An improved procedure for the cultivation and in situ chromosome preparation of monolayer cell cultures

A method for the cultivation of monolayer cell cultures on microslides in quadruple culture dishes together with a simple procedure for in situ chromosome preparation are described. The cells fixed to the slide can be stained according to standard procedures and analysed microscopically. The method is simple, rapid and reliable and provides many advantages especially for cytogenetic diagnostics with fibroblasts and amniotic fluid cells. It simplifies the performance of cytogenetic mutagenicity testing with primary cultures and permanent cell lines, e.g. the analysis of chromosome aberrations, sister chromatid exchanges (SCEs) and induced aneuploidy, as well as large-scale cytogenetic experiments.     

Cytogenetics of in vitro cultured somatic cells and regenerated plants of barley (Hordeum vulgare L.)

Summary Cytogenetic analysis of immature embryoderived calli and regenerated plants of barley has demonstrated high heterogeneity of callus cultures and significant differences in cytogenetic processes between different callus lines. Regenerated plants usually have a normal chromosome complement (2n=14). Tetraploid plaints occur with a frequency of 1%. No chromosome aberrations have been detected by Feulgen staining. The phenomenon of chromosome stickiness recorded from the 2nd day of culture was discovered in a majority of callus lines as well as the phenomena of chromatin hypercondensation and chromosome supercoiling. A possible contribution of cytogenetic and molecular processes to somaclonal variation is discussed.     

Detection and chromosomal assignment of SV40-DNA integration in Chinese hamster cell lines by chromosome sorting and dot blot hybridization

A combination of cytometric (chromosome sorting), molecular (dot blot hybridization using radio-active and/or biotinylated DNA probes) and cytogenetic (G-banding) evaluation is described which allows the rapid identification of single copy and repetitive viral integrates and their assignment to chromosome groups or even individual chromosomes. In the case of Chinese hamster cell line CO 631 it could be demonstrated that SV40 DNA was solely integrated into a submetacentric marker chromosome. Such a cytometric/molecular/cytogenetic "identogram" may prove to be a useful tool in many areas of cell and tumor biology. Furthermore, amounts of chromosomes sufficient for analysis as well as subsequent cloning experiments can be accumulated.     

Recombination is suppressed over a large region of the rainbow trout Y chromosome

The previous genetic mapping data have suggested that most of the rainbow trout sex chromosome pair is pseudoautosomal, with very small X-specific and Y-specific regions. We have prepared an updated genetic and cytogenetic map of the male rainbow trout sex linkage group. Selected sex-linked markers spanning the X chromosome of the female genetic map have been mapped cytogenetically in normal males and genetically in crosses between the OSU female clonal line and four different male clonal lines as well as in outcrosses involving outbred OSU and hybrids between the OSU line and the male clonal lines. The cytogenetic maps of the X and Y chromosomes were very similar to the female genetic map for the X chromosome. Five markers on the male maps are genetically very close to the sex determination locus ( SEX ), but more widely spaced on the female genetic map and on the cytogenetic map, indicating a large region of suppressed recombination on the Y chromosome surrounding the SEX locus. The male map is greatly extended at the telomere. A BAC clone containing the SCAR (sequence characterized amplified region) Omy - 163 marker, which maps close to SEX , was subjected to shotgun sequencing. Two carbonyl reductase genes and a gene homologous to the vertebrate skeletal ryanodine receptor were identified. Carbonyl reductase is a key enzyme involved in production of trout ovarian maturation hormone. This brings the number of type I genes mapped to the sex chromosome to six and has allowed us to identify a region on zebrafish chromosome 10 and medaka chromosome 13 which may be homologous to the distal portion of the long arm of the rainbow trout Y chromosome.     

Cytogenetic anomalies and causes for their occurrence in somatic cells

Different types of cytogenetic anomalies used in classical cytogenetics for estimating the level of damage to the chromosome apparatus are considered. Possible causes for the occurrence of different types of cytogenetic anomalies as well as the range of methods of micronucleus testing are discussed. It is shown that different levels of organization of genetic material (nucleotide, chromosome, or suprachromosome material) have an effect on processes involved in realization of a defect in the nucleotide sequence into a cytogenetic anomaly.     

