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1.
Gene targeting for somatic cell manipulation: rapid analysis of reduced chromosome hybrids by Alu-PCR fingerprinting and chromosome painting. 总被引:3,自引:0,他引:3
J R Dorin E Emslie D Hanratty M Farrall J Gosden D J Porteous 《Human molecular genetics》1992,1(1):53-59
The techniques of reverse genetics rely heavily on parasexual methods for manipulating the human genome. However, the application of somatic cell genetics is severely limited by the availability of suitable endogenous selectable markers in the genome. We have addressed this problem by targeting a universally selectable marker into a predetermined region of the genome, using a stringent selection for homologous recombination. Correct gene targeting to human chromosome 7q11 was screened for by Southern blotting and confirmed by fluorescent in situ hybridization. Reduced chromosome 7 hybrids were generated by chromosome mediated gene transfer and selection for the neo gene. The resultant transgenomes were characterized by a combination of L1 fingerprinting, locus specific marker analysis, Alu-PCR and chromosome 'painting'. Alu-PCR and L1 'fingerprints' are complementary and mutually consistent. Chromosome 'painting' reflects and extends the results obtained for specific marker co-transfer. Thus Alu-PCR 'fingerprinting' and 'painting' combine to rapidly provide an accurate picture of transgenome content and complexity. Gene targeting, chromosome tagging and subsequent isolation can be applied to any region of the genome for which a molecular probe is available. 相似文献
2.
Kim MS Kim SH Kim HJ Hoang IN Oh WM Koh JT Park HO Jeong JY Kim WJ Lee EJ Koh JY Kim BY Jensen RH 《Cancer Genetics and Cytogenetics》2005,163(1):17-22
TSU-PR1 was originally reported as a prostatic carcinoma cell line derived from a lymph node metastasis. Recently, however, this cell line was reported to be derived from T24 bladder carcinoma cells, and thus further definition of its origin is needed. Conventional cytogenetic study of TSU-PR1 showed aneuploidy, ranging from 65 to 86 chromosome with a modal number of 80, and with 10 marker chromosomes, thus conventional cytogenetics cannot be used to determine which chromosomes or regions of chromosomes are critical in cancer development and progression of this cell line. The present study was conducted to characterize genetic changes of the cell line using comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), and flow cytometry. CGH results showed that green-to-red fluorescence ratios were within the range of 0.85-1.15, except for a few chromosomes, which reflected near tetraploidy in TSU-PR1. Flow cytometric analysis of TSU-PR1 revealed a DNA index of 3.46n, which is close to the 3.48n calculated from a modal number of 80. The copy numbers of chromosomes 4, 6, 7, 17, and 20 determined by the DNA index and the CGH analyses were 2.85 +/- 0.09, 3.22 +/- 0.77, 3.01 +/- 0.26, 4.05 +/- 0.44, and 4.99 +/- 0.48, respectively. These numbers are also in accordance with the chromosome copy numbers determined with FISH: 2.98 +/- 0.23, 2.91 +/- 0.44, 2.74 +/- 0.44, 3.93 +/- 0.38, and 5.05 +/- 0.78 for chromosomes 4, 6, 7, 17, and 20, respectively (P > 0.05). 相似文献
3.
Ostroverkhova NV Nazarenko SA Rubtsov NB Nazarenko LP Bunina EN 《American journal of medical genetics》1999,84(3):217-220
In order to identify genetic factors governing expansion of the CGG repeat in the FMR1 gene and to determine what predisposes or causes a normal stable allele to change to an unstable premutation allele, it is essential to study and understand the basis of normal variation. The aim of this study was to investigate genetic variation and intergenerational stability of the FMR1 CGG-repeat region in 100 unrelated three-generation families from the general population (651 meioses). The number of CGG-repeats in the FMR1 gene was determined in all 750 individuals from the 100 families (a total of 1,132 X-chromosomes), and the allele frequencies and variability were analyzed. Thirty-six different alleles (12-60 repeats) were seen with 30 (45.8%) as the most common allele; overall female heterozygosity was 73%. Most (>96%) of the normal array lengths were less than 40 repeats. Fifteen families with at least one allele equal to or greater than 40 repeats (40-60) were identified; in one of these families there was an increase of one triplet repeat during transmission from a mother to son. These findings, together with future molecular analyses, may provide data to test proposed models that attempt to explain the mutational process and the population dynamics of the triplet repeat region of the FMR1 gene, including the transition from normal to unstable alleles, or to test other putative cis-acting sequences that may be involved with instability in the FMR1 gene. 相似文献
4.
