Establishment and characterization of a novel human endometrial mesenchymal stem cell line from a patient with adenomyosis

Adenomyosis, previously termed “endometriosis interna,” is a widespread disease affecting the female reproductive system and frequently resulting in infertility in women. The aim of this work was to examine the properties of endometrial mesenchymal stem cells (eMSCs) from a patient with adenomyosis. We established the cell line from a patient with adenomyosis and compared the properties of these cells with cells derived from a healthy donor. It was found that patient-derived eMSCs and eMSCs from healthy donors had a fibroblast-like morphology and did not differ in expression of surface markers and adipogenic potential. Karyotype analysis of G-banded metaphase chromosomes was performed in cells of both lines at the six or seventh passages. Cells from the healthy donor mostly had normal karyotype. Karyotype of eMSCs from the patient with adenomyosis usually had chromosomal abnormalities. The abnormalities concerned aneuploidy and nonrandom chromosomes breaks more often involving chromosomes 7 and 11. Although karyotype instability may be a sign of cell transformation, the patient-derived eMSCs stopped cycling after about 26 passages and entered into replicative senescence. This shows that the karyotypic abnormalities that we observed in adenomyosis-derived eMSCs are not relevant to the cell transformation and immortalization in vitro.     

Mesenchymal stem cells from human endometrium do not undergo spontaneous transformation during long-term cultivation

Human-endometrium mesenchymal stem cells (eMSCs) are a promising source of stem cells for regenerative medicine. A large amount of these cells accumulated by in vitro cultivation are usually required for transplantation into patients. We established several cell eMSC lines and cultivated them over a long period to examine the possibility of spontaneous transformation. All cell lines exhibit limited lifespan, undergo replicative senescence, and die. Karyotypic analysis upon different passages reveals that most cells display karyotypic stability. Thus, extended in vitro cultivation of eMSCs does not lead to spontaneous transformation, which makes therapeutic application of these cells safe for patients. During long-term cultivation, eMSCs maintain the expression of surface markers.     

Chromosomal aberrations after induced pluripotent stem cells reprogramming

Induced pluripotent stem cells (iPSCs) are generated from adult cells that have been reprogrammed to pluripotency. However, in vitro cultivation and genetic reprogramming increase genetic instability, which could result in chromosomal abnormalities. Maintenance of genetic stability after reprogramming is required for possible experimental and clinical applications. The aim of this study was to analyze chromosomal alterations by using the G-banding karyotyping method applied to 97 samples from 38 iPSC cell lines generated from peripheral blood or Wharton’s jelly. Samples from patients with long QT syndrome, Jervell and Lange-Nielsen syndrome and amyotrophic lateral sclerosis and from normal individuals revealed the following chromosomal alterations: acentric fragments, chromosomal fusions, premature centromere divisions, double minutes, radial figures, ring chromosomes, polyploidies, inversions and trisomies. An analysis of two samples generated from Wharton’s jelly before and after reprogramming showed that abnormal clones can emerge or be selected and generate an altered lineage. IPSC lines may show clonal and nonclonal chromosomal aberrations in several passages (from P6 to P34), but these aberrations are more common in later passages. Many important chromosomal aberrations were detected, showing that G-banding is very useful for evaluating genetic instability with important repercussions for the application of iPSC lines.     

Characterization of supernumerary rings and giant marker chromosomes in well-differentiated lipomatous tumors by a combination of G-banding,CGH, M-FISH,and chromosome- and locus-specific FISH

