The behavior of allocyclic chromosomes in Bloom's syndrome

The behavior of individual allocyclic chromosomes has been analyzed in lymphocytes of a sister and a brother with Bloom's syndrome. Of 4,633 diploid cells, 115 showed allocyclic chromosomes, and 74 of these had 44, 45 or 46 normal metaphase chromosomes accompanied by one or two allocyclic chromosomes. Of 56 tetraploid cells, 9 contained such chromosomes. The allocyclic chromosomes appeared pulverized or extended corresponding to S or G₂ PCC. We have proposed the hypothesis that individual allocyclic chromosomes do not, as a rule, come from micronuclei, as has often been assumed, but have been left behind in their cycle. This would be caused by a mutation or deletion of a hypothetical coiling center situated near the centromere of each chromosome arm. The following observations agree with our explanation but less well or not at all with the idea of micronuclei: (1) In only 9.6% of the cells does the allocyclic chromosome lie at the edge of the metaphase plate. (2) In 24 cells a part of a chromosome is pulverized while the rest is in metaphase. (3) Both a pulverized and an extended chromosome were present in the same cell. (4) A pulverized acrocentric is often nose-to-nose with a normal D or G chromosome. (5) No allocyclic chromosomes corresponding to G₁ PCC have been found in our material. (6) When a ring is replaced by an allocyclic chromosome, it is usually a member of a 46-chromosome complement. Furthermore, the occurrence of allocyclic chromosomes is correlated with that of other chromosome anomalies which do not follow a Poisson distribution. Allocyclic chromosomes are also more frequent (16%) in tetraploid than in diploid cells (2%).     

Computerized analysis of chromosomal parameters in karyotype studies

Summary A study is presented of the possibilities and limitations of semi-automated karyotype analysis on the basis of chromosome length and centromere index. A number of computer programs have been developed for 1) quick and precise measurements of chromosome arm length with the help of a graphics tablet, 2) computing (relative) length and centromere index and statistical analyses of the data, and 3) representation of these chromosomal parameters in two-dimensional scattergrams. An ellipse representing 95% of the probability mass is drawn around the bivariate mean of each chromosome. The size and orientation of the axes are calculated from repeated measurements of the chromosomes of one metaphase plate. If there is a correlation between length and centromere index, which is often the case, the axes of the ellipse are tilted. Incorporation of such a covariance analysis proved to be of great importance for an accurate karyotype analysis. The Computer Aided Karyotyping package does not contain routines for an automated classification of the chromosomes. The main reason is that the variation in length and centromere index of a given chromosome in different cells is often much larger than the variation between nonhomologous chromosomes. In addition, it was our aim to develop universal karyotyping aids which can be used regardless of the species studied.     

Sequence of centromere separation: Analysis of mitotic chromosomes in man

Summary Mitotic chromosomes from human peripheral lymphocytes studied at the junction of metaphase and anaphase show that the centromeres of various chromosomes separate in a nonrandom, apparently genetically controlled sequence. It does not depend upon the position of the centromere in the chromosome, the length of the chromosome or total amount of detectable C-chromatin. In man, several chromosomes e.g. 18, 17, 2, separate very early. Such early cells do not include nos. 1, 13, 14, 15, and Y and very rarely nos. 21 and 22. The last separating chromosomes are those from group D, G, no. 1, 16, and Y. The possible implication of these findings in evolution, non-disjunction and the control of centromere separation sequences is discussed.This study was supported by a grant from National Institure of Health (GM 24591)     

