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%).     

Premature centromere division of a translocation-carrier autosome

Summary Premature centromere division of an aberrant chromosome 3 was found in a newborn girl with fetal hydrops and in her healthy father, both bearing a familial balanced 3p;19q translocation. The out-of-phase separation of a structurally abnormal chromosome may be of pathogenetic significance, the nature of which is not yet understood.     

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.     

Chromosomal unit fibers in Drosophila

Chromosomal unit fibers consisting of long, regular fibers of about 0.40 m diameter were obtained from disintegrated, isolated chromosomes of two Drosophila melanogaster cell lines. In one cell line with an essentially normal karyotype, three clearly defined size classes of 15, 13, and 11 m length were observed corresponding to the three larger chromosomes of Drosophila. In a cell line carrying an additional translocation between the two largest chromosomes a 19 m fiber derived from the translocation chromosome was observed. Direct determinations of the DNA content per m length of Drosophila unit fibers show that DNA is contracted by a factor of about 1400x in agreement with calculations based on the length of the unit fibers and the known DNA content of the individual Drosophila chromosomes. These findings support our previously proposed model for the unit fiber sub-structure of chromosomes as being derived by a hierarchy of coiling with the corresponding contraction ratios being 7 (100 Å string of nucleosomes), 5 to 6 (250–300 Å thick nucleohistone fiber), and about 40 (unit fiber), resulting in a total contraction of DNA in unit fibers in the order of 1400x.     

A genetic and molecular analysis of P-induced mutations at the glucose-6-phosphate dehydrogenase locus in Drosophila melanogaster

Summary We examined P factor induced mutations of the Zw gene of Drosophila melanogaster in order to learn more about the site specificity of such mutations. Approximately 70000 chromosomes were screened using a powerful positive selection scheme. As only two mutants were discovered, Zw is a cold spot for transposable element insertion. One mutation involved a complex P element associated chromosomal rearrangement which was used to define the orientation of the gene with respect to the centromere of the X chromosome. The second mutation was either a simple, non-dysgenically induced point mutation or a very unstable insertion.     

X heterochromatin subdivision and cytogenetic analysis in Sciara coprophila (diptera,sciaridae)

The so-called controlling element (CE), which normally programs the curious behavior of the sex chromosome in this genus, has been localized in the short right arm of the polytene X in S. coprophila. The localization was accomplished by use of five X-autosome translocations whose break points define three blocks of heterochromatin (heterochromomeres) extending from the X centromere to the very end (right) of the chromosome. The behavior of the translocation chromosomes at the crucial second spermatocyte division was examined and the precocious chromosome identified in all five cases. Then, knowing the heterochromomere make-up of each chromosome, the position of the CE could be mapped; it is located in heterochromomere H2, the same block of heterochromatin that contains 50% of the ribosomal RNA cistrons. — The question of whether the CE can manipulate any centromere in the nucleus has been only partially answered. It can manipulate translocation chromosomes which possess the centromere of the metacentric autosome (salivary chromosome IV) or that of the shorter rod (salivary chromosome II); but the longer rod (salivary chromosome III) whose proximal end, as seen in the polytene nucleus, is heavily laden with heterochromatin of its own, has not been brought under CE control. — In one of the translocations, T23, the precocious chromosome is a very large metacentric chromosome which resembles the peculiar V-shaped X of S. pauciseta. This peculiarity is not observed in the J-shaped precocious chromosome of T29. These points are discussed.Dedicated to Professor Hans Bauer on the occasion of his 75th birthday.     

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     

Identification of a 1B/1R wheat-rye chromosome translocation

Summary The common wheat selection 79-4045 was identified as a wheat-rye 1B/1R chromosome translocation line, by means of C-banding patterns and test cross with Chinese Spring double-ditelosomic line. The translocation chromosome consisted of the long arm of wheat chromosome 1B, including its centromere, and the short arm of rye chromosome 1R or tis portion.     

Cytological and molecular characterization of a chromosome interchange and addition lines in Cadet involving chromosome 5B of wheat and 6Ag of Lophopyrum ponticum

Efforts to transfer wheat curl mite (Eriophyes tulipae Keifer) resistance from Lophopyrum ponticum 10X (Podb.) Love to bread wheat (Triticum aestivum L.) have resulted in the production of a number of cytogenetic stocks, including an addition line of 6Ag, a ditelo addition line, and a wheat-Lophopyrum translocation line. Characterization of these lines with C-banding, in situ hybridization with a Lophopyrum species-specific repetitive DNA probe (pLeUCD2), and Southern blotting with pLeUCD2 and a 5S ribosomal DNA probe (pScT7) confirmed that the distal portion of the short arm of 6Ag was translocated onto the distal portion of 5BS (5BL. 5BS-6AgS). It was also determined that the ditelo addition was an acrocentric chromosome of 6AgS.     