A transgenomic cytogenetic sorghum (Sorghum propinquum) bacterial artificial chromosome fluorescence in situ hybridization map of maize (Zea mays L.) pachytene chromosome 9, evidence for regions of genome hyperexpansion

A cytogenetic FISH map of maize pachytene-stage chromosome 9 was produced with 32 maize marker-selected sorghum BACs as probes. The genetically mapped markers used are distributed along the linkage maps at an average spacing of 5 cM. Each locus was mapped by means of multicolor direct FISH with a fluorescently labeled probe mix containing a whole-chromosome paint, a single sorghum BAC clone, and the centromeric sequence, CentC. A maize-chromosome-addition line of oat was used for bright unambiguous identification of the maize 9 fiber within pachytene chromosome spreads. The locations of the sorghum BAC-FISH signals were determined, and each new cytogenetic locus was assigned a centiMcClintock position on the short (9S) or long (9L) arm. Nearly all of the markers appeared in the same order on linkage and cytogenetic maps but at different relative positions on the two. The CentC FISH signal was localized between cdo17 (at 9L.03) and tda66 (at 9S.03). Several regions of genome hyperexpansion on maize chromosome 9 were found by comparative analysis of relative marker spacing in maize and sorghum. This transgenomic cytogenetic FISH map creates anchors between various maps of maize and sorghum and creates additional tools and information for understanding the structure and evolution of the maize genome.     

Characterization of a (Y;4) translocation by DNA hybridization

Summary A phenotypically normal male with azoospermia was found to have a translocation between the short arm of the Y chromosome and the distal long arm of a chromosome 4. By cytogenetic analysis it could not be determined whether the translocation was reciprocal, nor whether it was balanced. In situ DNA hybridization with two pseudoautosomal and one Y-specific probe demonstrated that the breakpoint was on distal Yp and that there was Y chromosome material on 4q. Thus the translocation was reciprocal and could be characterized as t(Y;4)(pll;q32). There was no evidence for loss of Y-DNA sequences as judged by Southern blotting with Y-DNA probes. Thus the translocation may be balanced. We conclude that DNA hybridization can be used to refine considerably the cytogenetic analysis of such translocations.     

Uniparental disomy as a mechanism for human genetic disease.

A female with cystic fibrosis and short stature was investigated for molecular or cytogenetic abnormalities that might explain the combined phenotype. Analysis with polymorphic DNA markers indicated that the father did not contribute alleles to the propositus for markers near the CF locus or for centromeric markers on chromosome 7. High-resolution cytogenetic analysis was normal, and the result could not be explained on the basis of nonpaternity or a submicroscopic deletion. All of the data indicate that the propositus inherited two identical copies of maternal sequences for much or all of chromosome 7. The occurrence of uniparental disomy could be explained by models postulating postfertilization error, gamete complementation, monosomic conception with subsequent chromosome gain, or trisomic conception followed by chromosome loss. Uniparental disomy in an individual with a normal chromosome analysis is a novel mechanism for the occurrence of human genetic disease.     

Tools and methodologies for cytogenetic studies of plant chromosomes

A brief overview is presented in advances in cytogenetic methodology and development of aneuploid stocks since the 1920s. The methodologies range from first reports of chromosome numbers of major organisms, the development of chromosome karyotypes, then aneuploid stocks in the major crop plants. Molecular inputs included chromosome banding techniques, molecular marker maps and in situ hybridization methodologies. All of the new techniques greatly increased the degree of resolution obtained from cytogenetic studies.     