Quantitative studies on the arrangement of human metaphase chromosomes

Summary The spatial relationships between the homologous pairs of chromosomes in the normal human colcemid-treated metaphase plate were tested by two different mathematical approaches: (a) determination of the distances between the centromeres of the homologous chromosomes compared to the mean distance of all centromeres of the mitosis in question; (b) measuring the distances of the different chromosomes from the center of the mitosis.The following results were obtained: (1) The arrangement of human metaphase chromosomes does not follow a normal distribution; the distribution is narrower and taller, probably due to an impairment of free chromosome spreading by the cell membrane. We believe that only in membraneless mitotic cells should the chromosome-spread correspond to a normal distribution under the same preparation conditions. (2) There is a positive correlation between decreasing chromosome size and decreasing mean distance between homologous chromosomes. (3) A close positive correlation exists between increasing chromosome size and increasing distance to the barycenter of the mitosis. (4) There is also a close positive correlation between the distance of homologous chromosome pairs and their distance from the center of the mitosis, i.e., with increasing distance from the center of the mitosis, the distance between the homologous partners increases. (5) The following statistically significant deviations from these rules could be established: (a) The large acrocentric chromosomes are closer associated, as one would expect from their size, probably due to their participation in the nucleolus organization; (b) in the female cell one of the two X chromosomes has an extremely peripheral localization; the X chromosomes are furthest apart of all pairs of homologous chromosomes; (c) the chromosome pairs 6 and 8 are relatively close together in spite of their peripheral position, suggesting a truc close association of the homologus partners; (d) the chromosome pair 18 has a more peripheral position than expected, and a relatively large mean distance between the homologous partners; (e) the chromosome pair 1 has a much more central position and a closer association than is expected from its size.     

Dicentric chromosomes and the inactivation of the centromere

Summary The origin and behavior of human dicentric chromosomes are reviewed. Most dicentrics between two non-homologous or two homologous chromosomes (isodicentrics), which are permanent members of a chromosome complement, probably originate from segregation of an adjacent quadriradial; such configurations are the result of a chromatid translocation between two nonhomologous chromosomes, or they represent an adjacent counterpart of a mitotic chiasma. The segregation of such a quadriradial may also give rise to a cell line monosomic for the chromosome concerned (e.g., a 45,X line). Contrary to the generally held opinion, isodicentrics rarely result from an isolocal break in two chromatids followed by rejoining of sister chromatids. In this case the daughter centromeres go to opposite poles in the next anaphase, and the resulting bridge breaks at a random point. This mechanism, therefore, leads to the formation of an isodicentric chromosome only if the two centromeres are close together, or if one centromere is immediately inactivated. Observations on the origin of dicentrics in Bloom syndrome support these conclusions. One centromere is permanently inactivated in most dicentric chromosomes, and even when the dicentric breaks into two chromosomes, the centromere is not reactivated. The appearance and behavior of the acentric X chromosomes show that their centromeres are similarly inactivated and not prematurely divided. Two Bloom syndrome lymphocytes, one with an extra chromosome 2 and the other with an extra chromosome 7, each having an inactivated centromere, show that this can also happen in monocentric autosomes.     

Nondisjunction of the X chromosomes in females ofDrosophila hydei

An investigation of nondisjunction inDrosophila hydei has disclosed that spontaneous primary nondisjunction of the X chromosomes occurs with a frequency of 1/13000, and secondary nondisjunction with a frequency of 1/3500. These rates are much lower than the ones previously reported forDrosophila melanogaster which are about 1/1000 for primary nondisjunction and 1/50 for secondary nondisjunction.The low rate of secondary nondisjunction inhydei is attributed to the much greater genetic length of the X chromosome and the corresponding reduction in noncrossover X's available for distributive pairing with the Y chromosome.The low rate of primary nondisjunction is attributed to both a reduction in noncrossover X chromosomes, and to the large heterochromatic arm of the X chromosome which, it is suggested, makes the X centromere a strong centromere. Thus, it is further suggested, the reduction in noncrossover chromosomes reduces the opportunity for nonhomologous distributive pairing and nondisjunction of the type involving noncrossover chromosomes. Nondisjunction of the type involving crossover chromosomes then is prevented by the success of the strong centromeres in overcoming entanglements that would lead to nondisjunction in the case of ordinary or weak centromeres.This investigation was supported in part by U.S. Public Health research grant GM 12093 and in part by a National Science Foundation research grant 14200.     