Unequal X Chromosomes in Bradysia Hygida (Diptera:Sciaridae) Females: Karyotype Assembly and Morphometric Analysis

The mitotic chromosomes of Bradysia hygida(Diptera:Sciaridae) neuroblast cells are described together with their morphometric data. Giemsa-stained neuroblast chromosomes from female and male larvae confirm the chromosome number of this species, 2n=8 (XX) and 2n=7 (XO), respectively. The karyotype assembly reveals two metacentric autosomic pairs, the A and B chromosome; a subtelocentric, the C chromosome, the smallest one; and a sexual unequal metacentric pair, X chromosome, in female karyotype and a one sexual metacentric X chromosome in male. The implications of the unequal X chromosome pair are discussed.     

Rearrangements between irradiated chromosomes in three-species radiation hybrid cell lines revealed by two-color in situ hybridization

A human-hamster hybrid cell line containing only the human X chromosome (GM06318B) was exposed to 6,000–7,000 rad of X-rays and fused with a mouse cell line (CL1D,TK^-). Three radiation hybrids, LXKC40, LXKC50, and LXKC56, were selected among 39 independent clones containing human material. Two-color in situ hybridization with total genomic DNA probes (cotl human DNA and hamster total genomic DNA) was used to analyse the irradiated chromosome rearrangements. With this three-species model system (human-hamster-mouse) and the chromosome painting process it was possible to determine the origin of each chromosomal fragment in metaphase and interphase. The results obtained indicate preferential rearrangement between irradiated human and hamster chromosomes. Whole, apparently intact hamster chromosomes were observed in all the mitoses. We suggest that these chromosomes could be neoformated from random fragments after irradiation. Hamster and human minichromosomes were also detected. While the integration of human material into the mouse genome was exceptional, the integration of hamster material into mouse chromosomes was more frequent. During interphase the irradiated chromosome domains were often at the periphery of the nucleus. Irradiated material protruded at the periphery of the nuclei. Micronuclei containing hamster material were detected in the vicinity of these protrusions.     

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.     

Molecular cytogenetic analysis of Agropyron elongatum chromatin in wheat germplasm specifying resistance to wheat streak mosaic virus

Summary Three lines derived from wheat (6x) x Agropyron elongatum (10x) that are resistant to wheat streak mosaic virus (WSMV) were analyzed by chromosome pairing, banding, and in situ hybridization. Line CI15321 was identified as a disomic substitution line where wheat chromosome 1D is replaced by Ag. elongatum chromosome 1Ae-1. Line 87-94-1 is a wheat-Ag. elongatum ditelosomic addition 1Ae-1L. Line CI15322 contains an Ag. elongatum chromosome, 1Ae-2, that substitutes for chromosome 1D. The short arm of 1Ae-2 paired with the short arm of 1Ae-1 at metaphase I (MI) in 82% of the pollen mother cells (PMCs). However, the long arms of these two chromosomes did not pair with each other. In CI15322, the long arm of chromosome 4D has an Agropyron chromosome segment which was derived from the distal part of 1Ae-1L. This translocation chromosome is designated as T4DS·4DL-1L. T4DS·4DL-1Ae-1L has a 0.73 m distal part of the long arm of 4D replaced by a 1.31 m distal segment from 1Ae-1L. The major WSMV resistance gene(s) in these lines is located on the distal part of 1Ae-1L.Contribution No. 92-599-J from the Kansas Agricutural Experiment Station, Kansas State University, Manhattan, Kansas, USA     

Human lens γ-crystallin sequences are located in the p12-qter region of chromosome 2

Summary The human -crystallin genes constitute a multigene family whose members are only expressed in the eye lens. The chromosomal location of these sequences has been determined by screening a panel of human/rodent hybrid cell lines containing overlapping subsets of human chromosomes for the presence of human -crystallin sequences. By correlating these genomic hybridization data with the chromosomal constitution of the somatic cell hybrids, all human -crystallin sequences could be assigned to chromosome 2. The use of human/hamster cell hybrids derived from human Burkitt lymphoma cells carrying a reciprocal translocation between human chromosomes 2 and 8, allowed a further localization of the sequences to the region 2p12-qter.     