Supernumerary ring chromosomes and/or giant marker chromosomes are often seen in soft-tissue tumors of low-grade or borderline malignancy, such as well-differentiated liposarcomas or atypical lipomas. Classic cytogenetic banding techniques have proved insufficient to identify the genomic composition and structure of such rings and markers, but fluorescent in situ hybridization (FISH) studies have shown that they consist mainly of amplified material from chromosome 12, more specifically from bands 12q13-->q15. We have used the new FISH-based screening techniques comparative genomic hybridization (CGH) and multicolor-FISH (M-FISH) in combination with G-banding and analysis by chromosome- and locus-specific fluorescent in situ probes to examine in detail the karyotypic characteristics of 22 lipomatous tumors, most of them classified histologically as well-differentiated liposarcomas, selected because they had been shown to harbor rings and/or marker chromosomes. M-FISH, in contrast to G- banding, was found to be informative with regard to the chromosomal origin of the rings and other markers present, whereas CGH and hybridizations with locus-specific probes helped identify which subchromosomal regions were involved. We found that chromosome bands 12q15-->q21 were always gained, with 12q15-->q21 being amplified (i.e., a green-to-red ratio >2 by CGH) in 14 of 22 tumors. In three tumors, two distinct but close amplicons in 12q could be identified, corresponding to bands 12q13-->q15 and 12q21. The genomic segment 1q21-->q23 was gained in 12 cases, reaching the level of amplification in seven. Bands 6q24 and 7p15, whose pathogenetic involvement in liposarcomas has not been reported previously, were gained in three cases each. In addition, the rings and giant markers often contained interspersed sequences from several other chromosomes that did not give an equally clear impression of being nonrandomly involved.     

High-resolution analysis of genetic stability of human adipose tissue stem cells cultured to senescence

The potential use of human mesenchymal stem cells for therapeutic applications implies large scale in vitro culture, increasing the probability of genetic instability and transformation. We examine here the incidence of unbalanced and balanced chromosome rearrangements in polyclonal and single cell-derived cultures of human adipose stem cells to senescence. G-banding karyotyping of the polyclonal cultures shows a normal karyotype. In addition, high-resolution microarray-based comparative genomic hybridization analyses relative to uncultured adipose stem cells from the same donors reveal overall genomic stability in long-term (approximately 6 months) polyclonal and clonal culture. One adipose stem cell clone displayed minor deletions in gene-rich telomeric and sub-telomeric regions on three chromosomes in early passage. This however, was detected only in a sub-population of cells that was subsequently spontaneously eliminated from the culture. Apparent pericentromeric instabilities are also occasionally detected in specific chromosomes. Our results indicate that clonal chromosomal aberrations may arise transiently in early passage adipose stem cells (ASC) cultures. Nonetheless, incidence of these aberrations seems to be negligible in the majority of long-term ASC cultures, at least under the culture conditions used here.     

Multipotent mesenchymal stem cells of desquamated endometrium: Isolation,characterization, and application as a feeder layer for maintenance of human embryonic stem cells

In this study, we characterize new multipotent human mesenchymal stem cell lines (MSCs) derived from desquamated (shedding) endometrium of menstrual blood. The isolated endometrial MSC (eMSC) is an adhesive to plastic heterogeneous population composed mainly of endometrial glandular and stromal cells. The established cell lines meet the criteria of the International Society for Cellular Therapy for defining multipotent human MSCs of any origin. The eMSCs have positive expression of CD13, CD29, CD44, CD73, CD90, and CD105 markers and lack hematopoietic cell surface antigens CD19, CD34, CD45, CD117, CD130, and HLA-DR (class II). Multipotency of the established eMSCs is confirmed by their ability to differentiate into other mesodermal lineages, such as osteocytes and adipocytes. In addition, the isolated eMSCs partially (over 50%) express the pluripotency marker SSEA-4. However, they do not express Oct-4. Immunofluorescent analysis of the derived cells revealed the expression of the neural precursor markers nestin and β-III-tubulin. This suggests a neural predisposition of the established eMSCs. These cells are characterized by a high proliferation rate (doubling time 22–23 h) and a high colony-forming efficiency (about 60%). In vitro, the eMSCs undergo more than 45 population doublings without karyotypic abnormalities. We demonstrate that mitotically inactivated eMSCs are perfect feeder cells for maintenance of human embryonic stem cell lines (hESCs) C612 and C910. The eMSCs, being a feeder culture, sustain the hESC pluripotent status that verified by expression of Oct-4, alkaline phosphatase and SSEA-4 markers. The hESCs cocultured with the eMSCs retain their morphology and proliferative rate for more than 40 passages and exhibit the capability for spontaneous differentiation into embryoid bodies comprising three embryonic germ layers. Thus, an easy and noninvasive isolation of the eMSCs from menstrual blood, their multipotency and high proliferative activity in vitro without karyotypic abnormalities demonstrate the potential of use of these stem cells in regenerative medicine. Using the derived eMSCs as the feeder culture eliminates the risks associated with animal cells while transferring hESCs to clinical setting.     