Arrangement of prematurely condensed chromosomes in cultured cells and lymphocytes of the Indian muntjac

Premature chromosome condensation (PCC) was induced in order to study the arrangement of muntjac chromosomes in the interphase nuclei of proliferating and resting cells with respect to their polarity and the spatial relationship between them. The data were compared with the situation in in situ fixed and colcemid blocked metaphases. It appears that in rapidly dividing cells almost all G₁- and G₂ interphase chromosomes exhibit the Rabl type polarized orientation. This pattern still predominates in G₀ lymphocytes which may have been arrested at this stage for some months or even years. — The location of the small chromosome Y₂ was found to be central in normal metaphases but peripheral in colcemid blocked mitoses. The behavior in the premature condensed chromosome preparations was intermediate. Measurements of centromere distances between all possible pairs of chromosomes as well as on the relative position of chromosomes in circular spreads revealed no evidence for homologous somatic association during interphase and metaphase or any other suprachromosomal ordering principle. Interphase chromosome orientation seems to be solely the result of chromosome arrangement of the foregoing anaphase. Association between heterochromatic regions or the nucleolus organizers did not substantially influence this pattern. There is no support for speculations that in mammalian cells close proximity of homologoues sites is instrumental in functional cooperation.     

B chromosome variation in Euphydryas colon (Lepidoptera: Nymphalidae)

Cytogenetic analysis of an Idaho population of the checkerspot butterfly, Euphydryas colon, has revealed considerable inter- and intra-individual variation in chromosome number which turns out to be a classic case of B chromosome variation. The basic chromosome complement of the species is n (, )=31. The A chromosomes were aligned equatorially at mitotic metaphase and metaphase II, and axially at metaphase I, indicating a restriction of centric activity at the first meiotic division. No failure of pairing between homologous A chromosomes was observed and, although a marked asynchrony of chromatid separation was found to be characteristic of mitotic telophase and telophase II, the frequency of macrospermatid formation was low. The B chromosomes were at least partly heterochromatic but exhibited some variation in both pycnosity and size. Mitotically stable B-containing individuals showed a preponderance of unpaired Bs at first metaphase and these divided at either first or second anaphase. The presence of Bs was associated with a heightened production of abnormal spermatids particularly in individuals with high numbers of B chromosomes. Among the 25 individuals sampled, 21 carried from 1–6 B chromosomes, and of these 14 were mitotically stable. In all 7 unstable individuals the mean number of B chromosomes per cell exceeded the modal number. Assuming that the modal number represents the zygotic number, these results suggest that a mechanism to boost the number of B chromosomes exists in males of E. colon.     

Relationship between measured chromosome distribution parameters and Ag-staining of the nucleolus organizer regions

Summary Trypsin-banded metaphase plates provided by one whole blood culture of a normal adult female were analyzed as to the chromosome distribution by measuring: (1) distances between centromeres; (2) angles formed between a centromere, the gravity center of the metaphase plane, and a second centromere; and (3) the measured tendency to associate, as defined by Galperin (1969b). These data are correlated with Ag-NOR staining findings obtained from 72 cells from another culture of the same individual. In these cells, the chromosome pairs are identified using a simultaneous Ag-NOR staining and acridine orange banding technique. The silver precipitation is also correlated with the scored satellite associations in these cells. The results show a correlation between all concerned parameters, indicating that the nucleolar function of the human acrocentric chromosomes, as demonstrated by the silver precipitation technique, is probably one of the major determinants of the proximity of these chromosomes. There is a pronounced correlation of the Ag-NOR findings with those measured parameters which describe best the preferential small distances between chromosomes (angle analysis and tendency to associated data). Moreover, the association patterns of the acrocentrics with small amounts of NOR provide some evidence for the interference of other determinants cogoverning the position of the human D-and G-group chromosomes.To whom offprint requests should be sent     

Chromosome distribution studies after inorganic lead exposure

Summary We studied the chromosome distribution in persons professionally exposed to inorganic lead. The degree of lead exposure was evaluated by biochemical measurements and cytogenetic analysis. The chromosome distribution was analyzed from trypsin banded karyotypes; in particular we studied centromere distances ² and centromere-metaphase-center distances (d²) which were obtained by computer-aided mathematical transformation of the individual metaphase coordinates.Higher concentrations of blood lead and urine -ALA and a statistically significant increase in aneuploidy, hypoploidy, and type-B chromosome aberrations revealed appreciable exposure although none of the subjects showed signs of excessive lead absorption.However study of the chromosome distribution showed no major differences with that of the controls indicating that lead acts preferentially (directly or indirectly) on the chromosomes rather than on the spindle apparatus. A dissociation of the acrocentric chromosomes was observed in the lead group when compared with the controls. This is thought to reflect a secondary action of lead on the nucleolar organiser regions.     

