNA dissected from GTG-banded human chromosomes. The amplification products are cloned into plasmid vectors, which allow the rapid characterization of recombinant clones. Starting from 20–40 chromosome fragments, several thousand independent clones detecting single-copy sequences can be obtained. Although these libraries comprise only a few percent of the dissected DNA, they provide narrowly spaced anchor clones for the molecular characterization of chromosome bands and the identification of gene sequences. Here we describe the construction and characterization of DNA libraries for the Langer-Giedion syndrome chromosome region (LGCR, 8q23–24.1), Wilms tumor chromosome region 1 (WT1, 11p13), Prader-Willi syndrome/Angelman syndrome chromosome region (PWCR/ANCR, 15q11.2–12), meningioma chromosome region (MGCR, 22q12–13), and fragile X chromosome region (FRAXA, Xq27.3).     

Chromosome numbers of some species of Salvia (Lamiaceae) from the Sichuan Province, China

The present study reports the chromosome number of 12 accessions belonging to 10 species of Salvia from the Sichuan Province in China. Most accessions have the chromosome number 2n=2x=16. However, three species ( S. evansiana, S. przewalskii and S. brevilabra ) are tetraploid with a chromosome number of 2n=4x=32. A B-chromosome was observed in S. tricuspis . The basic chromosome number x=8 was inferred for all accessions studied. The chromosome number of all the species was determined for the first time, except for S. evansiana, S. przewalskii , S. flava and S. miltiorrhiza . The chromosomes in this genus are mostly small (0.46–2.94 μm). The small size of the chromosomes, together with their unclear centromeres, has hampered a detailed karyotype analysis.     

Genetic dissection of centromere function.

A system to detect a minimal function of Saccharomyces cerevisiae centromeres in vivo has been developed. Centromere DNA mutants have been examined and found to be active in a plasmid copy number control assay in the absence of segregation. The experiments allow the identification of a minimal centromere unit, CDE III, independently of its ability to mediate chromosome segregation. Centromere-mediated plasmid copy number control correlates with the ability of CDE III to assemble a DNA-protein complex. Cells forced to maintain excess copies of CDE III exhibit increased loss of a nonessential artificial chromosome. Thus, segregationally impaired centromeres can have negative effects in trans on chromosome segregation. The use of a plasmid copy number control assay has allowed assembly steps preceding chromosome segregation to be defined.     

Chromosomal differentiation of Hyla nana and Hyla sanborni (Anura,Hylidae) with a description of NOR polymorphism in H. nana

Specimens of Hyla nana and Hyla sanborni from a syntopic population were studied cytogenetically. These species are morphologically very similar and are frequently misidentified, confused with each other. Both species had a diploid chromosome number, 2n = 30. However, the karyotypes of H. nana and H. sanborni differed considerably from each other in the number of submetacentric and telocentric chromosomes. The two species also differed in their primary NOR-bearing chromosomes (metacentric pair 13 in H. nana and telocentric pair 12 in H. sanborni). Additional nucleolus organizer regions (NORs) were detected by Ag-NOR staining and FISH in chromosome pairs 1, 5, 6, 12, and 14 in seven specimens of H. nana. Thus, a total of six patterns of NOR were identified. These differences in karyotype and in NOR location allowed the unambiguous identification of syntopic individuals of the two species. However, the chromosomal morphology of both species differed from that reported for populations from other geographic regions, suggesting that a systematic reevaluation of this group of Hyla may be necessary.     

A chromosome study of twenty species of Spanish carabid beetles (Coleoptera)

The chromosome number of 20 Spanish species of carabid beetles belonging to 12 tribes varies between 2n=20 and 59. Results corroborate that many tribes of Carabidae have a peculiar pattern of karyotypic evolution, causing a great diversity of chromosome number and/or chromosome morphology. Together with numberically stable groups (Carabini, Bembidiini) others like Nebriini and Licinini are found in which there are marked karyotypic differences even between related species. Trends towards packing the genetic material may become extreme in groups such as Brachinini, whereas dissociations lead to marked numerical increases in groups such as Zabrini. A number of these karyotypic conclusions are of great interest for the systematics of Carabidae.     

A PCR-based method of identifying species-specific repeated DNAs.

A PCR-based method is described to facilitate the identification of DNA fragments that are highly repeated and species-specific. The precision of the technique was demonstrated by cloning a fragment that occurred in a high number of copies in the plant species Medicago granadensis but in a low number of copies in 17 other Medicago species and Melilotus officinalis. This method should greatly accelerate the isolation and cloning of short and long interspersed nuclear elements with species-specific distributions. Such clones should prove useful in studies of phylogenetic relationships in the identification of interspecific hybrids, as in situ chromosome markers and other applications.     

Flow cytometry measurements of human chromosome kinetochore labeling

A method for the preparation and measurement of immunofluorescent human chromosome centromeres in suspension is described using CREST antibodies, which bind to the centromeric region of chromosomes. Fluorescein isothiocyanate (FITC)-conjugated antihuman antibodies provide the fluorescent label. Labeled chromosomes are examined on microscope slides and by flow cytometry. In both cases a dye which binds to DNA is added to provide identification of the chromosome groups. Sera from different CREST patients vary in their ability to bind to chromosome arms in addition to the centromeric region. Flow cytometry and microfluorimetry measurements have shown that with a given CREST serum the differences in kinetochore fluorescence between chromosomes are only minor. Flow cytometry experiments to relate the number of dicentric chromosomes, induced by in vitro radiation of peripheral blood cells to the slightly increased number of chromosomes with above-average kinetochore fluorescence did not produce decisive radiation dosimetry results.     

三种食肉目动物的核型分析

本文报道了产于我国华北地区的艾鼬、貉和果子狸的核型。其中艾鼬2n=36。组型由20条双臂染色体、14条单臂染色体和一对性染色体组成。貉2n=65,它与wuster等报道过的貉的2n=42的核型表现出同种动物核型间的多态现象。果子狸2n=44,其c带带型中具有一对带有较大异染色质的中部着丝点染色体。     

Two new reciprocal translocations in the German cockroach. Cytology and genetics of T(3;12) and T(7;12).

Two new reciprocal translocations in the German cockroach have been analyzed. They were identified cytologically to be T(3;12) and T(7;12). Linkage studies showed that groups XI, IX, and IV are on chromosomes 12, 3, and 7, respectively, and clearly demonstrated sex differences in recombination. Each of these chromosomes have distinctive morphological features that facilitate their identification, and permit breakpoint and centromere localization. A sex difference in fecundity is associated with T(7;12), but not T(3;12). About 40 percent mortality occurred when T(3;12) males or females and T(7;12) females were outcrossed. Outcrossing T(7;12) males produced the expected 50 percent mortality. Cell counts at metaphase I revealed that T(3;12) males exhibit directed segregation, while T(7;12) males do not. Tests for homozygosity indicated that the T(7;12) homozygote is viable. A map of chromosome 12 is presented showing the tentative placement of linkage group XI with respect to interchange breakpoints and chromosome morphology. The results are discussed in relation to possible sex differences in chiasma localization.