Direct analysis of radiation-induced chromosome fragments and rings in unstimulated human peripheral blood lymphocytes by means of the premature chromosome condensation technique

Development of the procedure to stimulate peripheral blood lymphocytes has greatly facilitated the understanding of chromosome aberration formation and repair mechanisms in human cells. Yet, because radiation induces far more initial chromosome breaks than are observed as aberrations in metaphase, it has not been possible to examine the kinetics of primary chromosome breakage and rejoining with this procedure. An improved method to induce premature chromosome condensation in unstimulated lymphocytes has been used to study primary chromosome breakage, rejoining, and ring formation at various times after irradiation with up to 800 rad of X-rays. The dose-response relations for chromosome fragments analyzed immediately or 1, 2, or 24 h after exposure were found to be linear. Rapid rejoining of chromosome fragments, which takes place in the first 3 h after X-ray exposure, was not correlated with a simultaneous increase in the formation of rings. The yield of rings per cell scored 24 h after irradiation, however, increased significantly and fit a linear quadratic equation. Both chromosome fragment rejoining and ring formation were completed about 6 h after irradiation. The frequency distributions of rings among cells followed a Poisson distribution, whereas chromosome fragments were overdispersed.     

Molecular cytogenetic analysis of eight inversion duplications of human chromosome 13q that each contain a neocentromere

Neocentromeres are fully functional centromeres that have arisen in previously noncentromeric chromosomal locations on rearranged chromosomes. The formation of neocentromeres results in the mitotic stability of chromosomal fragments that do not contain endogenous centromeres and that would normally be lost. Here we describe a unique collection of eight independent patient-derived cell lines, each of which contains a neocentromere on a supernumerary inversion duplication of a portion of human chromosome 13q. Findings in these patients reveal insight into the clinical manifestations associated with polysomy for portions of chromosome 13q. The results of FISH and immunofluorescent analysis of the neocentromeres in these chromosomes confirm the lack of alpha-satellite DNA and the presence of CENtromere proteins (CENP)-C, -E, and hMAD2. The positions of the inversion breakpoints in these chromosomes have been placed onto the physical map of chromosome 13, by means of FISH mapping with cosmid probes. These cell lines define, within chromosome 13q, at least three distinct locations where neocentromeres have formed, with five independent neocentromeres in band 13q32, two in band 13q21, and one in band 13q31. The results of examination of the set of 40 neocentromere-containing chromosomes that have thus far been described, including the 8 neocentromere-containing chromosomes from chromosome 13q that are described in the present study, suggest that chromosome 13q has an increased propensity for neocentromere formation, relative to some other human chromosomes. These neocentromeres will provide the means for testing hypotheses about sequence requirements for human centromere formation.     

Three unusual but cytogenetically similar cases with up to five different cell lines involving structural and numerical abnormalities of chromosome 18.

We report two prenatal and two postnatal diagnosed cases (the latter monozygotic twins) with ring chromosomes after GTG banding. All four, de novo r(18), cases turned out to be more complex after application of high-resolution molecular cytogenetics techniques such as use of fluorescence in situ hybridization, centromeric probes, multicolor banding, and locus-specific probes for chromosome 18. All four cases are mosaics involving chromosome 18 in up to five different cell lines, including 46,r(18); 46,dr(18); 47,r(18)x2; 46,mar(18); and 45,-18. Mosaicism sharing both numerical and structural anomalies is rare, but rings often appear as mosaics due to their mitotic instability. Overall, patients with ring chromosome 18 usually share clinical features of 18q- syndrome and, less frequently, those of 18p- syndrome. High-resolution molecular cytogenetics techniques were useful in the characterization of cases with dynamic mosaicism and in establishing the relationship between loss or gain of chromosomal material and the phenotype.     