Chizuko Nishida Junko Ishijima Ayumi Kosaka Hideyuki Tanabe Felix A. Habermann Darren K. Griffin Yoichi Matsuda 《Chromosome research》2008,16(1):171-181
Karyotypes of most bird species are characterized by around 2n = 80 chromosomes, comprising 7–10 pairs of large- and medium-sized
macrochromosomes including sex chromosomes and numerous morphologically indistinguishable microchromosomes. The Falconinae
of the Falconiformes has a different karyotype from the typical avian karyotype in low chromosome numbers, little size difference
between macrochromosomes and a smaller number of microchromosomes. To characterize chromosome structures of Falconinae and
to delineate the chromosome rearrangements that occurred in this subfamily, we conducted comparative chromosome painting with
chicken chromosomes 1–9 and Z probes and microchromosome-specific probes, and chromosome mapping of the 18S–28S rRNA genes
and telomeric (TTAGGG)
n
sequences for common kestrel (Falco tinnunculus) (2n = 52), peregrine falcon (Falco peregrinus) (2n = 50) and merlin (Falco columbarius) (2n = 40). F. tinnunculus had the highest number of chromosomes and was considered to retain the ancestral karyotype of Falconinae; one and six centric
fusions might have occurred in macrochromosomes of F. peregrinus and F. columbarius, respectively. Tandem fusions of microchromosomes to macrochromosomes and between microchromosomes were also frequently observed,
and chromosomal locations of the rRNA genes ranged from two to seven pairs of chromosomes. These karyotypic features of Falconinae
were relatively different from those of Accipitridae, indicating that the drastic chromosome rearrangements occurred independently
in the lineages of Accipitridae and Falconinae. 相似文献
5.
6.
Using chromosome painting, a study of chromosomal abnormalities was performed in six gastric carcinoma cell lines (SNU-484, 601, 620, 638, 668, 719) from Korean patients. Each carcinoma cell line had unique modal karyotypic characteristics and showed a variable number of numerical and structural clonal cytogenetic aberrations. SNU-484, SNU-620, and SNU-668 had near-triploidy; SNU-601, SNU-638, and SNU-719 had near-diploidy. The origins of the marker chromosomes of these cell lines were identified by fluorescence in situ hybridization with constructed painting probes. In all of six cell lines, rearrangement of chromosome 17 resulting in partial deletion of 17p (and/or partial duplication of 17q) was found. The most frequent marker was a partial gain of chromosome 7 with the breakpoints on 7q22 and 7q31. The nonrandom rearrangements of chromosomes were also determined on 1q32, 5q11-q22, 8q, 14q22, 14q34, and 15q15; suggesting that they may be the candidate regions for the isolation of the genes related to gastric cancer. 相似文献
7.