Molecular cytogenetic characterization of pancreas cancer cell lines reveals high complexity chromosomal alterations

Karyotype analysis can provide clues to significant genes involved in the genesis and growth of pancreas cancer. The genome of pancreas cancer is complex, and G-band analysis cannot resolve many of the karyotypic abnormalities seen. We studied the karyotypes of 15 recently established cell lines using molecular cytogenetic tools. Comparative genomic hybridization (CGH) analysis of all 15 lines identified genomic gains of 3q, 8q, 11q, 17q, and chromosome 20 in nine or more cell lines. CGH confirmed frequent loss of chromosome 18, 17p, 6q, and 8p. 14/15 cell lines demonstrated loss of chromosome 18q, either by loss of a copy of chromosome 18 (n = 5), all of 18q (n = 7) or portions of 18q (n = 2). Multicolor FISH (Spectral Karyotyping, or SKY) of 11 lines identified many complex structural chromosomal aberrations. 93 structurally abnormal chromosomes were evaluated, for which SKY added new information to 67. Several potentially site-specific recurrent rearrangements were observed. Chromosome region 18q11.2 was recurrently involved in nine cell lines, including formation of derivative chromosomes 18 from a t(18;22) (three cell lines), t(17;18) (two cell lines), and t(12;18), t(15;18), t(18;20), and ins(6;18) (one cell line each). To further define the breakpoints involved on chromosome 18, YACs from the 18q11.2 region, spanning approximately 8 Mb, were used to perform targeted FISH analyses of these lines. We found significant heterogeneity in the breakpoints despite their G-band similarity, including multiple independent regions of loss proximal to the already identified loss of DPC4 at 18q21.     

Molecular cytogenetic analysis of follicular lymphoma (FL) provides detailed characterization of chromosomal instability associated with the t(14;18)(q32;q21) positive and negative subsets and histologic progression

We analyzed a cohort of 61 follicular lymphomas (FL) with an abnormal G-banded karyotype by spectral karyotyping (SKY) to better define the chromosome instability associated with the t(14;18)(q32;q21) positive and negative subsets of FL and histologic grade. In more than 70% of the patients, SKY provided additional cytogenetic information and up to 40% of the structural abnormalities were revised. The six most frequent breakpoints in both SKY and G-banding analyses were 14q32, 18q21, 3q27, 1q11-q21, 6q11-q15 and 1p36 (15-77%). SKY detected nine additional sites (1p11-p13, 2p11-p13, 6q21, 8q24, 6q21, 9p13, 10q22-q24, 12q11-q13 and 17q11-q21) at an incidence of >10%. In addition to the known recurring translocations, t(14;18)(q32;q21) [70%], t(3;14)(q27;q32) [10%], t(1;14)(q21;q32) [5%] and t(8;14)(q24;q32) [2%] and their variants, 125 non-IG gene translocations were identified of which four were recurrent within this series. In contrast to G-banding analysis, SKY revealed a greater degree of karyotypic instability in the t(14;18) (q32;q21) negative subset compared to the t(14;18)(q32;q21) positive subset. Translocations of 3q27 and gains of chromosome 1 were significantly more frequent in the former subset. SKY also allowed a better definition of chromosomal imbalances, thus 37% of the deletions detected by G-banding were shown to be unbalanced translocations leading to gain of genetic material. The majority of recurring (>10%) imbalances were detected at a greater (2-3 fold) incidence by SKY and several regions were narrowed down, notably at gain 2p13-p21, 2q11-q21, 2q31-q37, 12q12-q15, 17q21-q25 and 18q21. Chromosomal abnormalities among the different histologic grades were consistent with an evolution from low to high grade disease and breaks at 6q11-q15 and 8q24 and gain of 7/7q and 8/8q associated significantly with histologic progression. This study also indicates that in addition to gains and losses, non-IG gene translocations involving 1p11-p13, 1p36, 1q11-q21, 8q24, 9p13, and 17q11-q21 play an important role in the histologic progression of FL with t(14;18)(q32;q21) and t(3q27).     