Presence of telomeric and subtelomeric sequences at the fusion points of ring chromosomes indicates that the ring syndrome is caused by ring instability

In situ hybridization of a telomeric (TTA-GGG) _n sequence to metaphases from three cases of ring chromosome, involving respectively chromosomes 4, 16, and 20, showed the presence of the cognate sequences in all three rings. To investigate whether these ring chromosomes originated by telomere-telomere fusion, we determined, by in situ hybridization, whether telomere-associated sequences and/or specific distal sequences were still present in the ring chromosomes. The finding that these sequences were preserved in all the ring chromosomes strongly indicates that they originated by telomere-telomere fusion. All three subjects carrying the ring chromosomes are affected by the so-called ring syndrome, with failure to thrive, minor dysmorphic signs and no major anomalies. The r(4) patient has the ring in mosaic form with a normal cell line and has normal intelligence. The r(16) and the r(20) patients have moderate mental retardation and suffer from seizures. We conclude that the ring syndrome, even in its more severe manifestation, is caused by ring chromosome instability.     

Assembly of the FtsZ ring at the central division site in the absence of the chromosome

The FtsZ ring assembles between segregated daughter chromosomes in prokaryotic cells and is essential for cell division. To understand better how the FtsZ ring is influenced by chromosome positioning and structure in Escherichia coli , we investigated its localization in parC and mukB mutants that are defective for chromosome segregation. Cells of both mutants at non-permissive temperatures were either filamentous with unsegregated nucleoids or short and anucleate. In parC filaments, FtsZ rings tended to localize only to either side of the central unsegregated nucleoid and rarely to the cell midpoint; however, medial rings reappeared soon after switching back to the permissive temperature. Filamentous mukB cells were usually longer and lacked many potential rings. At temperatures permissive for mukB viability, medial FtsZ rings assembled despite the presence of apparently unsegregated nucleoids. However, a significant proportion of these FtsZ rings were mislocalized or structurally abnormal. The most surprising result of this study was revealed upon further examination of FtsZ ring positioning in anucleate cells generated by the parC and mukB mutants: many of these cells, despite having no chromosome, possessed FtsZ rings at their midpoints. This discovery strongly suggests that the chromosome itself is not required for the proper positioning and development of the medial division site.     

Chromosomal origin of small ring marker chromosomes in man: characterization by molecular genetics.

Ten cases of small ring chromosomes which did not stain with distamycinA/DAPI and did not possess satellite regions associated with nucleolus-organizing regions are described. In situ hybridization with a battery of biotinylated pericentric repeat probes specific either for individual chromosomes or for groups of chromosomes allowed the identification of the chromosomal origin of these marker chromosomes. There was one example of a marker derived from each of chromosomes 1, 3, 6, 14, 16, 18, 20, 13 or 21, and the X, and there were two examples of markers derived from chromosome 12. One case possessed two markers, one derived from chromosome 6, and one derived from the X. The mechanism of generation of ring marker chromosomes is discussed. Five of seven cases who could be phenotypically assessed were abnormal. Three of these--the first with a ring chromosome derived from chromosome 1; the second with two markers, one derived from chromosome 6 and the other from the X chromosome; and the third with a ring chromosome derived from chromosome 20--each possessed distinctive facies. Additional cases with identified rings may allow the delineation of new chromosomal syndromes.     

125IdUrd-induced chromosome fragments, assayed by premature chromosome condensation, and DNA double-strand breaks have similar repair kinetics in G1-phase CHO-cells

The effect of 125I-decay on cell lethality, and induction of chromosome and DNA damage, was studied in synchronous non-cycling, G1-phase CHO-cells. For this purpose a population of mitotic cells was allowed to divide and progress through S-phase in the presence of 125IdUrd. Cells were subsequently transferred to conditioned medium (C-med) obtained from plateau-phase cultures that allowed cells to divide and accumulate in G1-phase in a non-cycling state. To accumulate 125I-induced damage, cells were kept frozen at -80 degrees C. Freezing was carried out using a new method that optimally preserves cell integrity. After various times of cold storage, cells were thawed and assayed for survival, DNA and chromosome damage, either immediately or after various times in C-med. Neutral filter elution was used to assay repair of DNA double-strand breaks (dsbs), and premature chromosome condensation was used to assay repair of chromosome fragments and induction of ring chromosomes. The results indicate very little repair at the cell survival level (repair of PLD). At the DNA level an efficient repair of DNA dsbs was observed, with kinetics similar to those observed after exposure to X-rays. At the chromosome level a fast repair of prematurely condensed chromosome fragment was observed, with a concomitant increase in the number of ring chromosomes induced. The repair kinetics of chromosome fragments and DNA dsbs were very similar, suggesting that DNA dsbs may underlie chromosome fragmentation.     

