Distribution of (TTAGGG)n telomeric sequences in karyotypes of the Xenopus species complex.

The chromosomal distribution of the conserved vertebrate telomeric (TTAGGG)(n) sequence was studied by fluorescence in situ hybridization (FISH) in four Xenopus species and the triploid Silurana tropicalis. As expected, hybridization signals were observed at the distal ends of every chromosome in all species. In addition, the hybridization pattern demonstrates varied organization of (TTAGGG)(n) sequences in the different karyotypes. In X. borealis and X. muelleri hybridization signals intensely labeled one end of a homologous chromosome pair that coincides with the sites containing ribosomal RNA gene clusters. The karyotype of X. clivii remarkably differs from other Xenopus karyotypes in displaying numerous interstitial telomeric sites (ITS). C-banding analysis shows that the non-telomeric sites appear to correspond to the interstitially located constitutive heterochromatin. This suggests that interstitial telomeric sites in X. clivii do not necessarily represent the relic of ancestral telomeres resulting from the fusion of chromosomes, but their occurrence is due to the fact that (TTAGGG)(n) repeat arrays may be a constituent of highly repetitive DNA.

A haploid-diploid-triploid mosaic of the Amazon molly, Poecilia formosa.

We report the finding of the first haploid-diploid-triploid mosaic fish from the family Poeciliidae. The animal was derived from a laboratory cross of a female F1 hybrid of Poecilia mexicana and P. latipinna with a male from an ornamental strain derived from P. mexicana and P. sphenops (Black molly). It was identified because of its unusual pigmentation pattern and molecular methods (flow cytometry, NOR staining) confirmed its mosaic genotype. The mode of mosaic formation and the possible importance for poeciliid fish evolution are discussed.

Disruption of the FA/BRCA pathway in bladder cancer.

Bladder carcinomas frequently show extensive deletions of chromosomes 9p and/or 9q, potentially including the loci of the Fanconi anemia (FA) genes FANCC and FANCG. FA is a rare recessive disease due to defects in anyone of 13 FANC genes manifesting with genetic instability and increased risk of neoplasia. FA cells are hypersensitive towards DNA crosslinking agents such as mitomycin C and cisplatin that are commonly employed in the chemotherapy of bladder cancers. These observations suggest the possibility of disruption of the FA/BRCA DNA repair pathway in bladder tumors. However, mutations in FANCC or FANCG could not be detected in any of 23 bladder carcinoma cell lines and ten surgical tumor specimens by LOH analysis or by FANCD2 immunoblotting assessing proficiency of the pathway. Only a single cell line, BFTC909, proved defective for FANCD2 monoubiquitination and was highly sensitive towards mitomycin C. This increased sensitivity was restored specifically by transfer of the FANCF gene. Sequencing of FANCF in BFTC909 failed to identify mutations, but methylation of cytosine residues in the FANCF promoter region was demonstrated by methylation-specific PCR, HpaII restriction and bisulfite DNA sequencing. Methylation-specific PCR uncovered only a single instance of FANCF promoter hypermethylation in surgical specimens of further 41 bladder carcinomas. These low proportions suggest that in contrast to other types of tumors silencing of FANCF is a rare event in bladder cancer and that an intact FA/BRCA pathway might be advantageous for tumor progression.

SNP-Array genotyping and spectral karyotyping reveal uniparental disomy as early mutational event in MSS- and MSI-colorectal cancer cell lines.

In this study nine colorectal cancer cell lines were analysed by 10K SNP-arrays and spectral karyotyping (SKY). Complex chromosomal alterations and breakpoints of deleted or translocated fragments found by SKY could further be characterized by SNP-array analysis. Interestingly many monoallelic regions identified by SNP-array analysis display no copy number alterations, representing uniparental disomy (UPD). It was demonstrated that UPD seems to be involved in activation of early-acting tumor suppressor genes in MSS- (APC, CDKN2A) and MSI- (MLH1, MSH2, APC, CDKN2A) colorectal cancer cell lines. Genes involved later on in the adenoma-carcinoma sequence (i.e. TP53/SMAD4) were not found to be inactivated by UPD. Furthermore, identified amplified monoallelic regions may include oncogenes activated by allele-specific-amplification (i.e. Cyclin D1). However, at present, the majority of the monoallelic regions located in the present study have not yet been associated with known tumor suppressor genes and oncogenes. Further studies are warranted to identify relevant genes in the respective regions and to further verify the results presented here.