Molecular cytogenetic characterization of a case of primary amenorrhea with intrachromosomal triplication of the X chromosome q arm

This is a unique case of intrachromosomal triplication of the X chromosome q arm detected with cytogenetic and spectral karyotyping in a 21-year-old woman with primary amenorrhea, who had been referred because of primary hypergonadotropic hypogonadism and Mullerian hypoplasia. Intrachromosomal triplications are rare rearrangements resulting in partial tetrasomy. Since 1993, at least 34 cases of intrachromosomal triplications involving 9 different chromosomes have been reported. The vast majority of the reported triplications are on the 15th chromosome, arised de novo and had middle inverted repetitions. In this report the genotype-fenotype correlation in a case of primary amenorrhea associated with triplication of the X chromosome q arm and the possible mechanisms of this rearrangement are discussed. Further the clinical usability of SKY analysis as a molecular cytogenetic tool in searching for genomic instability arising from cytogenetic rearrangements is highlighted.     

In vitro growth and chromosome constitution of placental cells. II. Hydatidiform moles

Histopathological features, in vitro growth, and cytogenetic characteristics of tissue samples from molar placentae were studied. Tissue from complete moles is often degenerate, making them more difficult to establish in culture. However, if viable stromal cells are present, complete moles can be cultured as easily as other placental tissue. Longitudinal cytogenetic studies of molar cultures showed the emergence of clonal chromosome abnormalities to be a common feature in both complete and partial moles. The distribution of chromosome abnormalities among these clones was nonrandom, with a high proportion having an additional chromosome 20; among the complete moles, 9 of the 14 clones studied were trisomic for chromosome 20.     

Tools and methodologies for cytogenetic studies of plant chromosomes

A brief overview is presented in advances in cytogenetic methodology and the development of aneuploid stocks since the 1920s. The methodologies range from first reports of chromosome numbers of major organisms, the development of chromosome karyotypes, then aneuploid stocks in major crop plants. Molecular inputs include chromosome banding techniques, molecular marker maps, and in situ hybridization methodologies. All of the new techniques have greatly increased the degree of resolution obtained from cytogenetic studies. The text was submitted by the authors in English.     

Effect of a viral infection on the cellular chromosome apparatus of mice in vitro and in vivo against a background of hydrocortisone action]

A cytogenetic investigation of murine bone marrow after hydrocortison injection has been made. High doses of hormone (50 mg/kg) provoke deteriorations in bone marrow both in the structure and in the chromosome number. A dose of 5 mg/kg has no such effect. The Koksak A13 virus does not induce cytogenetic deteriorations in mice, however, it is able to produce a big mutagenic effect on the hydrocortison background. The vaccine strain of measles virus -- Leningrad-16 -- also increases its mutagenic action on the bone marrow cell chromsome apparatus of mice affected with hydrocortison. At the same time, in the cell culture of murine kidney, hydrocortison does not induce chromosome deteriorations and even lowers the frequency of cells with deteriorations in the chromosome set during the initial days after injecting the virus culture with measles virus.     

Chromosome anomalies in human azoospermia

The anomalies of genome were found as a result of cytogenetic study of three azoospermic men. In two cases, the circular Y chromosome was revealed. Different methods of chromosome staining demonstrated complete loss of heterochromatic portion of the long arm of the Y chromosome in one case, and the absence of the euchromatic region in another. A balanced translocation among the chromosomes 1 and 15 was observed in the third case. A question concerning disturbances of spermatogenesis having chromosomal etiology is discussed.     

Cytogenetic alterations in field workers routinely exposed to pesticides in Bogota flowers farms

Frequency of cytogenetic alterations (micronuclei and chromosome aberrations), DNA repair deficiencies and acetylcholinesterase activity was determined for field workers in Bogotá, Colombia. These workers were regularly exposed to organophosphate and carbamate insecticides while employed on farms for flower growing. Interviews were conducted with 31 workers associated with occupational risk of pesticides exposure and 30 without exposure. A standard cytogenetic assay was used to determine chromosome aberrations and micronuclei frequencies. In addition, a challenge assay assessed response to gamma-rays as an indication of DNA repair deficiencies--cells were exposed to gamma-rays in vitro and the frequencies of chromosome aberrations in post-irradiation metaphase cells were quantified. The data were evaluated for percentage of aberrant cells, cells with chromosome aberrations and frequencies of chromatid breaks per 100 metaphase cells in each worker. The exposed group had a significantly higher frequency of cells with chromosome aberrations and micronuclei as compared with the non-exposed group (p = 0.02). However, the challenge assay did not indicate a significant difference (p > 0.1). These findings require confirmation by further analytical studies involving larger sample. Cytogenetic and toxicological studies, in conjunction with thorough clinical examination are recommended.     