A Teratocarcinoma-Like Human Embryonic Stem Cell (hESC) Line and Four hESC Lines Reveal Potentially Oncogenic Genomic Changes

The first Swiss human embryonic stem cell (hESC) line, CH-ES1, has shown features of a malignant cell line. It originated from the only single blastomere that survived cryopreservation of an embryo, and it more closely resembles teratocarcinoma lines than other hESC lines with respect to its abnormal karyotype and its formation of invasive tumors when injected into SCID mice. The aim of this study was to characterize the molecular basis of the oncogenicity of CH-ES1 cells, we looked for abnormal chromosomal copy number (by array Comparative Genomic Hybridization, aCGH) and single nucleotide polymorphisms (SNPs). To see how unique these changes were, we compared these results to data collected from the 2102Ep teratocarcinoma line and four hESC lines (H1, HS293, HS401 and SIVF-02) which displayed normal G-banding result. We identified genomic gains and losses in CH-ES1, including gains in areas containing several oncogenes. These features are similar to those observed in teratocarcinomas, and this explains the high malignancy. The CH-ES1 line was trisomic for chromosomes 1, 9, 12, 17, 19, 20 and X. Also the karyotypically (based on G-banding) normal hESC lines were also found to have several genomic changes that involved genes with known roles in cancer. The largest changes were found in the H1 line at passage number 56, when large 5 Mb duplications in chromosomes 1q32.2 and 22q12.2 were detected, but the losses and gains were seen already at passage 22. These changes found in the other lines highlight the importance of assessing the acquisition of genetic changes by hESCs before their use in regenerative medicine applications. They also point to the possibility that the acquisition of genetic changes by ESCs in culture may be used to explore certain aspects of the mechanisms regulating oncogenesis.     

Chromosomal rearrangements and their effects in spontaneous immortalization and transformation of rat embryo cells in vitro

G-banding analysis of LRec-1 and LRec-3, spontaneously immortalized cell lines from rat embryo fibroblast, revealed diploid karyotypes with specific clonal structural rearrangements of chromosomes 7 and 19 - del(7)(q11.2q22.1), t(7;19)(q11.1;q12) in malignant stage. Both clonal abnormalities of chromosomes 7 and 19 were also revealed in LRec-1k clone and LRec-1 sf cell line. Previous study of LRec-1 and LRec-3 cells showed the presence of karyotypes with pseudodiploid modal chromosome number, partial trisomy of chromosome 7 and same clonal structural rearrangements of chromosomes 7 and 19 in immortalized stage. In malignant stage, the trisomy 6 and new clonal structural rearrangements of chromosomes 1, 2, 11, 15, 18, 19 and of chromosomes 10, 20 were also found in LRec-1 sf and LRec-1 cells, accordingly. There were no new clonal structural chromosome rearrangements in LRec-1 k and LRec-3 cells. We compared locies of chromosomes involved in rearrangements with mapped genes on these chromosomes according to RATMAP. Supposedly these genes are involved in spontaneous immortalization of rat embryo fibroblast and malignant transformation of LRec-1 and LRec-3 cells and rearrangements of chromosomes 1, 2, 11, 15 and 18 facilitate expression of growth factors of LRec-1 sf cells.     