Characterization of chromosomal aberrations in diffuse large B-cell lymphoma (DLBL) by G-banding and spectral karyotyping (SKY).

Cytogenetic chromosome analysis by classical G-banding was supplemented by spectral karyotyping (SKY) in 12 cases of diffuse large B-cell lymphoma (DLBL). SKY is a fluorescence in-situ-based, genome-wide screening technique allowing identification of genetic material even in highly condensed metaphase chromosomes of poor morphology. By simultaneous hybridization of whole chromosome painting probes onto tumor chromosome spreads genetic rearrangements are visualized permitting the clarification of even complex karyotype alterations and the identification of genetic material of previously unknown origin, so-called marker chromosomes. Taking the SKY results into account, we reevaluated the G-banding karyotypes initially carried out, thus generating a more precise karyotype in ten of twelve (83%) cases investigated. In particular, thirteen chromosomal rearrangements not correctly recognized by classical cytogenetics were identified, the genetic origin of seven marker chromosomes was elucidated and three structural genetic rearrangements were redefined. We found SKY to be a valuable technique to establish a definite karyotype in addition to classical cytogenetics.

Cytogenetics of the bleak (Alburnus alburnus), with special emphasis on the B chromosomes.

Some of the largest B chromosomes so far discovered in vertebrates are present in the cyprinid fish Alburnus alburnus. Previous cytogenetic analyses revealed a diploid chromosome number of 2n = 50. In addition, in some individuals one or two unusually large B chromosomes are present. Two morphologically different types of B chromosomes were observed. The frequency of animals bearing a supernumerary chromosome was found to vary considerably between different populations. A more detailed analysis of the A and B chromosomes of A. alburnus by conventional banding techniques, as well as fluorescence in-situ hybridization (FISH) with the telomeric DNA repeats (GGGTTA)7/(TAACCC)7, 18S + 28S rDNA and 5S rDNA were performed in the present study. Furthermore, a B chromosome-specific DNA probe obtained by amplified length polymorphism (AFLP) was hybridized on metaphases of A. alburnus carrying supernumerary B chromosomes. The banding analyses showed that the B chromosomes are completely heterochromatic, consist of GC-rich DNA sequences, replicate their DNA in the very late S-phase of the cell cycle and are composed mainly of a specific retrotransposable DNA element. Finally, blood probes from A. alburnus were collected for DNA-flow cytometric measurements. It could be shown that the huge supernumerary chromosomes represent nearly 10% of the total genome size of A. alburnus.

Mosaicism for an ectopic NOR at 8pter and a complex rearrangement of chromosome 8 in a patient with severe psychomotor retardation.

We describe a 3-year-old girl with severe delays in mental and motor skills, a history of generalized seizures, and subtle dysmorphic features. Conventional cytogenetics revealed a mosaic karyotype. A de novo ectopic NOR at the telomeric region of the short arm of one chromosome 8 (8ps) was found in 90% of lymphocyte and in 98% of fibroblast metaphases. A small NOR-bearing marker chromosome and a large derivative chromosome 8 without short arm satellites (der(8)) were present in the remaining cells. FISH with a probe specific for centromeres 14 and 22 labeled both the telomeric region of 8ps and the small marker centromere. Der(8) included an inverted duplication of 8p and a rearranged duplication of 8q but lacked a second centromere. A subtelomeric probe for 8p revealed a cryptic deletion in 8ps and der(8). Thus, the karyotype represents a combination of submicroscopic partial monosomy 8pter and mosaic trisomy 8.

Chromosome banding in Amphibia. XXX. Karyotype aberrations in cultured fibroblast cells.