Telomere and centromere association tendencies in the human male metaphase complement

Summary Generalized distances between centromeres and between telomeres were statistically analyzed (x ² tests) in 100 trypsin-banded metaphase figures derived from normal males.Analysis of association tendencies on the first column of obtained c-c, p-p, q-p, and p-q histograms showed significant heterochromatin attraction not only between nonacrocentrics and acrocentrics but also between two nonacrocentric chromosome pairs (1 and 16). However since, not all c-heterochromatin-rich chromosomes were involved in associations (pair 5), and conversely, since chromosomes without an important centromeric heterochromatin block were involved in associations (pairs 8 and 11), it is probable that centromeric heterochromatin is not the only factor responsible for chromosome association. Moreover associations occur not only at the centromeres; in our circle analysis of the binding capacity of the telomeres or centromere of one chromosome pair with the telomeres or the centromeres of all other chromosome pairs, we also found preferential associations for T(4,13), T(9,15), T(11,15), T(13,19) T(15,19), T(17,18), T(17,22), and T(19,20).We therefore suggest that heterochromatin is not the only reason for chromosome association and that telomeres may also play an important part in this process.     

Assignment of the developmentally regulated gene NEDD1 to human chromosome 12q22 by fluorescence in situ hybridization

The developmentally regulated mouse gene Nedd 1 encodes a protein showing similarities with the -subunit of heterotrimeric GTP-binding proteins and has growth suppressing activity when overexpressed in various cultured cell types. We have mapped the human homolog (NEDD1) of the mouse gene to chromosome 12q22 by fluorescence in situ hybridization using R-banded human (pro)metaphase chromosomes.     

Interstitial deletions of repetitive DNA blocks in dicentric human Y chromosomes

Cytogenetic analysis of aberrant human Y chromosomes was done by fluorescence in situ hydbridization (FISH) with Y specific repetitive DNA probes. It revealed an interstitial deletion of different DNA blocks in two dicentric chromosome structures. One deletion includes the total alphoid DNA structure of one centromeric region. The second deletion includes the total repetitive DYZ5 DNA structure in the pericentromeric region of one short Y arm. Both dicentric Y chromosomes were iso(Yp) chromosomes with break and fusion point located in Yq11, the euchromatic part of the long Y arm. Their phenotypic appearance was abnormal, resembling small monocentric Yq-chromosomes in metaphase plates. Mosaic cell lines, usually included in karyotypes with dicentric Y chromosomes, were not observed. It is assumed that both deletion events suppress the kinetochore activity in one Y centromeric region and thus stabilize its dicentric structure. Local interstitial deletion events had not been described in dicentric human Y chromosomes, but are common in dicentric yeast chromosomes. This raises the question of whether deletion events in dicentric human chromosomes are rare or restricted to the Y chromosome or also represent a general possibility for stabilization of a dicentric chromosome structure in human.     

DNA replication and inactivation patterns in structural abnormality of sex chromosomes