Preleukemic syndromes.

Acute nonlymphocytic leukemia (ANLL) is preceded by a hematologic illness representing the "preclinical" stages of the disease in many patients. This "preclinical stage" or preleukemic stage is difficult to recognize by conventional hematologic morphologic techniques. A prospective study was carried out to determine whether cytogenetic studies would be helpful in the recognition of preleukemic states and whether the presence of cytogenetic abnormalities would have prognostic significance. A study of 284 patients with suspected preleukemia has yielded 62 patients with progression to overt ANLL. Cytogenetic abnormalities were found in 30% of suspected preleukemic patients, whereas 53% of the patients progressing to acute leukemia had cytogenetic abnormalities. These studies show that the presence of cytogenetic abnormalities aid in the recognition of preleukemia but are not specific for early leukemia. Patients with cytogenetic abnormalities are more likely to develop overt ANLL. Banded chromosome studies demonstrated cytogenetic abnormalities in the preleukemic phase in 13 of 26 patients. A variety of clonal chromosomal abnormalities were observed.     

Phenotypic correlations in a patient with ring chromosome 22

Ring chromosome 22, a rare cytogenetic anomaly, has been described in over 60 cases in the medical literature. The aim of this report was to present a case carrying ring chromosome 22, and her family.It is a case report of a patient presented at Medical Faculty of ?ukurova University in Turkey.An 8-year-old girl with ring chromosome 22 and her family were evaluated cytogenetically and clinically.A chromosome analysis of the proband revealed a de novo 46, XX, r(22)(p11.2;q13) karyotype. Our subject demonstrated the prominent features of this syndrome including profound mental retardation, language impairment, dysmorphic features, lack of speech, hyperactivity, and behavioral disorders.There is lack of consistency between the physical abnormalities that we observed in our subject and those observed for such patients in the literature. The wide range of manifestations observed in patients with this cytogenetic alteration is probably due to size differences in the deleted region.     

Immunogold labeling of metaphase cells

A method is presented for the phenotypic identification of metaphase cells stained for chromosome aberration and SCE analysis. The cells are labeled in suspension with antibodies conjugated with colloidal gold, and then chromosome preparations are made using a cytocentrifuge. A silver development (IGSS) procedure is used to enhance the gold labeling for light microscopy. A variety of fixatives may be employed, permitting various cytogenetic and cytochemical staining procedures to be used.     

Nondisjunction of chromosome 21: comparisons of cytogenetic and molecular studies of the meiotic stage and parent of origin.

In the present report, we summarize studies aimed at examining the reliability of chromosome heteromorphisms in analyses of chromosome 21 nondisjunction. We used two cytogenetic approaches--fluorescent in situ hybridization (FISH) to repetitive sequences on 21p and traditional Q-banding--to distinguish chromosome 21 homologues and then compared the results of these studies with those obtained by DNA markers. Using a conservative scoring system for Q-banding and FISH heteromorphisms, we were able to specify the parental origin of trisomy in 10% of cases; in contrast, DNA marker studies were informative for parental origin in almost all cases. The results of the molecular and cytogenetic studies of parental origin concurred in all cases in which assignments were made independently using both techniques. However, in 4 of 13 cases in which the molecular studies contributed to the interpretation of the cytogenetic findings, the two results did not agree with respect to the meiotic stage of nondisjunction. A relatively high frequency of crossing-over on either the short arm or proximal long arm of chromosome 21 could explain these results and may be a mechanism leading to nondisjunction.