The present study reports for the first time on the numerical and structural chromosome anomalies that spontaneously arise in aging cultured fibroblast cells of Amphibia. The analyses were conducted on kidney fibroblasts of three anuran species with extremely divergent genome sizes (Bufo rubropunctatus, Scaphiopus holbrooki, Gastrotheca riobambae), in the sixth up to the 14th culture passage. The chromosomal rearrangements were identified by means of the 5-bromodeoxyuridine/deoxythymidine (BrdU/dT) replication banding technique. The aberrations can be either confined to a single chromosome, or else involve all chromosomes of the karyotype. The most frequent structural aberrations in the cell cultures of S. holbrooki and G. riobambae are tandem fusions between two or more chromosomes. These tandem fusions originating in vitro in long-termed cell cultures reflect the chromosome mutations which also took place during amphibian phylogenesis.

Chromosome banding in Amphibia. XXVII. DNA replication banding patterns in three anuran species with greatly differing genome sizes.

The mitotic chromosomes of three anuran species, Scaphiopus holbrooki, Litoria infrafrenata and Odontophrynus americanus, were analyzed by means of the 5-bromodeoxyuridine/deoxythymidine (BrdU/dT) replication banding technique. These species exhibit large differences in their genome sizes: S. holbrooki possesses one of the smallest genomes among vertebrates, L. infrafrenata has a genome size near the modal DNA value of most Amphibia, whereas O. americanus is a tetraploid species. BrdU/dT labeling induces reproducible and reliable R- and G-replication bands along the metaphase chromosomes of all three species. Irrespective of the genome size of the species considered, the number of early (R-) and late (G-) replicating bands per haploid karyotype is nearly the same. The chromosomes of the autotetraploid O. americanus can be arranged into sets of four homologous chromosomes (quartets). C-bands and BrdU/dT replication bands reveal heterogeneity within the quartets 1, 3 and 4 that are interpreted as the initiation of a diploidization process.

Chromosome banding in Amphibia. XXVIII. Homomorphic XY sex chromosomes and a derived Y-autosome translocation in Eleutherodactylus riveroi (Anura, Leptodactylidae).

Extensive cytogenetic analyses on a population of the leptodactylid frog Eleutherodactylus riveroi in northern Venezuela revealed the existence of multiple XXAA male/XYAA female/XAA(Y) female sex chromosomes. The XAA(Y) karyotype originated by a centric (Robertsonian) fusion between the original, free Y chromosome and an autosome. 46.2% of the male individuals in this population are carriers of this Y-autosome fusion. In male meiosis the XAA(Y) sex chromosomes pair in the expected trivalent configuration. In the same population 53.8% of the male animals still possess the original, free XY sex chromosomes. E. riveroi is only the second vertebrate species discovered in which a derived Y-autosome fusion coexists with the ancestral free XY sex chromosomes. The free XY sex chromosomes, as well as the multiple XA(Y) sex chromosomes are still in a very primitive (homomorphic) stage of differentiation. With no banding technique applied it is possible to distinguish the Y from the X. Various banding techniques and in situ hybridizations have been carried out to characterize the karyotypes. DNA flow cytometric measurements show that the genome size of E. riveroi resembles that of other Eleutherodactylus species. The cytogenetic data obtained in E. riveroi are compared with those of the sole other vertebrate known to possess the extremely rare, multiple XXAA male/XYAA female/XAA(Y) female sex chromosomes. Surprisingly enough, this vertebrate again is a frog belonging to the genus Eleutherodactylus [E. ((maussi) biporcatus] which lives exactly in the same habitat in northern Venezuela as does E. riveroi.

Chromosome banding in Amphibia. XXIX. The primitive XY/XX sex chromosomes of Hyla femoralis (Anura, Hylidae).

The karyotype of the pine woods treefrog, Hyla femoralis, is characterized by primitive XY female/XX male sex chromosomes. The sole difference between the X and the Y is the presence of a nucleolus organizer region (NOR) in the X. Due to a deletion of the NOR in the Y, this chromosome is distinctly smaller than the X. Since no autosomal NORs exist in the karyotype of this species, the NOR deletion in the Y results in a sex-specific difference in the number of ribosomal RNA genes, with a female:male ratio of about 2:1. Interphase nuclei of male animals contain always one silver-stained nucleolus, whereas most nuclei of female specimens exhibit two nucleoli. This is in agreement with the absence of dosage compensation for sex-linked genes in amphibian cells. The consequences of the loss of about 50% of ribosomal RNA genes for the viability of male individuals and spermatogenesis are discussed.

The giant B chromosome of the cyprinid fish Alburnus alburnus harbours a retrotransposon-derived repetitive DNA sequence.