Microfluorimetric analysis of dynamics of genomic changes of Chinese hamster fibroblasts CHL V-79 RJK with multidrug resistance

Results of karyological analysis of cells CHL V-79 RJK selected for resistance to ethidium bromide (EB) causing multidrug resistance (MDR) (line Vebr-5) were compared with the data of microfluorimetric determination of DNA content in individual chromosomes of the karyotype. The analysis was performed at the 11th and 88th passages. Karyotyping of Vebr-5 has shown the presence of an additional genetic material (ADM) in the form of homogenously or differentially stained regions (HSRs and DSRs, respectively) in two chromosomes (Z1 and Z6, loci 1 p29-31 and 1q26, respectively). HSRs in Z6, in the region of localization of the wild type of gene mdr, had unstable length and structure characteristic of morphological markers of amplification of genes of the family mdr. During long cultivation of Vebr-5 in the presence of EB (88 passages), the instability of HSRs in Z6 increased. Results of microfluorimetric analysis of Vebr-5 at the 11th passage have shown an increase in the DNA content not only in chromosomes Z1 and Z6 marked by HSRs, but also in three chromosomes (Z5, Z12 and Z13) that have no visual morphological changes. The corresponding analysis at the 88th passage has also revealed non-random changes in the DNA content in four more chromosomes: an increase in Z14, while a decrease in chromosomes 8, Z7, and Z9. A decrease of the DNA content in chromosomes is considered to be a result of a partial loss of genetic material, while its increase is a result of its translocation and (or) amplification. Coefficient of variation of the DNA content changes for large chromosomes amounted to about 9%. while for small chromosomes it is about 26%, which indicates that small chromosomes have greater potential for instability than the large ones. The data obtained not only confirm, but also enlarge the concept of directions and character of destabilization of the cell genetic apparatus in the process of neoplastic transformation due to the MDR acquisition by cells.     

Microfluorimetric analysis of dynamics of genomic changes in Chinese hamster fibroblasts CHL V-79 RJK with multiple drug resistance

Results of a karyological analysis of cells CHL V-79 RJK selected for their resistance to ethidium bromide (EB) causing multidrug resistance (MDR) (subline Vebr-5) were compared with data from the microfluorimetric determination of the DNA content in individual chromosomes of the karyotype. The analysis was performed at the 11th and 88th passages. Karyotyping of Vebr-5 has shown the presence of an additional genetic material (AGM) in the form of homogenously or differentially stained regions (HSR or DSR, respectively) in two chromosomes (Z1 and Z6, loci 1p29-31 and 1q26, respectively). HSR in Z6, in the site of localization of the mdr gene of the wild type had unstable length and structure, which is characteristic of the morphological markers of the amplification of genes of the mdr family. During the long cultivation of Vebr-5 in the presence of EB (88 passages), the instability of HSR (DSR) in Z6 increased. Results of a microfluorimetric analysis of Vebr-5 at the 11th passage have shown a statistically significant increase of the DNA content not only in chromosomes Z1 and Z6 marked by HSR (DSR), but also in three chromosomes (Z5, Z12, and Z13) that have no visual morphological changes. The corresponding analysis at the 88th passage has also revealed nonrandom changes in the DNA content in four more chromosomes, including an increase in Z14 and a decrease in chromosomes 8, Z7, and Z9. A decrease in the DNA content in chromosomes is considered to be the result of a partial loss of genetic material, while its increase is considered to be the result of its translocation and/or amplification. While the coefficient of the variation of the DNA content changes about 9% for large chromosomes, it amounted to about 26% for the small ones, indicating that small chromosomes to have a greater potential for instability than large chromosomes. The obtained data not only confirm, but also enlarge, the concept of the directions and character of the destabilization of the cell genetic apparatus in the process of neoplastic transformation due to MDR acquisition by these cells.     

