Determination of the DNA content of human chromosomes by flow cytometry

The mean relative DNA content of each human chromosome was calculated from flow karyotypes of ethidium bromide-stained chromosomes obtained from healthy, normal individuals. These values were found to correlate closely with previously published data obtained by photometric scanning of stained, fixed chromosomes. Calculations of the normal variation in DNA content of each human chromosome indicated that chromosomes 1, 9, 16, and Y (chromosomes with large centric heterochromatic regions) were the most variable, followed by the acrocentrics, 13, 14, 15, 21, and 22. Chromosomes 2, 3, 18, and 19 were also found to vary significantly in DNA content. Chromosomes from a number of subjects with extreme heteromorphisms were flow karyotyped to obtain an estimate of the extent of variation in DNA content of each chromosome. The greatest difference between extreme variants was found for chromosome 1 (which differed by 0.82% of the total genomic DNA), followed by 16 and 9. The largest Y-chromosome variant was 85.9% bigger than the smallest. The precise karyotype analysis produced by flow cytometry resolved many differences between chromosome homologs, including some that cannot be readily distinguished cytogenetically. The implications of these findings for detection of chromosome abnormalities by flow karyotype analysis are discussed.     

Chromosome heteromorphisms in the gorilla karyotype. Analyses with distamycin A/DAPI, quinacrine and 5-azacytidine

The chromosomes of one male and three female gorillas were extensively studied with various regional banding methods. The chromosomes were stained with the fluorescent dyes quinacrine mustard and distamycin A/DAPI (DA/DAPI), which label different subsets of heterochromatin in the chromosome complement. Furthermore, lymphocyte cultures were treated with the cytidine analog 5-azacytidine (5-azaC). The 5-azaC-induced undercondensations were found in most of the DA/DAPI-bands as well as in many telomeric C-bands. The karyotype of the gorilla exhibits a considerable number of heterochromatin variants. Of the different types of heteromorphisms noted, the most striking is that involving the short arm regions of chromosomes 12 to 16 and 23 (satellite stalk regions) and the paracentromeric heterochromatin of chromosomes 17 and 18. There also are numerous heteromorphic C-bands localized in the telomeric regions of homologous chromosome arms. In comparison, only few heteromorphisms occur between C-bands in the centromeric and pericentromeric regions of homologs. Finally, a variability in the fluorescence intensity of quinacrine-bright satellites in the short arms of chromosomes 12 to 16, 22, and 23 is observed.     

Karyotyping and identification of human chromosome polymorphisms by single fluorochrome flow cytometry

Summary Frequency distributions of fluorescence intensity of ethidium bromide stained human chromosomes from nine phenotypically normal males are cross correlated and autocorrelated following repeated flow cytometric measurements. It is shown that each individual donor produces a fluorescence profile which is both visually and numerically different from those of other individuals in the set. The wide variety of chromosome heteromorphisms which occur to varying degrees for chromosomes 1, 9, 13, 14, 15, 16, 21, 22 and Y give rise to the uniqueness of a given fluorescence profile. Estimates of chromosome heteromorphisms for each individual in the set were made and then compared with parallel results obtained from inspection of Q-banded and C-banded conventional metaphase preparations. Fluorescence profiles identifiable with each individual were also obtained for Hoechst 33258 stained chromosomes.     

Co-occurrences of polymorphic heterochromatin regions of chromosomes and effect on reproductive failure