Sgura A Stronati L Gullotta F Pecis A Cinelli S Lascialfari A Tanzarella C Pacchierotti F 《Environmental and molecular mutagenesis》2005,45(5):419-426
Chromosomal aberrations are a measure of genomic instability, which is known to play a key role in the initiation and promotion of carcinogenesis. Stable reciprocal translocations are of particular importance since they are often involved in neoplastic transformation and tumor cell clonal evolution. In this study, chromosome painting analysis was used to test for stable aberrations induced in the bone marrow of C57BL/6J and FVB mice exposed for 4 weeks to 2 or 4 mg/kg of melphalan (MLP), a chemotherapeutic agent with carcinogenic potential. To compare the chemical-induced damage in different tissues, chromosome aberrations were also analyzed by chromosome painting in the spleen of C57BL/6J mice. At the 2 mg/kg dose, MLP induced comparable levels of chromosome-type aberrations in bone marrow cells of both mouse strains and in splenocytes of C57BL/6J mice. At 4 mg/kg, no further increase in aberrations was detected in bone marrow, while a dose-effect relationship was found in spleen cells. This different response may result from a negative selection against highly damaged bone marrow cells during mitotic proliferation. The results indicate that chromosome painting is a useful tool for detecting stable chromosome aberrations in somatic cells exposed to MLP and possibly to other genotoxic chemical carcinogens. 相似文献
8.
J. Hindkjær C. A. Brandt J. Koch T. B. Lund S. Kølvraa L. Bolund 《Chromosome research》1995,3(1):41-44
In situ techniques for the detection of specific chromosomes using centromeric probes and the decoration of entire chromosomes using chromosome painting are well established. However, in the deciphering of complex chromosomal aberrations it is valuable to be able to detect the centromere and the entire DNA of a specific chromosome in different colours simultaneously on the same metaphase. In this report we describe a combination of the primedin situ labelling (PRINS) technique and chromosome painting for simultaneous visualization of centromere-specific oligonucleotides and chromosome painting libraries. A key feature is that the denaturation step in the PRINS reaction is sufficient to keep the chromosomes denatured for chromosome painting. This means that PRINS and consecutive chromosome painting can be performed as a single procedure (PRINS-painting) 相似文献
9.
Beatriz Crispino Horacio Cardoso Adriana Mimbacas Virginia Mndez 《American journal of medical genetics. Part A》1995,55(1):27-29
The combined use of high resolution banding and chromosome painting techniques allowed us to identify a reciprocal translocation involving chromosomes 3 and 20 and simultaneous interstitial deletion of chromosome 3 in a patient with several minor anomalies of the face and hands. His karyotype is described as 46,XY,t(3;20) (p14.2;p12.2),del(3)(p11-p14.1). © 1995 Wiley-Liss, Inc. 相似文献
10.
Irradiation-reduced human chromosome 21 hybrids 总被引:4,自引:0,他引:4
Sharon Graw Jeffrey Davidson James Gusella Paul Watkins Rudolph Tanzi Rachael Neve David Patterson 《Somatic Cell and Molecular Genetics》1988,14(3):233-242
Rodent-human somatic cell hybrids have been constructed which contain fragments of human chromosome 21 as their only human material. This was done by irradiating rodent-human somatic cell hybrids containing a complete chromosome 21 to fragment the genome and then rescuing human GAR synthetase and various amounts of flanking chromosome 21 DNA by fusing with GAR synthetase-deficient hamster cells and selecting for growth in purine-free medium. Four irradiation-reduction hybrids were produced by this method and contain the distal, proximal, and central portions of the long arm of human chromosome 21, all centered about GAR synthetase. These irradiation-reduction hybrids were used as a panel to regionally map single-copy and individual copies of repetitive sequences. Using these hybrids along with another independently constructed hybrid, the GAR synthetase gene was mapped distal to SOD-1 and proximal to CP21G1(D21S60). Of special interest is the regional mapping of the gene for the amyloid -protein distal to pPW236B(D21S11) and proximal to SOD-1. 相似文献
11.