Detection of DNA copy number changes in human endometriosis by comparative genomic hybridization

Endometriosis is characterized by infertility and pelvic pain in 10-15% of women of reproductive age. The genetic events involved in endometriotic cell expansion remain in large part unknown. To identify genomic changes involved in development of this disease, we examined a panel of 18 selected endometriotic tissues by comparative genomic hybridization (CGH), a molecular cytogenetic method that allows screening of the entire genome for chromosomal gains and/or losses. The study was performed on native, nonamplified DNA extracted from manually dissected endometriotic lesions. Recurrent copy number losses on several chromosomes were detected in 15 of 18 cases. Loss of chromosome 1p and 22q were detected in 50% of the cases. Additional common losses occurred on chromosomes 5p (33%), 6q (27%), 7p(22%), 9q (22%), 16 (22%) as well as on 17q in one case. Gain of DNA sequences were seen at 6q, 7q and 17q in three cases. To validate the CGH data, selective dual-color FISH was performed using probes for the deleted regions on chromosomes 1, 7 and 22 in parallel with the corresponding centromeric probes. Cases showing deletion by CGH all had two signals at 1p36, 7p22.1 and 22q12 in less than 30% of the nuclei in comparison to the double centromeric labels found in more than 85% of the cells. These findings indicate that genes localized to previously undescribed chromosomal regions play a role in development and progression of endometriosis.     

Genomic imbalances in 61 renal cancers from the proximal tubulus detected by comparative genomic hybridization

Comparative genomic hybridization (CGH) has been applied to characterize 61 primary renal cell carcinomas derived histogenetically from the proximal tubulus. The tumor samples comprised 46 clear-cell renal cell carcinomas (ccRCCs) and 15 papillary renal cell carcinomas (pRCCs). Changes in the copy number of entire chromosomes or subregions were detected in 56 tumors (92%). In ccRCCs, losses of chromosome 3 or 3p (63%); 14q (30%); 9 (26%); 1 and 6 or 6q (17% each); 4 and 8 or 8p (15% each); 22 (11%); 2 or 2q and 19 (9% each); 7q, 10, 16, 17p, 18, and Y (7% each); and 5, 11, 13, 15, and 21 (4% each) were detected. Most frequent genomic gains in ccRCC were found on chromosome 5 (63%); 7 (35%); 1 or 1q (33%); 2q (24%); 8 or 8q, 12, and 20 (20% each); 3q (17%); 16 (15%); 19 (13%); 6 and 17 or 17q (11% each); and 4, 10, 11, 21, and Y (9% each). In pRCCs, gains in the copy number of chromosomes 7 and 17 (7/15, each) and 16 and 20 (6/15, each) were frequent. One pRCC showed amplification of subchromosome regions 2q22-->q33, 16q, 17q and the entire X chromosome. In pRCC, losses were less frequently seen than gains. Losses of chromosomes 1, 14, 15, and Y (3/15 each) and 2, 4, 6, and 13 (2/15 each) were observed. In ccRCCs, statistical evaluation revealed significant correlations of chromosomal imbalances with tumor stage and grade, i.e., a gain in copy number of chromosome 5 correlated positively with low tumor grade, whereas a gain of chromosomes 10 and 17 correlated positively with high tumor grade. Furthermore, loss of chromosome 4 correlated positively with high tumor stage.     

Homologous alpha satellite sequences on human acrocentric chromosomes with selectivity for chromosomes 13, 14 and 21: implications for recombination between nonhomologues and Robertsonian translocations.

We report a new subfamily of alpha satellite DNA (pTRA-2) which is found on all the human acrocentric chromosomes. The alphoid nature of the cloned DNA was established by partial sequencing. Southern analysis of restriction enzyme-digested DNA fragments from mouse/human hybrid cells containing only human chromosome 21 showed that the predominant higher-order repeating unit for pTRA-2 is a 3.9 kb structure. Analysis of a "consensus" in situ hybridisation profile derived from 13 normal individuals revealed the localisation of 73% of all centromeric autoradiographic grains over the five acrocentric chromosomes, with the following distribution: 20.4%, 21.5%, 17.1%, 7.3% and 6.5% on chromosomes 13, 14, 21, 15 and 22 respectively. An average of 1.4% of grains was found on the centromere of each of the remaining 19 nonacrocentric chromosomes. These results indicate the presence of a common subfamily of alpha satellite DNA on the five acrocentric chromosomes and suggest an evolutionary process consistent with recombination exchange of sequences between the nonhomologues. The results further suggests that such exchanges are more selective for chromosomes 13, 14 and 21 than for chromosomes 15 and 22. The possible role of centromeric alpha satellite DNA in the aetiology of 13q14q and 14q21q Robertsonian translocations involving the common and nonrandom association of chromosomes 13 and 14, and 14 and 21 is discussed.     

