Flow cytometry isolation and improved visualization of sorted mouse chromosomes. Purification of chromosomes X and ISO-1 from cell lines with Robertsonian translocations

While analysis and sorting of human chromosomes by flow cytometry has been widely used, isolation of a pure mouse chromosome remains very difficult, since most murine chromosomes are quite similar in size. To overcome this problem, we have analysed mouse cell lines having either Robertsonian translocations or isochromosomes. The resulting metacentric chromosomes are very different in size and in morphology from normal mouse acrocentric chromosomes. These characteristics have been analysed by computer-monitored flow cytometry, facilitated by improvements in the chromosome extraction procedure. Signals characteristic of the iso-lq chromosome in cell line PCC4 azaR1, and of the normal X chromosome in the mouse strain 22CD have thus been obtained. These chromosomes have been sorted and can be easily recognized by fluorescence microscopy when collected onto serum-albumin-coated microscope slides. The technical modifications made, coupled with the existence of a great diversity of metacentric chromosomes resulting from Robertsonian translocations, should allow the purification of a number of different mouse chromosomes.     

Construction of microcell hybrid clones containing specific mouse chromosomes: application to autosomes 8 and 17.

Fibroblast cultures prepared from mice homozygous for a Robertsonian translocation (centric fusion) between autosomes 8 and 17 [Rb(8.17)] were used as donors in microcell-mediated chromosome transfer experiments. By using hamster recipient cells deficient in adenine phosphoribosyltransferase (APRT-) and selecting for expression of murine APRT (a chromosome 8 marker), microcell hybrids were isolated which retained only the mouse Rb(8.17) translocation in addition to the hamster chromosome complement. The translocation was stable in cells maintained under APRT+ selective pressure, and mouse marker traits encoded by genes on both chromosomes 8 and 17 segregated concordantly. A second family of hybrid clones was constructed by fusing microcells derived from wild-type mouse fibroblasts with APRT- hamster cells. Four of six clones analyzed retained only mouse chromosome 8. These studies demonstrated that microcell hybrids containing specific Robertsonian translocations as the only donor-derived genetic material can be obtained. Furthermore, a number of Robertsonian translocations between chromosomes which carry selectable markers (chromosomes 3, 8, and 11) and other autosomes have been described. By using fibroblast cultures prepared from mice containing these translocations as donors in microcell fusions, 18 of the 20 mouse chromosomes could be selectively fixed in different hybrid clones. Thus, a collection of 20 hybrid clones, each containing a single, specific mouse chromosome, can be constructed by using the strategy described in this report. The potential utility of such a monochromosomal hybrid panel is discussed.     

Identification of uniparental disomy following prenatal detection of Robertsonian translocations and isochromosomes

Rearrangements of the acrocentric chromosomes (Robertsonian translocations and isochromosomes) are associated with an increased risk of aneuploidy. Given this, and the large number of reported cases of uniparental disomy (UPD) associated with an acrocentric rearrangement, carriers are presumed to be at risk for UPD. However, an accurate risk estimate for UPD associated with these rearrangements is lacking. A total of 174 prenatally identified acrocentric rearrangements, including both Robertsonian translocations and isochromosomes, were studied prospectively to identify UPD for the chromosomes involved in the rearrangements. The overall goal of the study was to provide an estimate of the risk of UPD associated with nonhomologous Robertsonian translocations and homologous acrocentric rearrangements. Of the 168 nonhomologous Robertsonian translocations studied, one showed UPD for chromosome 13, providing a risk estimate of 0.6%. Four of the six homologous acrocentric rearrangements showed UPD, providing a risk estimate of 66%. These cases have also allowed delineation of the mechanisms involved in producing UPD unique to Robertsonian translocations. Given the relatively high risk for UPD in prenatally identified Robertsonian translocations and isochromosomes, UPD testing should be considered, especially for cases involving the acrocentric chromosomes 14 and 15, in which UPD is associated with adverse clinical outcomes.     

The behavior of nucleolus organizers in structurally changed karyotypes of barley

Two standard karyotype barley lines and 18 lines with karyotypes reconstructed by means of induced reciprocal translocations have been studied with respect to nucleolus formation. The standard karyotype contains two pairs of satellite chromosomes (pairs 6 and 7). Five of the structurally changed karyotypes contain, as a result of reciprocal translocations between the standard satellite chromosomes, only one satellite chromosome pair, each chromosome with two satellites and two nucleolus organizing regions. Under these circumstances, only two of the four NORs are active in nucleolus formation while the other two — probably the transposed ones — remain inactive; hence the maximum number of primary nucleoli per nucleus is two. — When NORs are translocated to chromosomes with no NOR in the standard karyotyp, the normal pattern of nucleolus formation remains unchanged. The same is true after transposition of segments from other chromosomes to the satellites of the standard SAT-chromosome pairs 6 and 7. The results obtained are discussed with respect to effects of translocations on the activity and behaviour of nucleolus organizing regions.     