Although the polymorphic heterochromatin regions of chromosomes (heteromorphisms) have been extensively studied for their phenotypic effects on humans, co-occurrences of chromosome 1, 9, 16 and Y heteromorphisms and of acrocentric variants have never been studied on humans with an objective scoring system. Here we compared the frequencies of individual heteromorphisms on a total of 602, 768 and 224 patients with the indications of infertility, recurrent miscarriage and in vitro fertilization (IVF) failure, respectively and on 272 controls. Then we examined whether there were significant co-occurrences between heteromorphisms within and between the groups. There were no statistically significant differences in the frequencies of heteromorphisms between the groups. Both statistically significant and non-significant correlations were observed within the non-acrocentric and certain acrocentric heteromorphisms in each group. When these co-occurrences were examined between the groups, a 2.2 fold increased risk of IVF failure in males in the presence of either chromosome 13 or chromosome 21 variants was observed (95 %CI:1.1–4.2). We conclude that the simultaneous manifestations of heteromorphisms have no effect on reproductive failure. There seems to be a correlation between the non-acrocentric heteromorphisms (1qh+, 9qh+, 16qh + and Yqh+/-), which might be the result of complex interactions of formation of these heterochromatin regions. The correlations observed between certain acrocentric chromosomes might be related to satellite association and nucleolus formation. The increased risk observed in males with IVF failure in the presence of either chromosome 13 or 21 variants should be interpreted cautiously due to the heterogeneity of the group.     

Optimising human chromosome separation for the production of chromosome-specific DNA libraries by flow sorting

Summary A number of cell lines, some containing chromosomes with distinctive heteromorphisms, have been flow karyotyped using a single laser flow sorter in an attempt to select those suitable for sorting all human chromosomes individually. Using the non-base-specific DNA stain ethidium bromide, chromosomes 3,4,5, and 6 form individual peaks in practically all normal subjects, while the right combination of heteromorphisms enables chromosomes 1, 2, 8, 9, 13, 16, 17, 18, 19, 20, 21, 22, and Y to be sorted separately. Two male cell lines, one containing a duplication and one a deletion of the X, produce flow karyotypes suitable for sorting chromosomes 7 and 8. The use of numerical chromosome abnormalities to enrich the sex chromosomes and the autosomes 18 and 21 is also illustrated. The DNA stain Hoechst 33258 binds preferentially to AT base pairs. Flow karyotypes produced with this fluorochrome separate some chromosomes not well separated with ethidium bromide. Chromosomes 5, 6, 8, 13, 14, 15, 17, and 20, and Y can be sorted individually with Hoechst 33258 with the right combination of heteromorphisms. Using these techniques, all human chromosomes apart from 10, 11, and 12 have been found as individual flow karyotype peaks, suitable for sorting with a high degree of purity.     

Q Band Chromosomal Polymorphisms in Lake Trout (SALVELINUS NAMAYCUSH)

Q banding of chromosome preparations from lake trout revealed the presence of heteromorphic quinacrine bright bands on several chromosomes. All of the metacentric chromosome pairs can be distinguished on the basis of number, position and intensity of the quinacrine bright bands and chromosome size. These bands appear to represent heterochromatin, since they are darkly staining with the C band technique. Since all of the fish examined had consistent heteromorphisms at several of the quinacrine bright bands, these chromosome markers should be useful in genetic comparisons between different trout stocks and populations.     

Binding of quinacrine to the human Y chromosome

Human chromosome spreads were stained with 3H-quinacrine and their fluorescence observed. The exact location of specific spreads on each slide was noted and photographs taken. Autoradiographs were then prepared so that the quinacrine fluorescence of any specific chromosome could be compared directly with the distribution of grains over the same chromosome on the autoradiograph. The Y chromosome fluoresced much more intensely than any of the other chromosomes, but there were no more grains over the Y chromosome than over the other chromosomes. Therefore the enhanced fluorescence of the human Y chromosome is not due to an increased binding of quinacrine.     

Identification of several chromosome aberrations in man by the fluorescent method using quinacrine yprite]

The applications of the fluorescent staining of chromosomes with quinacrine mustard allowed to identify a dicentric Y-chromosome in two patients with defected external gynaetalies: a boy of 15 years old and a girl of 2 years old. Both the patients had mosaicism of sex chromosomes: 45, x/46, x dic (Y). The dicentric Y-chromosome, resembling chromosome, 16, had bright luminescence of the thelomeric regions characteristic of the normal Y-chromosome. Besides, a balanced autosomic translocation t (1, 14) (q 31, q 3) was found in the girl identified also with quinacrine mustard fluorescent staining.     