Molecular systematics of Lachancea yeasts

This work presents the results of molecular-genetic investigation of a new yeast genus, Lachancea Kurtzman (2003). Analysis of rRNA sequences and molecular karyotyping have shown genetic homogeneity of the genus Lachancea. Yeasts of this genus have an identical haploid number of chromosomes equal to eight, whereas limiting chromosome sizes significantly differ in various species. The largest range of chromosome bands was registered in L. cidri strains (400-2800 kb), while the smallest was found in L. waltii (1400-2800 kb). The intra- and interspecies polymorphism of Lachancea chromosomes is discussed.     

Genetic Polymorphism and Recombination in the Subtelomeric Region of Chromosome 14q

Subtelomeric regions of human chromosomes are the sites of increased meiotic recombination and have a male-to-female recombination ratio that is higher than elsewhere in the genome. We isolated two novel, polymorphic CA repeat markers from the distal part of the immunoglobulin heavy chain gene cluster, approximately 90 and 200 kb from the telomere of chromosome 14q. The 14q telomere was unambiguously located by physical mapping of telomeric YACs andBal31 exonuclease digestion of genomic DNA. We then constructed haplotypes using genotype data from these markers and data from sCAW1 (D14S826) for use as a highly polymorphic genetic marker. Linkage analysis using the 40 pedigree CEPH reference panel and genotype data from these and other loci physically mapped to the terminal 1.5 Mb of chromosome 14q revealed an apparent increase in meiotic recombination within this region, relative to the average rate for the genome. Further, we found that recombination was higher in females than in males, indicating that the subtelomeric region of 14q differs from other human subtelomeric regions.     

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.     

Induced chromosome rearrangements and morphologic variation in Candida albicans.

We have isolated a mutant of Candida albicans that switches between colony morphologies at high frequencies in a strain with several genetic markers. This strain, 1183, has an altered karyotype with two extra chromosomes. The 1183 karyotype is unstable upon passage. Using DNA transformation with the URA3 gene flanked by sequences from the C. albicans repeat sequence 27A, we have marked individual chromosomes of 1183 and 1161, a related smooth, stable strain. Many transformants contained one or more extra chromosomes, ranging in size from 150 kb to 2.1 Mb. Most were less than 800 kb and appeared to be fragments of a single chromosome. All fragments tested derive from one of the two smallest chromosomes. Six of 13 fragments contained the URA3 gene. In some cases, URA3 was located at the end of a fragment with adjacent telomere repeats. The integrated copy of URA3 was unstable in some 1183 transformants. Our results suggest that 1183 has a mutation affecting genomic stability. A connection between karyotypic changes and morphologic variation has been suggested from studies of several C. albicans strains; however, we find that gross karyotypic and morphological changes are separable processes.     

Molecular cytogenetic evidence to characterize breakpoint regions in Robertsonian translocations

Four individuals carrying different Robertsonian translocations (13q;14q, 14q;21q, 14q;15q, and 13q;21q) were studied to determine the breakpoints involved in the generation of these derivative chromosomes. Sequential high-resolution G-banding, in situ hybridization using alphoid and ribosomal DNA probes, and C-banding were performed. In addition, silver staining was also used for visualization of the NOR region. The results provide direct molecular cytogenetic evidence that Robertsonian translocations can take place in different regions in both the short arm and proximal long arm of acrocentric chromosomes. Three different types of breakpoints were identified: between the ribosomal or alphoid sequences, as deduced from the banding and in situ hybridization results, and breaks in two seemingly unrelated regions on the two different chromosomes. The use of conventional cytogenetic techniques together with molecular studies allowed more precise evaluation of the breakpoints involved in Robertsonian translocations than either approach alone might have done.