The possible ways of the diploidization of the polyploid germinative stem cells in the mouse BALB/c line

Quantitative and qualitative chromosome rearrangements in the cell line G1 established from a genital ridge of the 12,5 dpc BALB/c mouse embryo were analysed. Cytogenetic analysis was performed on the 75th passage of in vitro cultivation. It has been shown that by this passage the cell population was heterogenous. It is suggested that such heterogeneity may be caused by realization of two simultaneous processes namely the cell polyploidization and their secondary diploidization. These processes were accompanied by some chromosome destructions, and the creation of small new acrocentric chromosomes and large aberrant chromosomes as well as Robertsonian translocations. The present study demonstrates in vitro karyotype evolution of the mouse cell line G1 including the increased instability of the chromosome apparatus.     

Robertsonian translocations in wheat arise by centric misdivision of univalents at anaphase I and rejoining of broken centromeres during interkinesis of meiosis II

The mechanism of origin of Robertsonian translocations was investigated in plants monosomic for chromosome 1A of wheat and 1H(t) of Elymus trachycaulus by GISH. Chromosomes 1A and 1H(t) stayed univalent in all metaphase I cells analyzed, suggesting that Robertsonian translocations do not originate from meiotic recombination in centromeric regions with shared DNA sequence homology. At ana-/telophase I, the 1H(t) and 1A univalents underwent either chromosome or chromatid segregation and misdivided in 6-7% of the pollen mother cells. None of the ana-/telophases I analyzed had Robertsonian translocations, which were only observed in 2% of the "half tetrads" at ana-/telophase II. The frequency of Robertsonian translocations observed at ana-/telophase II corresponds well with the number of Robertsonian translocations (1-4%) detected in progenies derived from plants monosomic for group-1 chromosomes of wheat (1A, 1B, and 1D) and 1H(t) of E. trachycaulus. Our data suggest that Robertsonian translocations arise from centric misdivision of univalents at ana-/telophase I, followed by segregation of the derived telocentric chromosomes to the same nucleus, and fusion of the broken ends during the ensuing interkinesis.     

非Robertsonian类型小黑麦易位系的研究

非Robertsonian类型小黑麦易位系的研究@胡含$中国科学院遗传研究所植物细胞与染色体工程国家重点实验室!北京100101小黑麦;;易位系     

Absence of selection against aneuploid mouse sperm at fertilization.

Is there selection against aneuploid sperm during spermatogenesis and fertilization? To address this question, we used male mice doubly heterozygous for the Robertsonian (Rb) translocations Rb(6. 16)24Lub and Rb(16.17)7Bnr, which produce high levels of sperm aneuploid for chromosome 16, the mouse counterpart of human chromosome 21. The frequencies of aneuploid male gametes before and after fertilization were compared by analyzing approximately 500 meiosis II spermatocytes and approximately 500 first-cleavage zygotes using fluorescence in situ hybridization with a DNA painting probe mixture containing three biotin-labeled probes specific for chromosomes 8, 16, and 17 plus a digoxigenin-labeled probe specific for chromosome Y. Hyperhaploidy for chromosome 16 occurred in 20.0% of spermatocytes and in 21.8% of zygotes. Hypohaploidy for chromosome 16 occurred in 17.0% and 16.7% of spermatocytes and zygotes, respectively. In addition, there was no preferential association between chromosome 16 aneuploidy and either of the sex chromosomes, nor was there an elevation in aneuploidy for chromosomes not involved in the Rb translocations. These findings provide direct evidence that there is no selection against aneuploid sperm during spermiogenesis, fertilization, and the first cell cycle of zygotic development.     

Quantitative studies on the arrangment of human metaphase chromosomes

Summary The pattern of association of acrocentric chromosomes was examined in ten and five carriers of a 15/21 and a 13/14 Robertsonian translocation, respectively, and was compared with that of the same numbers of relatives with normal karyotypes. In the carriers of 15/21 translocation, the number of large associations (involving more than two acrocentrics) and the association frequencies for individual acrocentric chromosomes, were significantly higher than in the control group. The mean number of associations of the single homologs of the translocation chromosomes was much higher than that of the other acrocentrics. In the carriers of 13/14 translocations, only the association frequency for chromosome 13 was higher than in the normal relatives. The uninvolved chromosomes homologous to those involved in translocations showed an insignificant increase in associations in comparison with the other acrocentrics. These results suggest that some mechanism within the cells compensates for the effect of missing acrocentrics or of acrocentrics lacking NORs on the number of associations. The possible relations of this phenomenon to the activity of the nucleolus organizing regions are discussed.     