无精和严重少精症患者的遗传缺陷分析——-附2例世界首报染色体异常核型

对217例无精和严重少精症患者外周血淋巴细胞染色体核型进行分析,并采用聚合酶链反应对7例Y染色体结构异常患者的AZFc区进行检测。发现187例无精症患者中检出异常核型77例（41.18%）（其中46,XY,t(6;14)(p21;p13),46,XY,t(8;12)(p21;q24)为世界首报核型）,主要涉及染色体异常（数目异常和结构异常）;染色体异态（Y染色体异态和9号染色体臂间倒位）及46,XX性反转;30例严重少精症患者中检出异常核型4例（13.33%）（结构异常和46,XX性反转）。由此可见,性染色体数目和结构异常是精子发生障碍的主要原因,其次常染色体的某些断裂点也可能影响精子发生。AZFc区的缺失与否与精子发生也有直接关系。     

The location of four human satellite DNAs on human chromosomes.

In situ hybridisation was carried out with ³H-cRNAs transcribed from four human satellite DNAs. The human metaphase chromosomes used were stained with quinacrine and photographed prior to hybridisation. This allowed accurate karyotyping of the autoradiographs. A method of quantitative analysis of grain distribution permitted identification of minor sites of hybridisation which could be distinguished from purely random grains. The hybridisation patterns for each of the transcribed satellite cRNAs were similar, with the C-band of chromosome 9 and the Y chromosome being the most heavily labelled sites. Other detectable sites of hybridisation were the centromeric regions of chromosomes 1, 5, 7, 10, 12, 13, 14, 15, 17, 20, 21 and 22. The cRNA transcribed from DNA satellite II, however, was the only one to hybridise to the centromere region of chromosome 16. The evolution of the human satellite DNAs and the validity of current models of satellite DNA function are discussed in the light of the present results.     

Y-chromosome length heteromorphisms in Delhi infants

One hundred and seventy normal male infants from Delhi were studied using the CBG technique to estimate Y-chromosome length heteromorphisms. The median class in Y/F [Y/F = total length of the Y chromosome/average total length of the F group chromosomes (19 and 20)] distribution was 0.75-0.79. The Y/F index in infants varied from 0.60 to 1.16 with a mean of 0.81 and a standard deviation of 0.09. A high incidence for very small (53.5 percent) and small (41.2 percent) categories of Y-chromosome length heteromorphisms was observed. Data were compared with other available reports; also possible mechanisms of the Y-chromosome length heteromorphisms and their role in ethnic/racial variation as well as in developmental disturbances are discussed. It is suggested there may be a need to redefine the long and short Y chromosome in a given population while studying different clinical disorders.     

Frequencies of centromeric heteromorphisms of human chromosomes 3 and 4 as detected by QFQ technique: can they be identified by RFA technique?

One hundred normal Caucasians were studied by sequential QFQ and RFA banding techniques in order to estimate the type and frequency of heteromorphisms in the centromeric regions of chromosome 3 and 4. Intensity variants were classified into 1 of 5 levels of QFQ banding. QFQ intensity heteromorphisms (greater than or equal to level 3) for chromosomes 3 and 4 were 62 and 15 percent respectively. The interrelationship between QFQ and RFA variants were also examined. When the centromere was brilliant by QFQ, it was found that it was deep red by RFA; when it was pale by QFQ, it was light red by RFA. Neverthless, a blind coded study could not pick up these color variants by RFA. QFQ banding showed variations of the centromeric regions of chromosomes 3 and 4 while RFA banding failed to demonstrate it. It was concluded that QFQ is the most useful technique in detecting the different intensity levels in the centromeric regions of chromsomes 3 and 4.     

Use of repetitive DNA for diagnosis of chromosomal rearrangements

Summary We have used two repeated DNA fragments (3.4 and 2.1 kb) released from Y chromosome DNA by digestion with the restriction endonuclease Hae III to analyze potential Y chromosome/autosome translocations. Two female patients were studied who each had an abnormal chromosome 22 with extra quinacrine fluorescent material on the short arm. The origin of the 22p+ chromosomes was uncertain after standard cytologic examinations. Analysis of one patient's DNA with the Y-specific repeated DNA probes revealed the presence of both the 3.4 and 2.1 kb Y-specific fragments. Thus, in this patient, the additional material was from the Y chromosome. Analysis of the second patient's DNA for Y-specific repeated DNA was negative, indicating that the extra chromosomal segment was not from the long arm of the Y chromosome. These two cases demonstrate that repeated DNA can distinguish between similar appearing aberrant chromosomes and may be useful in karyotypic and prenatal diagnosis.     