Radiation-induced cytogenetic damage in relation to changes in interphase chromosome conformation

The premature chromosome condensation (PCC) technique was used to study several factors that determine the yield of chromosome fragments as observed in interphase cells after irradiation. In addition to absorbed dose and the extent of chromosome condensation at the time of irradiation, changes in chromosome conformation as cells progressed through the cell cycle after irradiation affected dramatically the yield of chromosome fragments observed. As a test of the effect of chromosome decondensation, irradiated metaphase Chinese hamster ovary (CHO) cells were allowed to divide, and the prematurely condensed chromosomes in the daughter cells were analyzed in their G1 phase. The yield of chromosome fragments increased as the daughter cells progressed toward S phase and chromosome decondensation occurred. When early G1 CHO cells were irradiated and analyzed at later times in G1 phase, an increase in chromosome fragmentation again followed the gradual increase in chromosome decondensation. As a test of the effect of chromosome condensation, G0 human lymphocytes were irradiated and analyzed at various times after fusion with mitotic CHO cells, i.e., as condensation proceeded. The yield of fragments observed was directly related to the amount of chromosome condensation allowed to take place after irradiation and inversely related to the extent of chromosome condensation at the time of irradiation. It can be concluded that changes in chromosome conformation interfered with rejoining processes. In contrast, resting chromosomes (as in G0 lymphocytes irradiated before fusion) showed efficient rejoining. These results support the hypothesis that cytogenetic lesions become observable chromosome breaks when chromosome condensation or decondensation occurs during the cell cycle.     

Amplification of the Y chromosome in three murine tumor cell lines transformed in vivo by different human prostate cancers

Summary Conventional and molecular cytogenetic analyses of three murine cancer cell lines that had been induced in male athymic mice by the injection of three different human prostate cancer cell lines revealed selective amplification of the Y chromosome. In particular, analysis of metaphase and interphase nuclei by fluorescence in situ hybridization (FISH) with the mouse Y chromosome-specific DNA painting probe revealed the presence of various numbers of Y chromosomes, ranging from one to eight, with a large majority of nuclei showing two copies (46.5–60.1%). In Interphase nuclei, the Y chromosomes showed distinct morphology, allowing identification irrespective of whether the preparations were treated for 15 min or for 5 h with Colcemid, a chemical known to cause chromosome condensation. However, FISH performed on human lymphocyte cultures with chromosome-specific DNA painting probes other than the Y chromosome did not reveal condensed chromosome morphology in interphase nuclei even after 12 h of Colcemid treatment. Our FISH results indicate that (1) the Y chromosome is selectively amplified in all three cell lines; (2) the mouse Y chromosome number is comparable in both interphase and metaphase cells; (3) the Y chromosome number varies between one and eight, with a large majority of cells showing two or three copies in most interphase nuclei; (4) the condensation of the Y chromosome is not affected by the duration of Colcemid treatment but by its inherent DNA constitution; and (5) the number of copies of the Y chromosome is increased and retained not only in human prostate tumor cell lines but also in murine tumors induced by these prostate tumor cell lines.     