Summary High resolution chromosome analysis and bromodeoxyuridine (BrdUrd) incorporation have been applied to study patterns of chromosomal replication (inactivation) in two cases of unbalanced X-autosome translocations, seven cases of X and Y chromosome rings or fragments, and five cases of dicentric isochromosomes (Xq). Our results indicate the following: (1) In (X-A) translocations, detailed replicational analysis of the translocated autosomal segment is informative. Absence of spreading effect and partial-incomplete spreading effect are the most common observations. (2) Sex chromosome derived fragments and rings can be differentiated based on their replicational features. (3) Dicentric isochromosomes (Xq) can be classified based on intercentromeric distances, replicational asynchrony, and centromere inactivation. (4) A correlation between intercentromeric distance and degree of 45,X mosaicism was observed in dicentric i(Xq) chromosomes.Evidence for spreading effect based on our results and on the review of the literature has been critically analyzed and general rules in evaluating spreading effects (SE) proposed. The cytologic detection of active regions on the late replicating X chromosome and the inactivation capacity of the juxtacentromeric region of Xp is evaluated. It is proposed that centromere suppression and underreplication are related phenomena. Finally, the analysis of informative replicational stages is emphasized and the application of their analysis in basic and clinical cytogenetics demonstrated.     

Synaptonemal complex formation in hybrids of Lolium temulentum x Lolium perenne (L.)

Comparisons were made between two kinds of tetraploids derived from the hybrid Lolium temulentum x L. perenne. One hybrid behaves like an autotetraploid with multivalents at first metaphase of meiosis in pollen mother cells. The other behaves like an allotetraploid, in which pairing at first metaphase is restricted to bivalents comprised of strictly homologous chromosomes. The diploidisation of the latter form is controlled by determinants located on both the normal, A chromosomes and on supernumary B chromosomes. Reconstruction of synaptonemal complexes and their elements, from serial sections through pollen mother cell nuclei examined under the electron microscope, reveals that at zygotene pairing in both forms results in multivalent formation involving non-homologous as well as homologous chromosomes. The mechanism responsible for the diploidisation is, therefore, not based on a restriction of pairing at early meiosis to homologous chromosomes but on a correction or transformation of the multivalent chromosome associations to bivalents subsequent to zygotene. The transformation is not completed until late pachytene. In the multivalent-forming tetraploid a maximum of four chromosomes are associated at first metaphase. Yet configurations of a higher valency are found at zygotene. There is, therefore, a partial transformation of multivalents even in this autotetraploid form which restricts configurations at metaphase I to homologous and homoeologous chromosomes only. In both hybrids some homologous bivalents are not the product of resolution of multivalents but result from two-by-two pairing from the beginning of zygotene.     

Centromeric association of a microchromosome in a Turner syndrome patient with a pseudodicentric Y

A 12-year-old patient with Turner syndrome was found to have a complex mosaicism for a microchromosome (MC) and a psu dic(Y)(q11). The MC was smaller than Yp, appeared pale in G, C and late replicating bands, had a pair of small centromeric dots, was associated with other chromosomes in most metaphases, and was rather stable both in size and during mitosis. The psu dic(Y) was Cd-positive only at the active centromere, had two pericentromeric heterochromatic regions, and lacked the Yq12 band. No cells with both abnormal chromosomes were found. To evaluate the association of the MC with all ordinary chromosomes, 857 G-banded cells with the marker were screened. The MC was considered as associated whenever the distance between it and other chromosome(s) was equal to, or smaller than, 18p. Out of 848 associations registered, 489 (57.7%) were centromeric, 202 (23.8%) telomeric, and 157 (18.5%) interstitial; i.e., centromeric associations were overrepresented (P < 0.001) and showed a random distribution, except for an excessive involvement of chromosome 8. This association pattern, also exhibited by two similar MCs in human beings, the minute Y of a marsupial and certain B chromosomes in plants, probably reflects the Rabl orientation of chromosomes in interphase.     

Contribution of reciprocal translocations to an understanding of chromosome displacement: Inferences for studies of spatial order at metaphase

Summary Chromosome displacement was analysed in three different translocations. Two alternative hypotheses were examined: (1) Displacement is determined by the spatial ordering of chromosomes on the metaphase plate. (2) Displacement is a function of the gross physical property of chromosome size and does not reflect ordering. Predicted numbers of displacements were calculated for each chromosome for each of the two hypotheses and these were compared with the observed numbers of displacements using a ² analysis. In the analysis for two of the three translocations, the first hypothesis was rejected whilst the alternative hypothesis was supported by all three analyses. It is concluded that chromosome displacement is a function of chromosome size and does not reflect spatial ordering at metaphase. Furthermore, it is suggested that many studies of apparent ordering at metaphase may merely reflect chromosome displacement.The analysis of displacement rates in all other chromosomes of the complement was undertaken in one of the translocation carriers. This showed no alteration of relative displacement rates.     