DNA measurements of mitotic chromosomes of the rabbit (Orytolagus cuniculus)

DNA cytophotometry was used to obtain the relative DNA content of mitotic chromosomes of Orytolagus cuniculus. DNA ranking of the rabbit chromosomes is presented, and related to the quinacrine and Giemsa-banded karyotype. In addition, the relative DNA content of the short and long arms of each chromosome was calculated and the DNA-based centromeric index, (i.e., the ratio of long-arm DNA to total chromosomal DNA) determined. The Y chromosome is clearly a submetacentric chromosome on the basis of the DNA measurements.     

Individuality of human chromosome polymorphism revealed by Q-staining]

The G-polymorphism of metaphase chromosomes of peripheral human lymphocytes and its inheritance in 32 families (268 persons) and 315 unrelated persons after G-staining has been studied. The site of Q-heterochromatine, its size and the length of secondary constrictions were accepted as morphological signs of Q-polymorphic variants of chromosomes. All the three chromosome signs are shown to be inherited according to codominant type and are characteristic features of each separate chromosome. No identical patterns of Q-polymorphic chromosome variants are found among all the persons studied, except the monozygotic twins. According to the data obtained, the question of individualization of each chromosome in the karyotype (3, 4, 13--15, 21, 22) and of each personal individuality in relation to Q-polymorphism is discussed.     

Sex chromosomal dimorphisms narrated by X-chromosome translocation in a spiny frog (<Emphasis Type="Italic">Quasipaa boulengeri</Emphasis>)

Background

In the general model of sex chromosome evolution for diploid dioecious organisms, the Y (or W) chromosome is derived, while the homogametic sex presumably represents the ancestral condition. However, in the frog species Quasipaa boulengeri, heteromorphisms caused by a translocation between chromosomes 1 and 6 are not related to sex, because the same heteromorphic chromosomes are found both in males and females at the cytological level. To confirm whether those heteromorphisms are unrelated to sex, a sex-linked locus was mapped at the chromosomal level and sequenced to identify any haplotype difference between sexes.

Results

Chromosome 1 was assigned to the sex chromosome pair by mapping the sex-linked locus. X-chromosome translocation was demonstrated and confirmed by the karyotypes of the progeny. Translocation heteromorphisms were involved in normal and translocated X chromosomes in the rearranged populations. Based on phylogenetic inference using both male and female sex-linked haplotypes, recombination was suppressed not only between the Y and normal X chromosomes, respectively the Y and translocated X chromosomes, but also between the normal and translocated X chromosomes. Both males and females shared not only the same translocation heteromorphisms but also the X chromosomal dimorphisms in this frog.

Conclusions

The reverse of the typical situation, in which the X is derived and the Y has remained unchanged, is known to be very rare. In the present study, X-chromosome translocation has been known to cause sex chromosomal dimorphisms. The X chromosome has gone processes of genetic differentiation and/or structural changes by chance, which may facilitate sex chromosome differentiation. These sex chromosomal dimorphisms presenting in both sexes may represent the early stages of sex chromosome differentiation and aid in understanding sex chromosome evolution.

     

Preferential derivation of abnormal human G-group-like chromosomes from chromosome 15

Summary The marked binding of antibodies specific for 5-methylcytidine to the short arm of chromosome 15 distinguishes this chromosome from the other human acrocentrics. This method has been used to study over 60 individuals including 12 who did not have Down's syndrome, but who did have an extra G-group sized acrocentric chromosome. In six cases the extra chromosome did not show intensive binding of anti-5-methylcytidine. In the other six cases, the extra chromosome contained a 5-methylcytidine rich band at each end indicating that both ends were derived from chromosome 15 and contained centromeric heterochromatin normally present on the short arm of chromosome 15. The duplication of short arm material in the abnormal chromosomes was confirmed in all cases by quinacrine staining, nucleolar organizer (Ag-AS) staining or C-banding. In three cases, the abnormal chromosome appeared to arise from two different chromosomes 15. Several possible mechanisms for the production of the abnormal chromosome are discussed. The individuals with this abnormal chromosome all showed some degree of mental retardation, but few common physical findings.     