The cyprinid fish Alburnus alburnus possesses one of the largest supernumerary chromosomes in all vertebrates. In the present study, amplified fragment length polymorphism analyses (AFLP) and fluorescence in-situ hybridization (FISH) were performed in order to characterize these extraordinary chromosomes in detail. Sequence analysis of the B chromosome-specific DNA revealed a strong homology to a Drosophila Gypsy/Ty3 retrotransposon and also to a medaka (Oryzias latipes) one. The sequence is highly abundant on the B chromosome but undetectable in the normal A chromosome complement. It is also absent from the B chromosome of the closely related species, Rutilus rutilus, suggesting a specific spreading of the mobile element during evolution of the giant supernumerary chromosome within A. alburnus. Meitotic chromosomes were in-situ hybridized with the B chromosome-specific probe, documenting that the additional chromosome behaves as an autopaired ring chromosome in diakineses. Our results suggest that the supernumerary chromosome of A. alburnus is not derived from the normal chromosome complement but has evolved independently.

Multicolor spectral karyotyping of rat chromosomes.

Rat and mouse have become important animal models to study various human diseases such as cancer. Cytogenetic analysis of the respective karyotypes is frequently required to investigate the causative genetic defects and especially neoplastic cells often show complex chromosome aberrations and many different marker chromosomes. However, structural homogeneity of the chromosomes in these species as well as less pronounced differences in banding patterns make it difficult to assign genetic abnormalities to certain chromosomes by conventional banding techniques. Here we report for the first time the successful application of multicolor spectral karyotyping (SKY) to rat chromosomes, which allows unequivocal identification of all rat chromosomes with the exception of chromosomes 13 and 14 in different colors, thus enabling the elucidation of even complex rearrangements in the rat karyotype. Flow-sorted chromosome specific painting probes for all 22 rat chromosomes (20 autosomes, X, and Y) were combinatorially labeled by a set of five different fluorochromes and hybridized in situ to metaphase spreads of a healthy rat, to diakineses from testicular material, and to cells from a rat FAO hepatoma cell line. Measuring the complete spectrum at each image point by using the SpectraCube((R)) spectral imaging system and respective computer software allowed identification of the individual rat chromosomes by their specific emission spectra. Classification algorithms in the analysis software can then display the rat chromosomes in specific pseudo-colors and automatically order them in a karyotype table. After its successful application to human and mouse chromosomes, spectral karyotyping of rat chromosomes now also allows cytogenetic screening of the complete rat genome by a single hybridization.

Chromosome banding in Amphibia. XXIV. The B chromosomes of Gastrotheca espeletia (Anura, Hylidae).

The mitotic chromosomes of an Ecuadorian population of the marsupial frog Gastrotheca espeletia were analyzed by means of banding techniques and fluorescence in situ hybridization. This species is characterized by unusual supernumerary (B) chromosomes. The maximum number of B chromosomes is 9 and they occur in three different morphological types. Banding analyses show that the B chromosomes are completely heterochromatic, consist of AT base pair-rich repeated DNA sequences, replicate their DNA in very late S-phase of the cell cycle, and are probably derived from a centromeric or paracentromeric region of a standard (A) chromosome. Exceptionally, the B chromosomes carry 18S + 28S ribosomal RNA genes and the conserved vertebrate telomeric DNA sequence appears to be underrepresented. Flow cytometric measurements of the nuclear DNA content differentiate between individuals with different numbers of B chromosomes. Significantly more B chromosomes are present in female than in male animals.

Chromosome banding in Amphibia. XXV. Karyotype evolution and heterochromatin characterization in Australian Mixophyes (Anura, Myobatrachidae).

The mitotic chromosomes of the Australian ground frogs Mixophyes fasciolatus and M. schevilli were analyzed by means of banding techniques and restriction endonuclease digestions. Chromosomal differentiation in these two species occurred exclusively by considerable changes in the amount of telomeric and centromeric heterochromatin, whereas the sizes and locations of interstitial heterochromatic regions, the sizes of all euchromatic segments as well as the positions of centromeres remained nearly identical during karyotype evolution. The major heterochromatic regions in the karyotypes of M. fasciolatus and M. schevilli amount to 30.2% and 20.7%, respectively. They consist of AT base pair-rich repetitive DNA sequences that are brightly labeled by AT-specific fluorochromes and display quenched fluorescence after staining with GC-specific fluorochromes. The heterochromatic regions can be differentiated by treatment of metaphase chromosomes and interphase cell nuclei with various restriction enzymes which either disclose the complete set of C-band patterns in the karyotypes of both species, or else reveal several subsets of these C-bands.