Svetlana Derjusheva Anna Kurganova Felix Habermann Elena Gaginskaya 《Chromosome research》2004,12(7):715-723
Chicken chromosome paints for macrochromosomes 1-10, Z, and the nine largest microchromosomes (Griffin et al. 1999) were used to analyze chromosome homologies between chicken (Gallus gallus domesticus: Galliformes), domestic pigeon (Columba livia: Columbiformes), chaffinch (Fringilla coelebs Passeriformes), and redwing (Turdus iliacus: Passeriformes). High conservation of syntenies was revealed. In general, both macro- and microchromosomes in these birds showed very low levels of interchromosomal rearrangements. Only two cases of rearrangements were found. Chicken chromosome 1 corresponds to chromosome 1 in pigeon, but to chromosomes 3 and 4 in chaffinch and chromosomes 2 and 5 in redwing. Chicken chromosome 4 was shown to be homologous to two pairs of chromosomes in the karyotypes of pigeon and both passerine species. Comparative analysis of chromosome painting data and the results of FISH with (TTAGGG)n probe did not reveal any correlation between the distribution of interstitial telomere sites (ITSs) and chromosome rearrangements in pigeon, chaffinch and redwing. In chaffinch, ITSs were found to co-localize with a tandem repeat GS (Liangouzov et al. 2002), monomers of which contain an internal TTAGGG motif. 相似文献
12.
Pseudodicentric chromosome 18 diagnosed by chromosome painting and primed in situ labelling (PRINS).
C A Brandt B Djernes H Str?mkjaer M B Petersen S Pedersen J Hindkjaer J Brinch-Iversen G Bruun-Petersen 《Journal of medical genetics》1994,31(2):99-102
We report on a newborn white male infant with marked dysmorphic features and various congenital malformations. The initial clinical evaluation showed Crouzon-like features as well as some features of trisomy 18 syndrome and trisomy 13 syndrome. The results from conventional cytogenetic analysis showed a structurally abnormal chromosome replacing one normal chromosome 18, but only by applying molecular cytogenetic methods could the architecture of this abnormal chromosome be characterised clearly. The primed in situ labelling (PRINS) technique, using a newly synthesised alpha 18 oligonucleotide, showed the dicentric pattern and direct chromosome painting established the origin to be from chromosome 18. The combination of conventional cytogenetics and molecular cytogenetics showed the karyotype in the proband to be 45,XY,-14,-18,-21,+t(14;21),+psu dic(18) (qter-->cen-->p11.3: :p11.3-->psu cen-->qter). This was supported by molecular analysis using chromosome 18 specific DNA markers, which showed the paternal origin of the abnormal chromosome. 相似文献
13.
Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. 总被引:28,自引:0,他引:28
HKan Telenius Bruce A. J. Ponder Alan Tunnacliffe Adle H. Pelmear Nigel P. Carter Malcolm A. Ferguson-Smith Annemarie Behmel Magnus Nordenskjld Roswitha Pfragner 《Genes, chromosomes & cancer》1992,4(3):257-263
A novel polymerase chain reaction (PCR) technique has been combined with chromosome flow sorting to characterise two lymphoblastoid cell lines and one medullary thyroid carcinoma cell line carrying translocations close to the locus for multiple endocrine neoplasia type 2A (MEN 2A). Five hundred copies of the derivative chromosome(s) were flow sorted from each cell line and amplified by degenerate oligonucleotide-primed-polymerase chain reaction (DOP-PCR). This generated pools of DNA sequences corresponding to the abnormal chromosomes, which were then used as probes in fluorescence in situ hybridisation (FISH) experiments on normal metaphase cells. The resultant chromosome paints revealed the portions of the normal chromosomes related to those involved in the translocations. By this technique, translocation breakpoints in bands p15, q11.2, and q21 of chromosome 10 were defined in the above cell lines, in two cases refining previous cytogenetic data. This study shows that flow sorting of aberrant chromosomes and chromosome painting can be used as a rapid aid to cytogenetic analysis, particularly in cases of difficult karyotypes, such as tumours. Furthermore, the DOP-PCR technique described here will have applications to other areas of genome analysis, such as cloning of new markers; its design will allow a general and representative amplification to occur from any starting DNA in any species. 相似文献
14.