FtsZ ring formation without subsequent cell division after replication runout in Escherichia coli

In this report, we have investigated cell division after inhibition of initiation of chromosome replication in Escherichia coli. In a culture grown to the stationary phase, cells containing more than one chromosome were able to divide some time after restart of growth, under conditions not allowing initiation of chromosome replication. This shows that there is no requirement for cell division to take place within a certain time after initiation of chromosome replication. Continued growth without initiation of replication resulted in filamented cells that generally did not have any constrictions. Interestingly, FtsZ rings were formed in a majority of these cells as they reached a certain cell length. These rings appeared and were maintained for some time at the cell quarter positions on both sides of the centrally localized nucleoid. These results confirm previous findings that cell division sites are formed independently of chromosome replication and indicate that FtsZ ring assembly is dependent on cell size rather than on the capacity of the cell to divide. Disruption of the mukB gene caused a significant increase in the region occupied by DNA after the replication runout, consistent with a role of MukB in chromosome condensation. The aberrant nucleoid structure was accompanied by a shift in FtsZ ring positioning, indicating an effect of the nucleoid on the positioning of the FtsZ ring. A narrow cell length interval was found, under and over which primarily central and non-central FtsZ rings, respectively, were observed. This finding correlates well with the previously observed oscillatory movement of MinC and MinD in short and long cells.     

A third common allele in the transferrin system,Tf C3 , detected by isoelectric focusing

Summary The fluorochrome Hoechst 33258 which binds preferentially to A-T base pairs, drastically inhibits the condensation of A-T-rich centromeric heterochromatin regions in mouse cell lines. The condensation of all other regions of these chromosomes is also inhibited to some extent. The human Y chromosome contains a large heterochromatic region, which is also rich in A-T base pairs. This chromosome is not affected by Hoechst 33258 in human leukocyte cell cultures. On the other hand, condensation of the multiple copies of human Y chromosome in the mouse-human cell hybrid RH-28Y-23 is inhibited and the chromosomes appear distorted in Hoechst 33258-treated cells.     

Comparative study of pig-rodent somatic cell hybrids

The pig chromosome complement of six different types of pig-rodent hybrid cell lines was examined by means of fluorescence in situ hybridization with a porcine SINE probe. The cell lines were obtained by fusing pig lymphocytes with cells of the Chinese hamster cell lines wg3h, BK14-150 and E36, and of the mouse cell lines NSO, PU and LMTK^-. The hybrids were analysed with respect to: (1) the number of pig chromosomes, (2) the type of pig chromosomes, (3) the occurrence of pig-rodent chromosome trans-locations, and (4) the presence of pig chromsome fragments. The results show that the number of pig chromosomes varied within and among hybrid cell lines. The pig-hamster hybrids mainly retained nontelocentric pig chromosomes, whereas the pig-mouse hybrids also retained telocentric pig chromosomes. Pig-rodent chromosome translocations were found in all types of hybrids, but the incidence was in general low. Chromosome fragments were abundant in BK14-150 hybrids, and rare in most other hybrid cell lines. It is concluded that the SINE probe is a useful tool to make a preliminary characterization of the porcine chromosome complement of pig-rodent somatic cell hybrids. The results of this characterization can be used to select hybrids for further cytogenetic analysis. Furthermore, our data show that different rodent cell lines will have to be used as fusion partners for the production of hybrids when constructing a panel informative for all pig chromosomes.     

Nuclear asynchrony in multinucleate rat kangaroo cells

Multinucleate (MN) cells were induced in PtK1 cells by colcemid treatment. A large percentage of cells developed nuclear asynchrony both in relation to DNA synthesis and mitosis within one cell cycle. Asynchrony could be traced even in metaphase and anaphase cells in which interphase nuclei, PCC of S-phase nuclei and less condensed prophase-like chromosomes could be observed along with normally condensed chromosomes. The occurrence of such abnormalities in these large MN cells may be explained on the basis of an uneven distribution of inducer molecules of DNA synthesis and mitosis due to cytoplasmic compartmentation. The less condensed form of all the chromosomes except chromosome 4 could be traced in asynchronous metaphase. The failure of the less condensed chromosomes to undergo complete condensation does not always appear to result from late entry of nuclei containing these chromosomes into G2 phase. It is likely that chromosome 4 carries gene(s) for chromosome condensation, as this chromosome itself never appears in a less condensed form. The inducers for chromosome condensation may not always be available at equal concentrations to all chromosomes located in separate nuclei, thus they may sometimes fail to undergo complete condensation before other nuclei reach the end of prophase, when the nuclear envelopes of all nuclei present in the cell break down simultaneously.     