Chromosome banding in amphibia. XXIII. Giant W sex chromosomes and extremely small genomes in Eleutherodactylus euphronides and Eleutherodactylus shrevei (Anura, Leptodactylidae).

Highly differentiated, heteromorphic ZZ female symbol /ZW male symbol sex chromosomes were found in the karyotypes of the neotropical leptodactylid frogs Eleutherodactylus euphronides and E. shrevei. The W chromosomes are the largest heterochromatic, female-specific chromosomes so far discovered in the class Amphibia. The analyses of the banding patterns with AT- and GC base-pair specific fluorochromes show that the constitutive heterochromatin in the giant W chromosomes consists of various categories of repetitive DNA sequences. The W chromosomes of both species are similar in size, morphology and banding patterns, whereas their Z chromosomes exhibit conspicuous differences. In the cell nuclei of female animals, the W chromosomes form very prominent chromatin bodies (W chromatin). DNA flow cytometric measurements demonstrate clear differences in the DNA content of male and female erythrocytes caused by the giant W chromosome, and also shows that these Eleutherodactylus genomes are among the smallest of all amphibian genomes. The importance of the heteromorphic ZW sex chromosomes for the study of Z-linked genes, the similarities and differences of the two karyotypes, and the significance of the exceptionally small genomes are discussed.

Chromosome banding in Amphibia. XXII. Atypical y chromosomes in Gastrotheca walkeri and Gastrotheca ovifera (Anura, Hylidae).

The chromosomes of the rare South American marsupial frogs Gastrotheca walkeri and G. ovifera were extensively reexamined with various banding techniques. The karyotypes of both species are distinguished by a new category of XY female symbol /XX male symbol female sex chromosomes. The unusual Y chromosomes are characterized by containing the least amount of constitutive heterochromatin in the karyotypes. This is in contrast to all previously known amphibian Y chromosomes and does not fit the evolutionary model of early XY differentiation in vertebrates. In male meiosis, the heteromorphic XY chromosomes of both species still exhibit the same pairing configurations as the autosomes. DNA flow cytometric measurements show the nuclear DNA amount of G. walkeri to be 10.90 pg. The significance of the XY/XX sex chromosomes of these marsupial frogs, the various classes of constitutive heterochromatin detected, and the data obtained from meiotic analyses are discussed in detail.

Spectral karyotype analysis of colon cancer cell lines of the tumor suppressor and mutator pathway.

BACKGROUND AND AIMS: Microsatellite instability (MSI) is characterized by the size variation of microsatellites in tumor DNA as compared to matching normal DNA due to defects in the mismatch repair system. To examine the chromosomal differences in microsatellite-stable (MSS) and -unstable (MSI) tumors in detail, we analyzed MSS (Caco-2, Colo-205, SW948) and MSI (HCT-15, HCT-116, LoVo) cell lines by spectral karyotyping (SKY). METHODS: SKY is a sensitive method to detect chromosome aberrations by visualizing each chromosome in a different color. Metaphases were hybridized with a SKY probe mixture. Images were visualized with the SpectraCube system and analyzed with the SKYview imaging software. RESULTS: The average number of chromosomes was 49 in LoVo, 45 in HCT-116, 46 in HCT-15, 71 in Colo-205, 89 in Caco-2 and 66 in SW-948. Three aberrant chromosomes were detected in LoVo, three in HCT-116, two in HCT-15, seventeen in Colo-205, fourteen in Caco-2 and nine in SW948. CONCLUSION: The karyotypes of MSS colon cancer cells displayed complex numerical and structural aberrations. In contrast the chromosomes of MSI colon cancer cells were mostly unaltered but displayed a few isolated numerical and structural aberrations. We speculate that these isolated aberrations may be specifically involved in the pathogenesis of MSI tumors.