应用端粒区带涂染探针检测染色体微小结构重排

为了评估染色体端粒区带涂染探针在遗传诊断的应用价值,应用显微切割获得的11q、12q和22q等3个染色体端粒区涂染探针（11q23.3→qter,12q24.1→qter,22q13.1→qter）,通过荧光原位杂交技术分析两个疑有染色体末端微小易位的习惯性流产病例。结果显示,病例1和病例2分别为t(11;12)和t(11;22)长臂末端间的微小易位,结合G显带技术确定断裂位点位于11q23.3、12q24.1、22q13.1。结果表明特异性染色体端粒区带探针可以确诊染色体末端区域的微小结构异常,可作为一种检出隐匿易位携带者并确定断裂位点的方法。     

Chromosomal rearrangements in cattle and pigs revealed by chromosome microdissection and chromosome painting

A pericentric inversion of chromosome 4 in a boar, as well as a case of (2q-;5p+) translocation mosaicism in a bull were analysed by chromosome painting using probes generated by conventional microdissection. For the porcine inversion, probes specific for p arms and q arms were produced and hybridised simultaneously on metaphases of a heterozygote carrier. In the case of the bovine translocation, two whole chromosome probes (chromosome 5, and derived chromosome 5) were elaborated and hybridised independently on chromosomal preparations of the bull who was a carrier of the mosaic translocation. The impossibility of differentiating chromosomes 2 and der(2) from other chromosomes of the metaphases did not allow the production of painting probes for these chromosomes. For all experiments, the quality of painting was comparable to that usually observed with probes obtained from flow-sorted chromosomes. The results obtained allowed confirmation of the interpretations proposed with G-banding karyotype analyses. In the bovine case, however, the reciprocity of the translocation could not be proven. The results presented in this paper show the usefulness of the microdissection technique for characterising chromosomal rearrangements in species for which commercial probes are not available. They also confirmed that the main limiting factor of the technique is the quality of the chromosomal preparations, which does not allow the identification of target chromosomes or chromosome fragments in all cases.     

Detection of amplified DNA sequences by reverse chromosome painting using genomic tumor DNA as probe

A modification of reverse chromosome painting was carried out using genomic DNA from tumor cells as a complex probe for chromosomal in situ suppression hybridization to normal metaphase chromsome spreads. Amplified DNA sequences contained in such probes showed specific signals, revealing the normal chromosome positions from which these sequences were derived. As a model system, genomic DNAs were analyzed from three tumor cell lines with amplification units including the proto-oncogene c-myc. The smallest amplification unit was about 90 kb and was present in 16–24 copies; the largest unit was bigger than 600 kb and was present in 16–32 copies. Specific signals that co-localized with a differently labeled c-myc probe on chromosome band 8q24 were obtained with genomic DNA from each cell line. In further experiments, genomic DNA derived from primary tumor material was used in the case of a male patient with glioblastoma multiforme (GBM). Southern blot analysis using an epidermal growth factor receptor gene (EGFR) probe that maps to 7p13 indicated the amplification of sequences from this gene. Using reverse chromosome painting, signals were found both on band 7p13 and bands 12q13–q15. Notably, the signal on 12q13–q15 was consistently stronger. The weaker 7p13 signal showed co-localization with the major signal of the differently labeled EGFR probe. A minor signal of this probe was seen on 12q13, suggesting cross-hybridization to ERB3 sequences homologous to EGFR. The results indicate co-amplification of sequences from bands 12q13–q15, in addition to sequences from band 7p13. Several oncogenes map to 12q13–q15 providing candidate genes for a tumor-associated proto-oncogene amplification. Although the nature of the amplified sequences needs to be clarified, this experiment demonstrates the potential of reverse chromosome painting with genomic tumor DNA for rapidly mapping the normal chromosomal localization of the DNA from which the amplified sequences were derived. In addition, a weaker staining of chromosomes 10 and X was consistently observed indicating that these chromosomes were present in only one copy in the GBM genome. This rapid approach can be used to analyze cases where no metaphase spreads from the tumor material are available. It does not require any preknowledge of amplified sequences and can be applied to screen large numbers of tumors.     