Chromosome 18 replaced by two ring chromosomes of chromosome 18 origin

We here describe the first example of the replacement of an autosome by two ring chromosomes originating from the missing chromosome, presented in a patient with a single chromosome 18 and two additional ring chromosomes. Detailed fluorescence in situ hybridization (FISH) analysis revealed the chromosome 18 origin of both ring chromosomes and characterized the small and the large ring chromosome as derivatives of the short and long arm of chromosome 18, respectively. The loss of subtelomeric regions of the short and the long arm of chromosome 18 in the ring chromosomes was confirmed by FISH studies. Molecular studies showed the exclusive presence of the paternal alleles for microsatellite markers located distal to the short and long arm loci D18S843 and D18S474, respectively. This indicates the maternal origin of both rings and provides evidence for substantial deletions of the distal parts of both arms of chromosome 18 in the ring chromosomes. The dysmorphic features of the patient can be explained by these deletions in both chromosome arms, as the clinical findings partly overlap with observations in 18p- and 18q-syndrome and are similar to some cases of ring chromosome 18. Centromere misdivision is suggested as one mechanism involved in the formation of the ring chromosomes.     

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

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

Chromosomal instability of mouse pluripotent cells cultured in vitro

The perspectives of using embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSs) in clinics makes the karyological analysis of these cells an important issue. In the present study, using methods of classical and molecular cytogenetics of chromosome, we carried out a karyological study of two mouse ES and two iPS cell lines derived de novo. We obsererved the X chromosome monosomy in all studied ES and iPS cell lines, which makes the modal number of chromosomes in these cell lines equal to 39. The chromosomal instability (aneuploidy) was revealed in both studied iPS cell lines. Moreover, we have detected chromosomal rearrangements and chromosomal fragments in one of studied iPS. Our findings stress the importance of the careful cytogenetic evaluation of a pluripotent cell line, especially iPS cell lines, which should be carried out prior to any clinical use of these cells.     

Chromosomal in situ suppression hybridization of human gonosomes and autosomes and its use in clinical cytogenetics

Summary DNA libraries from sorted human gonosomes were used selectively to stain the X and Y chromosomes in normal and aberrant cultured human cells by chromosomal in situ suppression (CISS-) hybridization. The entire X chromosome was stained in metaphase spreads. Interphase chromosome domains of both the active and inactive X were clearly delineated. CISS-hybridization of the Y chromosome resulted in the specific decoration of the euchromatic part (Ypter-q11), whereas the heterochromatic part (Yq12) remained unlabeled. The stained part of the Y chromosome formed a compact domain in interphase nuclei. This approach was applied to amniotic fluid cells containing a ring chromosome of unknown origin (47,XY; +r). The ring chromosome was not stained by library probes from the gonosomes, thereby suggesting its autosomal origin. The sensitivity of CISS-hybridization was demonstrated by the detection of small translocations and fragments in human lymphocyte metaphase spreads after irradiation with ⁶⁰Co-gamma-rays. Lymphocyte cultures from two XX-males were investigated by CISS-hybridization with Y-library probes. In both cases, metaphase spreads demonstrated a translocation of Yp-material to the short arm of an X chromosome. The translocated Y-material could also be demonstrated directly in interphase nuclei. CISS-hybridization of autosomes 7 and 13 was used for prenatal diagnosis in a case with a known balanced translocation t(7;13) in the father. The same translocation was observed in amniotic fluid cells from the fetus. Specific staining of the chromosomes involved in such translocations will be particularly important, in the future, in cases that cannot be solved reliably by conventional chromosome banding alone.Dedicated to Professor Friedrich Vogel on the occasion of his 65th birthday     

On the continuity of chromosomal subunits: an analysis of induced ring chromosomes in Vicia faba