Mouse chromosome-specific painting probes generated from microdissected chromosomes

Using degenerate primer amplification of chromosomes microdissected from banded cytogenetic preparations, we constructed both whole chromosome painting probes for mouse Chromosomes (Chrs) 1, 2, 3, and 11 and a centromere probe that strongly paints most mouse centromeres. We also amplified a Robertsonian translocation chromosome microdissected from unstained preparations to construct a painting probe for Chrs 9 and 19. The chromosomes probes uniformly painted the respective chromosomes of origin. We demonstrated the utility of the Chr 11 probe in aberration analysis by staining mutants that we had previously identified as containing a Chr 11 translocation, and in some mutant cell lines we observed chromosome rearrangements not previously detected in stained cytogenetic preparations. The technology of microdissection and amplification applies to all mouse chromosomes or to specific subchromosomal regions and will be useful in mouse genetics, in aberration analysis, and for chromosome identification.     

Conventional karyotype, nucleolar organizer regions and genome size in five Mediterranean species of Syngnathidae (Pisces, Syngnathiformes)

Conventional karyotypes, NOR-bearing chromosomes by means of silver impregnation and genome size were investigated in five Mediterranean species in three genera of the Syngnathidae. A karyotype of 48 subtelocentric-acrocentric chromosomes was found in the seahorse Hippocampus hippocampus (FN=48) while a diploid value of 44 occurred in H. guttulatus (2 sm-m+42 a; FN=46) and the pipefish Syngnathus abaster (44 a; FN=44) and S. typhle (44 a; FN=44). The pipefish Nerophis ophidion , possessing a diploid chromosomal set of 58 made up of 50 meta-submetacentric and eight subteloacrocentric elements (FN=108) and a genome size three to four times larger than those known to date, differs cytogenetically from all other Syngnathids studied so far. A single pair of active NOR-bearing chromosomes was found in both species of the genus Hippocampus while in Syngnathus and Nerophis species more than two silver positive chromosomes were found to be involved in nucleolus organization giving rise to NOR-bearing chromosome polymorphism. The possible evolutionary routes of quantitative and qualitative changes in chromosome and DNA are discussed. The resulting phylogenetic scheme is shown to coincide with that constructed from morphological characters.     

Karyotypes of three species of the family Cottidae (Scorpaeniformes)

Karyotypes and cellular DNA contents of three species of the family Cottidae viz.Icelus cataphractus, Gymnocanthus intermedius andAlcichthys alcicornis were analyzed. Structural modifications within the family were supposedly by Robertsonian translocations. The diploid chromosome numbers were determined to be 48 inAlcichthys alcicornis, 44 inGymnocanthus intermedius and 40 inIcelus cataphractus. The DNA contents ranged from 1.46 to 1.50pg/cell in the three species. The karyotype ofIcelus cataphractus is unique in having the smallest chromosome number (2n = 40) and 14 large-sized chromosomes. From the chromosome number and the existence of some large chromosome pairs, Robertsonian translocations seem to have occurred frequently inIcelus cataphractus andGymnocanthus intermedius.     

Characterization of Robertsonian translocations by using fluorescence in situ hybridization.

Fluorescence in situ hybridization with five biotin-labeled probes (three alphoid probes, a probe specific for beta-satellite sequences in all acrocentric chromosomes, and an rDNA probe) was used to characterize 30 different Robertsonian translocations, including three t(13;13); one t(15;15), four t(21;21), three t(13;14), two t(13;15), two (13;21), two t(13;22), one t(14;15), eight t(14;21), two t(14;22), and two t(21;22). Of 8 de novo homologous translocations, only one t(13;13) chromosome was interpreted as dicentric, while 19 of 22 nonhomologous Robertsonian translocations were dicentric. The three monocentric nonhomologous translocations included both of the t(13;21) and one t(21;22). Two of 26 translocations studied using the beta-satellite probe showed a positive signal, while rDNA was undetectable in 10 cases studied. These results indicate that most homologous Robertsonian translocations appear monocentric, while the bulk of nonhomologous translocations show two alphoid signals. A majority of the breakpoints localized using this analysis seem to be distal to the centromere and just proximal to the beta-satellite and nuclear-organizing regions.     