Development of arm-specific and subtelomeric region-specific painting probes for Chinese hamster chromosomes and their utility in chromosome identification of Chinese hamster cell lines

Arm-specific and subtelomeric region-specific painting probes for Chinese hamster chromosomes have been generated by microdissection and use of the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). Fluorescence in situ hybridization (FISH) analyses using these probes demonstrated their specificity. These probes painted every chromosome arm and a total of 15 subtelomeric regions, namely, both ends of chromosomes 1, 2, 3, 4, and 8 and one end of chromosome arms 5q, 6q, 7q, 9p, and Xp. Many cryptic chromosomal rearrangements in the CHO-9 and V79 cell lines that were not detectable with whole chromosome paints could be recognized when these newly developed probes were used.     

Localization of 11q13 loci with respect to regional chromosomal breakpoints

We have employed two strategies to map 13 markers located at 11q13. First, we used pulsed-field gel electrophoresis of DNA fragments obtained with methylation-sensitive restriction enzymes. The markers used in this study were scattered over 8.4 Mb and, for most of them, could not be linked one to another. A second mapping strategy employed hybridization to either DNA of somatic hybrids containing various parts of the long arm of chromosome 11 or metaphase chromosomes of a B-cell line containing the t(11;14)(q13;q32) translocation. We were able to sort out the centromeric from the telomeric probes with respect to translocation breakpoints taken as reference chromosomal landmarks by this approach. BCL1, which corresponds to the region where the t(11;14)(q13;q32) translocation breakpoints are clustered, appears as a boundary between two areas of human/mouse homology present in conserved syntenic regions on mouse chromosomes 7 and 19.     

Fluorescent in situ identification of human marker chromosomes using flow sorting and Alu element-mediated PCR.

A novel approach to the identification of human chromosomes has been developed. Chromosomal in situ hybridization (or "chromosome painting") has been performed using Alu element-mediated PCR products from small quantities (250-500) of flow-sorted normal and abnormal chromosomes. Chromosome paints for various normal chromosomes, including 5, 6, 7, 14, 18, 19, 21, and 22, were generated and shown to be effective in the identification of the appropriate chromosomes. In addition, certain abnormal chromosomes, including a mental retardation-associated deletion chromosome 11 (q22-q23), the products of the constitutional translocation t(11;22), and the CML-associated t(9;22), were used to generate region-specific paints. In each case, the appropriate regions of the chromosomes were highlighted and this strategy is, therefore, well suited to the identification of previously unidentified marker chromosomes. A further direct consequence of this work is that chromosome paints specific for the common aberrant chromosomes, such as the Philadelphia chromosome, can be generated and made widely available. These may find particular use in the analysis of complex or masked chromosomal translocations.     

Telomeric sequences derived from laser-microdissected polytene chromosomes

Telomeric fragments from salivary gland squashes of Drosophila melanogaster Oregon R. were produced by a new microdissection technique, UV laser microbeam dissection. Microdissection, an essential step in microcloning procedures, is usually performed using micromanipulators and microneedles. Recently it has been shown that microdissection can be improved to very high precision if a laser coupled into a microscope is used. A laser microbeam, generated by an excimer pumped dye laser, allows chromosomes to be cut into slices of less than 0.5 m. Here it is shown, that single copy DNA probes prepared from Drosophila chromosomes by laser microdissection and microcloning relocalize to the chromosomal regions from which they are derived. The combination of laser technique and microcloning provides an advantageous approach for rapid genetic analysis with potential for the study of genetic diseases and genome mapping.     

Precise location of breakpoints in a frequent reciprocal translocation between chromosomes 11 and 22

One of the most frequent chromosomal translocations in human beings is 11q/22q, which results in the "partial trisomy of 22q syndrome." However, the breakpoint on the long arms of chromosomes 11 and 22 is still a matter of controversy. In the present study, we have used chromosomes from lymphocytes of a neonate who happened to have this classical abnormality, and by R-banding prometaphase chromosomes with acridine orange it has been possible to establish that the translocation between chromosomes 11 and 22 resulted from 3:1 meiotic maternal nondisjunction. A detailed analysis of the chromosome regions involved in this translocation revealed that the breakpoints on chromosomes 11 and 22 were at 11q23.3 and 22q11.1, respectively.     

PCR amplification of DNA microdissected from a single polytene chromosome band: a comparison with conventional microcloning.

A novel alternative to microcloning for the production of region specific chromosomal DNA is described. In this method, 'microamplification', single bands are dissected from polytene chromosomes and digested with Sau3A. Oligonucleotide adaptors are ligated to these fragments to provide convenient priming sites for polymerase chain reaction amplification. In this way, as much as 1 microgram of DNA can be amplified from a single band. Probes made from PCR amplified DNA from two such dissections have been used to probe cloned DNA form a 100 kb chromosome walk. Whereas conventional microcloning has generated cloned EcoRI fragments corresponding to 3-4 kb of the walk, the PCR probes cover greater than 90% of this chromosomal region. Thus microamplification is significantly more effective than microcloning in providing probes for establishing chromosomal walks.     