The proposition that subunits of a chromatid are continuous in a directional sense has been tested by observing the behaviour of induced ring chromosomes in Vicia faba. On the simplest hypothesis, that the subunits are the uninterrupted complementary strands of the DNA molecule, the polarity of rejoining should result in free separation of rings following replication in successive cell cycles. Centric and acentric ring chromosomes were separately assessed in both diploid and colchicine-accumulated tetraploid metaphase cells of primary root tips. Contrary to expectation large numbers of single and interlocked rings were observed in both cell cycles. Spontaneous sister chromatid exchanges and other breakage-reunion events can produce the configurations seen; with the postulated level of sister chromatid exchange equating that determined autoradiographically in rod chromosomes of V. faba. Unless the replication of ring chromosomes produces conditions unusual in rod chromosome replication, spontaneous breakage is probably common in replicating or post replication Vicia chromosomes. — A fundamental difference exists between the behaviour of centric and acentric ring chromosomes. Acentric ring chromosomes behave as if the chromatid arm were one DNA molecule, or a number of DNA molecules with identical directional sense. However, centric ring chromosomes behave as if there were a difference at the centromere in at least one (probably the metacentric) chromosome of the Vicia complement. That is, the two duplication-segregation subunits which extend the length of the chromosome, may contain a change in polarity at the centromere.     

The structure and dynamics of ring chromosomes in human neoplastic and non-neoplastic cells

Acquired ring chromosomes have been found in most types of human neoplasia, with a frequency approaching 10% in malignant mesenchymal tumours. In this study, the composition and dynamics of ring chromosomes were analysed in eight cases of acute myelogenous leukaemia, 17 solid tumours, and five cases with constitutional rings. Chromosomal banding and fluorescence in situ hybridisation were performed to determine the content and the structural heterogeneity of the rings. Telomeric repeats were detected using peptide nucleic acid probes or primed in situ labelling, whereas centromeric activity was evaluated by detection of kinetochore proteins. Mitotic instability was assessed by the frequency of anaphase bridges. The results suggest that human ring chromosomes can be structurally and functionally divided into two categories. In the first of these, size variation is minimal and rearrangement at cell division is uncommon. The majority of such rings contain subtelomeric sequences. Constitutional ring chromosomes and most rings in leukaemias belong to this group, whereas only a few mesenchymal tumours exhibit rings of this type. The second category consists of rings with amplified sequences, primarily from chromosome 12, characteristically occurring in atypical lipomatous tumours and other subtypes of low or borderline malignant mesenchymal neoplasms. Variation in size and number is extensive, and breakage-fusion-bridge events occur at a high frequency. Abnormalities in pericentromeric sequences are common and, in some cases, kinetochores assemble in the absence of alphoid DNA. We conclude that it is not only the ring structure per se or the neoplastic nature of the host cell that determines ring instability, but probably also the functional role of the genes carried in the ring. Received: 24 November 1998 / Accepted: 18 January 1999     

The use of cell surface antigens to characterize and select for fragments of human chromosomes retained by interspecies hybrids

We have used a mouse cell transformant generated by human chromosome-mediated gene transfer (CMGT) to explore the use of cell surface antigens in the identification of fragments of human chromosomes retained by somatic cell hybrids. The transformed line, 21-30b, contained an intact rear-ranged human chromosome, and could be shown by isozyme analysis to contain genetic material from chromosomes 9 and X. By using the transformant as an immunogen in mice, it was also possible to produce antiserum to human-specific surface antigens. Using genetically characterized human X rodent hybrid lines, the genes controlling expression of these antigens could be localized to 11per----11p13, segregating concordantly with surface antigen S3. These conclusions were possible despite the fact that the presence of chromosome 11 in the transformant was not detectable by the presence of chromosome specific isozyme LDH-A or surface antigens W6/34 and 4F2. Finally, the fluorescence-activated cell sorter (FACS) was used to fractionate the transformant cells into antigen positive and negative subpopulations. This resulted in the isolation and characterization of four additional chromosome rearrangements involving interspecies chromosome translocations. This work demonstrates the value of chromosome-specific surface antigens and the FACS in the evaluation of human chromosome fragments retained by interspecies hybrids.