James D. Tucker John W. Breneman Jane F. Briner Gerald G. Eveleth Richard G. Langlois Dan H. Moore 《Environmental and molecular mutagenesis》1997,30(3):264-272
In this article, we address the issue of persistence of chromosome exchanges following acute in vitro exposure of rat peripheral blood to 137Cs. Irradiation occurred 24 hr after culture initiation, and metaphase chromosomes were prepared 2, 3, 4, and 5 days later. Chromosomes 1, 2, and 4 were painted in unique colors and scored for structural aberrations. Dicentric chromosomes and acentric fragments diminished rapidly with time, as expected. Translocations exhibited greater persistence, but still showed a reduction in frequency, reaching a plateau of approximately 65 and 55% of their initial values, 4 days after exposure to 1 and 2 Gy, respectively. An exponentially declining model was fit to the combined dicentric, acentric fragment, and translocation frequencies, which showed that all three aberration types declined at equivalent rates. The frequencies of dicentrics and fragments declined to a plateau of zero, while translocations reached a plateau at frequencies significantly greater than zero. The decline in translocations with time is inconsistent with prevailing theoretical expectations, but is consistent with a model where some translocations are fully stable (persistent) and some are unstable (not persistent) through cell division. These results may have implications for radiation biodosimetry in humans. Environ. Mol. Mutagen. 30:264–272, 1997. © 1997 Wiley-Liss, Inc. 相似文献
15.
Characterization and use of somatic cell hybrids with interspecific translocations involving the human X chromosome 总被引:1,自引:0,他引:1
Y. BOYD 《Annals of human genetics》1987,51(1):13-26
Two hybrid cell lines, whose only human material was a portion of the X translocated on to a mouse chromosome, have been characterized by cytogenetics, in situ hybridization and Southern blotting. In one hybrid (HORL911R8B) the region Xpter to Xq2(2–4) was identified. In the other (PIP) the single human fragment was found to contain sequences from two separate X chromosomal regions (corresponding approximately to Xp11.4–Xp22.1 and Xq26–Xqter). These two hybrids in combination with a third (WAG 8) retaining Xqter to Xp21 as a human X-autosome translocation chromosome, form a mapping panel for rapid subregional assignments to the human X chromosome. This mapping panel has been used to provide information about the order of DNA sequences derived from the X chromosome and to provide an assignment for an anonymous DNA segment, M201γ, to Xp11.4–Xp21.1. 相似文献
16.
Katia-dni F. de Albuquerque Coêlho Masayuki Egashira Rumiko Kato Masahiro Fujimoto Naomichi Matsumoto Budsaba Rerkamnuaychoke Kyohko Abe Naoki Harada Hirofumi Ohashi Yoshimitsu Fukushima Norio Niikawa 《American journal of medical genetics. Part A》1996,63(3):468-471
A molecular cytogenetic method consisting of chromosome microdissection and subsequent reverse/forward chromosome painting is a powerful tool to identify chromosome abnormalities of unknown origin. We present 4 cases of chromosome structural abnormalities whose origins were ascertained by this method. In one MCA/MR patient with an add(5q)chromosome, fluorescence in situ hybridization (FISH), using probes generated from a microdissected additional segment of the add(5q) chromosome and then from a distal region of normal chromosome 5, confirmed that the patient had a tandem duplication for a 5q35-qter segment. Similarly, we ascertained that an additional segment of an add(3p) chromosome in another MCA/MR patient had been derived from a 7q32-qter segment. In a woman with a history of successive spontaneous abortions and with a minute marker chromosome, painting using microdissected probes from the whole marker chromosome revealed that it was i(15)(p10) or psu dic(15;15)(q11;q11). Likewise, a marker observed in a fetus was a ring chromosome derived from the paracentromeric region of chromosome 19. We emphasize the value of the microdissection-based chromosome painting method in the identification of unknown chromosomes, especially for marker chromosomes. The method may contribute to a collection of data among patients with similar or identical chromosome abnormalities, which may lead to a better clinical syndrome delineation. © 1996 Wiley-Liss, Inc. 相似文献
17.