应用染色体涂染技术分析两例染色体结构异常 Analysis of the Structure of Abnormal Karyotypes by Using Chromosome Painting

为了确定两例细胞遗传学提示染色体结构异常的核型,应用通过显微切割技 术构建的人类18号和7号染色体探针池,分别对这两例病例的中期分裂相进行染色体涂染,结合显带染色体,确定两者核型分别为46,XY,t(3;18) (q12;q21)和46,XX,dir ins(1;7)(p3104;q34q36)。染色体涂染技术是染色体显带技术的重要补充和发展,为染色体结构异常提供了一种直观、准确的检测手段,在遗传咨询和产前诊断方面有重要作用。 Abstract:In this study,chromosome painting technique was performed to analyse the abnormal karyotypes of two carriers.Chromosome 18 and 7 specific libraries,which were generated by chromosome microdissection technique,were used as probe pools to hybridize the carriers metaphase chromosomes respectively.Unlabled human genomic DNA was used to inhibit the hybridization of sequences in the library that bind to mutiple chromosomes.Structure abnormality was detected clearly in metaphase.Combined with the banding chromosomes,we concluded that their karytypes were 46,XY,t(3;18)(q12;q21)and 46,XX,dir ins(1;7)(p3104;q34q36).Chromosome painting,as a direct and concise method in analysing chromosome structure abnormality,is an important complement and development of chromosome banding technique,and has important application in genetic counselling and prenatal diagnosis.     

Jumping translocations in spontaneous abortions

Chromosome translocations involving one donor chromosome and multiple recipient chromosomes have been referred to as jumping translocations (JTs). Acquired JTs are commonly observed in cancer patients, mainly involving chromosome 1. Constitutional forms of JTs mostly involve the acrocentric chromosomes and their satellites and have been reported in patients with clinical abnormalities. Recognizable phenotypes resulting from these events have included Down, Prader-Willi, and DiGeorge syndromes. The presence of JTs in spontaneous abortions has not been previously described. The breakpoints of all JTs occur in areas rich in repetitive DNA (telomeric, centromeric, and nucleolus organizing regions). We report two different unstable chromosome rearrangements in samples derived from spontaneous abortions. The first case involved a chromosome 15 donor. The recipient chromosomes were 1, 9, 15, and 21, and the respective breakpoints were in either the heterochromatic regions or the centromeres. FISH studies confirmed that the breakpoints of the jumping 15 rearrangement did not involve the Prader-Willi region but originated at the centromere or in the proximal short arm. A second case of instability was observed with a rearrangement resulting from a presumed de novo 8;21 translocation. Three JT cell lines were observed. They consisted of a deleted 8p chromosome, a dicentric 8;21 translocation, and an 8q isochromosome. The instability regions appeared to be at the pericentromeric region of chromosome 8 and the satellite region of chromosome 21. Both cases proved to be de novo events. The unstable nature of the JT resulting in chromosomal imbalance most likely contributed to the fetal loss. It appears that JT events may predispose to chromosomal imbalance via nondisjunction and chromosomal rearrangement and, therefore, may be an unrecognized cause of fetal loss.     

Genomic homology of the domestic ferret with cats and humans

We used chromosome paints from both the domestic cat and humans to directly establish chromosomal homology between the genome of these species and the domestic ferret. The chromosome painting data indicate that the ferret has a highly conserved karyotype closer to the ancestral carnivore karyotype than that of the cat. The cat chromosome paints revealed 22 homologous autosomal regions in the ferret genome: 16 ferret chromosomes were hybridized by a single cat paint, while 3 ferret chromosomes were hybridized by two cat paints. In situ hybridization combined with banding showed that ferret Chromosome (Chr) 1 = cat A2p/C2, Chr 2 = F2/C1q, and Chr 3 = A2q/D2. Five ferret chromosomes are homologous to single arms of cat chromosomes: ferret 4 = A1q, 5 = B1q, 6 = C1p, 10 = A1p, and 12 = B1p. The human chromosome paints revealed 32 + XY homologous regions in the ferret genome: 9 ferret chromosomes were each hybridized by a single human paint, 7 by two paints, 3 by three paints. The 10 ferret chromosomes hybridized by multiple human paints produced the following associations: ferret 1 = human 19/3/21, 2 = 8q/2q, 3 = 10/7, 5 = 8/4, 8 = 15/14, 9 = 10/12/22, 11 = 20/2, 12 = 8/4, 14 = 12/22/18, 18 = 19/16. We present an index of genomic diversity, Z, based on the relative number of conserved whole chromosome and chromosome segments as a preliminary statistic for rapid comparison between species. The index of diversity between human-ferret (Z = 0.812) is slightly less than human-cat (Z = 0.843). The homology data presented here allow us to transfer gene mapping data from both cats and humans to the ferret. Received: 21 December 1999 / Accepted: 30 May 2000     