A comparison of male and female recombination frequency in wheat using RFLP maps of homoeologous group 6 and 7 chromosomes

A novel approach was used to compare male and female recombination rates in wheat. Doubled haploid lines were developed from an F₁ using two distinct approaches: the anther-culture technique and the Hordeum bulbosum system, from which sets of lines were developed from male and female meioses, respectively. The genotype of the lines was established at RFLP and isozyme markers polymorphic on chromosomes of homoeologous groups 6 and 7, and male and female linkage maps were calculated using this information. The markers in one segment of chromosome 6B exhibited disturbed segregation frequencies in the anther-culture population. The male and female maps differed significantly in recombination frequency between some markers on two chromosomes, and these were consistent in direction within chromosomes and inconsistent in direction between chromosomes. In two of the four chromosomes studied the male map was much longer than the female map. These results suggest that significant differences may exist in male and female recombination frequencies in bread wheat which are specific to certain chromosomal segments but are inconsistent in direction between chromosomes. Other factors, such as environmental influences, may also be important in creating differences.     

A possible mosaic form of delayed centromere separation and aneuploidy

Summary On average, a normal centromere separation sequence was seen in 3 neonates with trisomy 18 and in their parents. When evaluating individual mitoses, unusually late separating chromosomes 18 were found in a few cells of one of the parents in each family. A possible germline mosaicism of delayed separation in the parent may account for trisomy in the offspring.     

Structural abnormalities in chromosome 15 in cell lines with reduced expression of beta-2 microglobulin

Using somatic cell hybrids, the gene for beta-2 microglobulin has been assigned to human chromosome 15. We found it of interest to study a number of human lymphoid cell lines in light of this finding, to analyze whether spontaneously occurring loss or reduction of beta-2 microglobulin could be correlated with any aberration in chromosome 15. The Daudi cell line was shown to be devoid of any beta-2 microglobulin in total extracts. Chromosome analysis showed that one of the two chromosomes 15 was deleted in the region q14q21 on the long arm; in some metaphases, both chromosomes were deleted in this region. The K562 cell line was found to express very low (if any) membrane-associated beta-2 microglobulin. Chromosome analysis showed that this line was near-triploid, with two normal chromosomes 15 and one translocation chromosome t(15;18) involving the long arm of chromosome 15, whereby the segment proximal to the breakage point in band q15 was lost. The Namalwa cell line showed a reduction in membrane-associated and total beta-2 microglobulin. Chromosome analysis showed this line to contain one chromosome 15 which was shorter than its normal homolog. The deletion could be identified as such in the region q14q21 in Daudi cells, but is probably somewhat smaller than the one in Daudi cells. Since analyses of beta-2 microglobulin production and chromosomes 15 on several other human cells failed to reveal any abnormalities in either of these respects, we postulate that genes responsible for beta-2 microglobulin synthesis and membrane expression could be located in the region q14q21 on the long arm of chromosome 15. Since beta-2 microglobulin associated with the membrane was found to be absent in the K562 line, where total beta-2 microglobulin was nearly as high as in cell lines with normal membrane expression, it is suggested that membrane expression of beta-2 microglobulin can be regulated independentlyAbbreviations used in this paper are BSS Earle's balanced salt solution - Q banding bands obtained by fluorescence with Quinacrine Mustard - R banding bands obtained by fluorescence with Acridine Orange - FITC fluorescein isothiocyanate - EBV Epstein-Barr virus     

Morphological and chromosomal characterization of three new continuous cell lines of Drosophila melanogaster

Three continuous cell lines (GM₁, GM₂ and GM₃) were obtained from embryos of Drosophila melanogaster. Karyotypic analysis revealed characteristics distinguishing each line. Except for some minor variations GM₁ cells had an X and a centric heterochromatic fragment (which is a portion of the Y). GM₂ line was characterized by XO cells showing two new telocentric chromosomes while an autosome of the II pair was missing. GM₃ cells were XY; the Y chromosome, however, was shorter than the normal, having a deletion of the terminal section of the short arm. Several problems concerning the origin of these different genomes are discussed.This work was supported by a grant of the Consiglio Nazionale delle Ricerche, Roma.     

Evolution of karyotypic abnormalities and C-MYC oncogene amplification in human colonic carcinoma cell lines

Cell lines (COLO 320 DM and COLO 320 HSR), established from a human neuroendocrine tumor, contain an amplified cellular oncogene (c-myc). We have previously shown that the homogeneously staining regions (HSRs) of a marker chromosome in the COLO 320 HSR cells that evolved in culture from COLO 320 DM cells contain amplified c-myc. Molecular hybridization in situ has now been used to demonstrate that the HSRs are on both arms of what was once an X chromosome. We also show that amplified c-myc copies are present in the isolated double minute chromosomes (DMs) of the COLO 320 DM cells that were characteristic of the tumor cells initially established from the patient. The results suggest that the amplified c-myc appeared first as DMs and was subsequently transposed to engender HSRs on an X chromosome. The initial COLO 320 tumor cell may have acquired two early replicating (i.e., active) X chromosomes and lost the late replicating (i.e., inactive) X.     

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)