Genetic induction of chromosomal rearrangements in barley chromosome 7H added to common wheat

Chromosome 2C of Aegilops cylindrica induces chromosomal rearrangements in alien chromosome addition lines, as well as in euploid lines, of common wheat. To induce chromosomal rearrangements in barley chromosome 7H, reciprocal crosses were made between a mutation-inducing common wheat line that carries a pair of 7H chromosomes and one 2C chromosome and a 7H disomic addition line of common wheat. Many shrivelled seeds were included in the progeny, which was an indication of the occurrence of chromosome mutations. The chromosomal constitution of the viable progeny was examined by FISH (fluorescence in situ hybridization) using the barley subterminal repeat HvT01 as a probe. Structural changes of chromosome 7H were found in about 15% of the progeny of the reciprocal crosses. The aberrant 7H chromosomes were characterized by a combination of N-banding, FISH and genomic in situ hybridization. Mosaicism for aberrant 7H chromosomes was observed in seven plants. In total, 89 aberrant 7H chromosomes were identified in 82 plants, seven of which had double aberrations. More than half of the plants carried a simple deletion: four short-arm telosomes, one long-arm telosome, and 45 terminal deletions (23 in the short arm, 21 in the long arm, and one involving both arms). About 40% of the aberrations represented translocations between 7H and wheat chromosomes. Twenty of the translocations had wheat centromeres, 12 the 7H centromere, with translocation points in the 7HS (five) and in the 7HL (seven), and the remaining four were of Robertsonian type, three involving 7HS and one with 7HL. In addition, one translocation had a barley segment in an intercalary position of a wheat chromosome, and two were dicentric. The breakpoints of these aberrations were distributed along the entire length of chromosome 7H.     

Analysis of centromeric activity in Robertsonian translocations: implications for a functional acrocentric hierarchy

Approximately 90% of human Robertsonian translocations occur between nonhomologous acrocentric chromosomes, producing dicentric elements which are stable in meiosis and mitosis, implying that one centromere is functionally inactivated or suppressed. To determine if this suppression is random, centromeric activity in 48 human dicentric Robertsonian translocations was assigned by assessment of the primary constrictions using dual color fluorescence in situ hybridzation (FISH). Preferential activity/constriction of one centromere was observed in all except three different rearrangements. The activity is meiotically stable since intrafamilial consistency of a preferentially active centromere existed in members of six families. These results support evidence for nonrandom centromeric activity in humans and, more importantly, suggest a functional hierarchy in Robertsonian translocations with the chromosome 14 centromere most often active and the chromosome 15 centromere least often active.     

Synapsis and chiasma distribution in mice heterozygous for translocations in chromosomes 16 and 17

Electron microscopic analysis of synaptonemal complexes and analysis of chiasmata distribution in male mice heterozygous for Robertsonian translocation T(16; 17)7Bnr - (Rb7), for synaptonemal reciprocal translocation T(16;17)43H - (T43), in double heterozygotes for these translocations and in males with partial trisomy of the proximal region of chromosome 17 was carried out. Synaptic disturbances around the breakpoints of the translocations, such as asynapsis of homologous regions of partners and non-homologous synapsis of centromeric regions of acrocentric chromosomes, were revealed. Synaptic regularity in the proximal part of the chromosome 17 appeared to be affected by no t12 haplotype. Good coincidence between sizes of mitotic chromosomes and corresponding lateral elements of synaptonemal complexes was found for all chromosomes, with the exception of Rb7 in trisomics. In the latter karyotype, the proximal part of chromosome 17 involved in Robertsonian fusion seems to be shortened in the course of zygotene and never synapted with homologous segment of neither the acrocentric chromosome 17 nor large product of reciprocal translocation. Drastic increase in chiasmata frequency in the proximal part of chromosome 17 was revealed in heterozygotes for T43H and in trisomics, as compared with the double heterozygotes Rb7/T43. The latter finding was explained by the existence of two independent pairing segments in the former karyotypes.     

Induced Robertsonian fusions and tandem translocations in mammalian cell cultures.

Cultures of a cattle cell line and a Peromyscus eremicus cell line recovering from a pulse-treatment with mitomycin C, actinomycin D, 33258 Hoechst, and nitrosoguanidine exhibited translocations between chromosomes at the centromeric regions (Robertsonian fusions) as well as between centromere and telomere and between telomeres (tandem translocations). The frequency of Robertsonian fusions was found to be dose-dependent and duration-dependent with the mitomycin treatment. Biarmed chromosomes resulting from fusions may be monocentric or dicentric. Analyses of clones isolated from treated cells suggested that fused chromosomes may perpetuate in the cell populations.     

Human chromosome variation with two Robertsonian translocations

Summary A woman was found to have 42 autosomes due to engagement of both chromosomes 14 in Robertsonian rearrangements, one with a chromosome 21 and the other with a chromosome 22: t(14q21q) and t(14q22q). The two translocations appear monocentric and by silver staining have no rRNA activity. The t(14q21q) translocation is familial and was ascertained through a nephew with Down syndrome, while the origin of the t(14q22q) translocation was not established. In addition to these two translocations, the woman had XX/XXX sex chromosome mosaicism. She has had two recognized pregnancies, each resulting in the birth of a child with one of the two translocations. Both children are phenotypically normal, as is their mother, the first normal liveborn individual identified with two Robertsonian translocations.