Contribution of chromosomal imbalance to sperm selection and pre-implantation loss in translocation-heterozygous Chinese hamsters

Chinese hamster stocks with various structurally abnormal chromosomes have been produced by X irradiation. Among these stocks, 18 with various reciprocal translocations were used to investigate the participation of unbalanced gametes in fertilization and the development of unbalanced embryos. Among males as well as females heterozygous for the same translocation, there is no difference in the frequency of each disjunctional class. The participation of chromosomally unbalanced gametes in fertilization was investigated by chromosomal analysis of meiotic cells in heterozygotes for the 18 reciprocal translocations and pronuclei of fertilized ova obtained from crossing these heterozygotes. Compared with the expected frequencies from MII scoring, the frequencies of male pronuclei having a common deficiency of chromosome 1 (1q17-->1q42) or chromosome 3 (3p23-->3q31) decreased significantly in one-cell embryos. However, the frequencies of male pronuclei with other abnormalities were all consistent with those expected from MII scoring. In contrast, the frequencies of female pronuclei with any karyotype including the same abnormalities as those decreased in male pronuclei from the translocation heterozygotes were all consistent with those estimated from MII scoring. These results revealed clearly that most gametes with nullisomies as well as disomies for any chromosomal segments may participate in fertilization, whereas only male gametes nullisomic for certain segments of chromosomes 1 and 3 failed to participate in fertilization. The zygotic selection of chromosomal imbalance was also investigated by direct chromosomal and morphological analyses of preimplantation embryos from crosses between karyotypically normal females and male heterozygotes from the 18 stocks with various reciprocal translocations. These analyses revealed that some embryos were arrested in development at the two-cell stage. The karyotype of these two-cell embryos had a common deficiency in a segment of chromosome 1 or chromosome 2. Embryos with partial monosomy including chromosomes 1, 3, 4 and 5 showed arrested development at four- to eight-cell stages. Among day 4 embryos, some chromosomally unbalanced embryos, mainly with a deficiency of segments of chromosomes 1p, 1q, 2q, 5q, 7q and 8, had fewer blastomeres than karyotypically normal and balanced embryos. The homology between Chinese hamster and mouse chromosomes relating to abnormal embryogenesis at early stages has been partially confirmed from reported maps of chromosomes. The Chinese hamster is useful for further cytogenetic studies during the stages of meiosis and early embryogenesis.     

Microdissection and molecular manipulation of single chromosomes in woody fruit trees with small chromosomes using pomelo (<Emphasis Type="Italic">Citrus grandis</Emphasis>) as a model. I. Construction of single chromosomal DNA libraries

Construction of single chromosomal DNA libraries by means of chromosome microdissection and microcloning will be useful for genomic research, especially for those species that have not been extensively studied genetically. Application of the technology of microdissection and microcloning to woody fruit plants has not been reported hitherto, largely due to the generally small sizes of metaphase chromosomes and the difficulty of chromosome preparation. The present study was performed to establish a method for single chromosome microdissection and microcloning in woody fruit species using pomelo as a model. The standard karyotype of a pomelo cultivar (Citrus grandis cv. Guanxi) was established based on 20 prometaphase photomicrographs. According to the standard karyotype, chromosome 1 was identified and isolated with fine glass microneedles controlled by a micromanipulator. DNA fragments ranging from 0.3 kb to 2 kb were acquired from the isolated single chromosome 1 via two rounds of PCR mediated by Sau3A linker adaptors and then cloned into T-easy vectors to generate a DNA library of chromosome 1. Approximately 30,000 recombinant clones were obtained. Evaluation based on 108 randomly selected clones showed that the sizes of the cloned inserts varied from 0.5 kb to 1.5 kb with an average of 860 bp. Our research suggests that microdissection and microcloning of single small chromosomes in woody plants is feasible.Communicated by P. Langridge     