Somatic association of chromosomes in diploid and hexaploid Avena

The frequency distributions of certain homologous and non-homologous chromosomes in somatic cells of diploid and liexaploid species of Avena were studied and compared with the theoretical random distribution.All homologous chromosomes studied irrespective of shape and size showed non-random distribution in both diploid and hexaploid species. In all cases the homologous chromosomes were closer than would be expected with random distribution. Chromosomal characters such as size, shape, and presence or absence of nucleolar organizing regions did not exert appreciable influence on the somatic association of homologues.While the non-homologues followed the theoretical random distribution in diploid species, a significant deviation from the random curve was noted for non-homologues in hexaploid species. However, diploid and hexaploid non-homologous chromosomes had characteristic S-shaped cumulative frequency distributions which were distinct from the half-moon-shaped ones obtained for homologous chromosomes.The different regions (short arm, long arm, centromere and mid-points) of two pairs of homologous chromosomes (one of them being nucleolar) studied showed non-random distribution with the exception of the long arm of the non-nucleolar chromosome. From these results the role of the centromere or mutual attraction of homologous segments could not be assessed with certainty.Contribution No. 232 from the Research Station, Central Experimental Farm, Ottawa, Ontario, Canada.     

The location of DNA homologous to human satellite III DNA in the chromosomes of chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orang utan (Pongo pygmaeus)

Radioactive RNA with sequences complementary to human DNA satellite III was hybridised in situ to metaphase chromosomes of the chimpanzee (Pan troglodytes), the gorilla (Gorilla gorilla) and the orangutan (Pongo pygmaeus). A quantitative analysis of the radioactivity, and hence of the chromosomal distribution of human DNA satellite III equivalent sequences in the great apes, was undertaken, and the results compared with interspecies chromosome homologies based upon Giemsa banding patterns. In some instances DNA with sequence homology to human satellite III is present on the equivalent (homologous) chromosomes in identical positions in two or more species although quantitative differences are observed. In other cases there appears to be no correspondence between satellite DNA location and chromosome homology determined by banding patterns. These results differ from those found for most transcribed DNA sequences where the same sequence is located on homologous chromosomes in each species.     

Patterns of association in the human metaphase complement: Ring analysis and estimation of associativity of specific chromosome regions

Summary The pattern of metaphase chromosome association in the human complement was studied by two methods of statistical analysis of interchromosomal distances. Those methods included ring analysis in which a characteristic position of the centromere of each chromosome relative to the center of a two-dimensional representation of a metaphase complement was defined, and estimation of the capacity for associativity of each of three regions of each chromosome: the centromere (c) and the ends of each arm (p,q).The following information was obtained: 1. In general, the distance from the center is directly related to chromosome size. 2. The most notable deviation from that size-related progression is displayed by the X chromosomes. The markedly peripheral position of the X is characteristic of both X's of the female and the single X of the male. 3. The relative associativity of each chromosome of the complement is, in general, inversely related to size with an additional preferential capacity of associativity displayed by the acrocentric chromosomes. Analyses of the different inter-regional classes established that the supplementary associativity factor of the acrocentric chromosomes was inherent in their pericentromeric and p-arm regions and excluded the ends of the q arms from participation in that factor. 4. Those analyses demonstrated that the specific morphology or geometry of the acrocentric chromosomes contributes little to their high relative associativity. In addition to the tendency for the c/p regions of the acrocentric chromosomes to associate with each other, presumably because of their common function in nucleolar organization, those regions also displayed a propensity to associate with the distal regions of the arms of other chromosomes. A molecular basis for that propensity other than that of ribosomal DNA is postulated to be that of other fractions of highly reiterated DNA sequences. 5. Analysis of the relative associativities of each of the three regions of the Y chromosome revealed that the Yq displays a much stronger capacity to associate with the c's of other chromosomes than does the Yc or Yp.