Elisabeth Blennow> 《Chromosome research》2004,12(1):25-33
Reverse chromosome painting, as the opposite of forward chromosome painting, means that an abnormal chromosome of interest is recovered by flow sorting or by chromosome microdissection, amplified and labelled by DOP-PCR and hybridized onto normal metaphases of optimal quality. This provides rapid and unequivocal information about the chromosomal origin on the aberrant chromosome in one hybridization. Not only will the specific chromosome(s) involved be identified, but also the subchromosomal origin, including the breakpoints. The method has been used for over 10 years and has proven to be very useful for resolving complex chromosome rearrangements in a variety of different applications, both as a research tool and for clinical purposes in pre- and postnatal diagnosis. 相似文献
18.
Microdissection and chromosome painting of plant B chromosomes 总被引:1,自引:0,他引:1
Plant chromosome microdissection techniques together with different isolation and amplification methods of microisolated DNA are described. Such isolated DNA was used to 'chromosome paint' B chromosomes of the dicot Brachycome dichromosomatica and the monocot Secale cereale. It is demonstrated that the specific painting of the described chromosomes was possible because of enrichment for chromosome-specific repetitive sequences, rather than the chromosome specific low- and single-copy sequences which are responsible for the painting of mammalian chromosomes. The feasibility of 'chromosome painting' of standard chromosomes in plant species with relatively small or large genomes is discussed. 相似文献
19.
Chromosome evolution in bears: reconstructing phylogenetic relationships by cross-species chromosome painting 总被引:1,自引:0,他引:1
Ying Tian Wenhui Nie Jinhuan Wang Malcolm A. Ferguson-Smith Fengtang Yang 《Chromosome research》2004,12(1):55-63
Genome-wide homology maps among dog (Canis familiaris, CFA, 2n = 78), African lion (Panthera leo, PLE, 2n = 38), clouded leopard (Neofelis nebulosa, NNE, 2n = 38) and Malayan sun bear (Helartos malayanus, HMA, 2n = 74) have been established by chromosome painting using a complete set of dog probes. In total, chromosome-specific painting probes from the 38 dog autosomes reveal 69, 69 and 73 conserved segments in African lion, clouded leopard and Malayan sun bear, respectively. The chromosomal painting results show that the African lion and clouded leopard have an identical karyotype which, in turn, is similar to that previously published for the cat (Felis catus, FCA 2n = 38). The findings confirm and extend other studies that show felids to be karyotypically conserved. In contrast, ursids, including the Malayan sun bear, have a relatively highly rearranged karyotype in comparison with other carnivores. The 2n = 74 karyotype of the Malayan sun bear, which is believed to closely resemble the ancestral karyotype of the Ursidae, could have evolved from the 2n = 42 putative ancestral carnivore karyotype by an inversion and 16 centric fissions. Independent fusions of the acrocentric ancestral chromosomes have generated the unique karyotypes of the giant panda and the spectacled bear. 相似文献
20.
Nancy B. Spinner Joe N. Lucas Marilyn Poggensee Mary Jacquette Adele Schneider 《American journal of medical genetics. Part A》1993,45(5):609-613
We have studied an infant with multiple anomalies and a 46,XY,12p+ karyotype. Parental chromosomes were normal, and it was not possible to determine the identity of the extra material on chromosome 12 cytogenetically. Chromosome painting with probes from a chromosome 9 library identified this material as coming from chromosome 9, and cytogenetics established the duplication as 9q34→qter. Comparison of this patient with others reported with partial dup(9q) documented excellent concordance of minor anomalies, most notably dolichocephaly, “;deep-set”; eyes, short horizontal palpebral fissures, beaked nose, micrognathia, arachnodactyly, and developmental delay. Identification of cytogenetically indeterminate abnormalities by molecular cytogenetics is very important, as it permits prognosis to be offered for families of newborn infants with unbalanced karyo-types. © 1993 Wiley-Liss, Inc. 相似文献