Localization of the beta-globin gene by chromosomal in situ hybridization

A 3.7-kilobase (kb) genomic clone of the human beta-globin gene, including 1.5-kb upstream and approximately 0.5-kb downstream, was utilized in chromosomal in situ hybridization for precise mapping of the beta-globin locus on peripheral blood lymphocyte-derived metaphases from a normal male, and for further evaluation of a clonal t(7;11) (q22;p15) translocation on bone marrow-derived metaphases from a 46-year-old male with erythroleukemia. Analyses of 205 midmetaphases from a normal male hybridized with the tritium-labeled beta-globin probe and stained with quinacrine mustard dihydrochloride revealed approximately 12% of spreads to have silver-grain deposition over the p15 band of chromosome 11. Of the 365 silver grains observed to be located on or beside chromosomes, 25 (approximately 7%) grains were localized in band p15. Karyotype analysis of a bone marrow specimen from the patient with erythroleukemia revealed hypodiploidy with various unidentified marker chromosomes as well as a presumably balanced translocation between 7q and 11p . Chromosomal in situ hybridization showed localization of silver grains at the junction between chromosomes 7 and 11 as well as to the normal chromosome 11, indicating that the beta-globin locus had not been translocated in the chromosomal rearrangement. This case demonstrates the value of chromosomal in situ hybridization in the definition of chromosome rearrangements and provides further evidence for the localization of the beta-globin gene to 11p15 .     

A method for the rapid sequence-independent amplification of microdissected chromosomal material

We have developed a simple, efficient method by which microdissected material can be amplified directly in the collection container in a few hours. The procedure involves two initial rounds of DNA synthesis with T7 DNA polymerase, using a primer that contains a random pentanucleotide sequence at its 3' end and a defined sequence at its 5' end, followed by PCR amplification with the defined sequence as the primer. The resulting products can be biotinylated and used for fluorescence in situ hybridization (FISH) to confirm their chromosomal location. As few as 17 dissected chromosomal regions provide sufficient material for a specific FISH signal on the appropriate band of metaphase chromosomes. We have obtained a chromosome 6q25-qter-specific painting probe in this way.     

Mapping of genetic abnormalities of primary tumours from metastatic CRC by high-resolution SNP arrays

Background

For years, the genetics of metastatic colorectal cancer (CRC) have been studied using a variety of techniques. However, most of the approaches employed so far have a relatively limited resolution which hampers detailed characterization of the common recurrent chromosomal breakpoints as well as the identification of small regions carrying genetic changes and the genes involved in them.

Methodology/Principal Findings

Here we applied 500K SNP arrays to map the most common chromosomal lesions present at diagnosis in a series of 23 primary tumours from sporadic CRC patients who had developed liver metastasis. Overall our results confirm that the genetic profile of metastatic CRC is defined by imbalanced gains of chromosomes 7, 8q, 11q, 13q, 20q and X together with losses of the 1p, 8p, 17p and 18q chromosome regions. In addition, SNP-array studies allowed the identification of small (<1.3 Mb) and extensive/large (>1.5 Mb) altered DNA sequences, many of which contain cancer genes known to be involved in CRC and the metastatic process. Detailed characterization of the breakpoint regions for the altered chromosomes showed four recurrent breakpoints at chromosomes 1p12, 8p12, 17p11.2 and 20p12.1; interestingly, the most frequently observed recurrent chromosomal breakpoint was localized at 17p11.2 and systematically targeted the FAM27L gene, whose role in CRC deserves further investigations.

Conclusions/Significance

In summary, in the present study we provide a detailed map of the genetic abnormalities of primary tumours from metastatic CRC patients, which confirm and extend on previous observations as regards the identification of genes potentially involved in development of CRC and the metastatic process.     

猕猴、白眉长臂猿、人类染色体普通型脆性部位和G-带的比较研究

以BrdU、FdU、MTX诱导猕猴、白眉长臂猿和人类染色体普通型脆性部位的表达,并对染色体脆性部位和染色体进化的关系以及三种灵长类染色体的同源性进行了比较分析。结果表明,近缘动物染色体同源区内的脆性部位在进化上是保守的,可作为染色体具有共同起源的标志,结合G-带的比较,可以用以阐明近缘动物染色体的同源性和染色体进化。