应用染色体涂染法建立人和黑叶猴(Semnopithecus francoisi)的染色体同源性

应用荧光原位杂交技术中的染色体涂染法（Chromosomepainting）,以生物素标记的除Y染色体外的人全部整条染色体DNA特异性探针与黑叶猴的中期分裂相杂交,建立了人与黑叶猴之间的染色体同源性。除人的1、2、6、16和19号染色体特异探针分别与黑叶猴的2条非同源的染色体杂交外,其余人染色体特异探针均与黑叶猴的1条染色体杂交,其中有两对人染色体特异探针（14和15,21和22）分别杂交同一条黑叶猴染色体。在雌性黑叶猴的单倍染色体中,共检测到30个与人染色体具同源性的染色体和染色体片段。结果表明：黑叶猴的多数染色体与人染色体有高度同源性,仅有少数染色体发生了重排。将研究的结果与已报道的人染色体特异探针与其他灵长类的中期染色体杂交的结果进行比较,可以看出亚洲叶猴之间的相互关系较与非洲叶猴的更为密切。     

Genomic imbalances in 61 renal cancers from the proximal tubulus detected by comparative genomic hybridization

Comparative genomic hybridization (CGH) has been applied to characterize 61 primary renal cell carcinomas derived histogenetically from the proximal tubulus. The tumor samples comprised 46 clear-cell renal cell carcinomas (ccRCCs) and 15 papillary renal cell carcinomas (pRCCs). Changes in the copy number of entire chromosomes or subregions were detected in 56 tumors (92%). In ccRCCs, losses of chromosome 3 or 3p (63%); 14q (30%); 9 (26%); 1 and 6 or 6q (17% each); 4 and 8 or 8p (15% each); 22 (11%); 2 or 2q and 19 (9% each); 7q, 10, 16, 17p, 18, and Y (7% each); and 5, 11, 13, 15, and 21 (4% each) were detected. Most frequent genomic gains in ccRCC were found on chromosome 5 (63%); 7 (35%); 1 or 1q (33%); 2q (24%); 8 or 8q, 12, and 20 (20% each); 3q (17%); 16 (15%); 19 (13%); 6 and 17 or 17q (11% each); and 4, 10, 11, 21, and Y (9% each). In pRCCs, gains in the copy number of chromosomes 7 and 17 (7/15, each) and 16 and 20 (6/15, each) were frequent. One pRCC showed amplification of subchromosome regions 2q22-->q33, 16q, 17q and the entire X chromosome. In pRCC, losses were less frequently seen than gains. Losses of chromosomes 1, 14, 15, and Y (3/15 each) and 2, 4, 6, and 13 (2/15 each) were observed. In ccRCCs, statistical evaluation revealed significant correlations of chromosomal imbalances with tumor stage and grade, i.e., a gain in copy number of chromosome 5 correlated positively with low tumor grade, whereas a gain of chromosomes 10 and 17 correlated positively with high tumor grade. Furthermore, loss of chromosome 4 correlated positively with high tumor stage.     

Chromosome heteromorphism quantified by high-resolution bivariate flow karyotyping.

Maternal and paternal homologues of many chromosome types can be differentiated on the basis of their peak position in Hoechst 33258 versus chromomycin A3 bivariate flow karyotypes. We demonstrate here the magnitude of DNA content differences among normal chromosomes of the same type. Significant peak-position differences between homologues were observed for an average of four chromosome types in each of the karyotypes of 98 different individuals. The frequency of individuals with differences in homologue peak positions varied among chromosome types: e.g., chromosome 15, 61%; chromosome 3, 4%. Flow karyotypes of 33 unrelated individuals were compared to determine the range of peak position among normal chromosomes. Chromosomes Y, 21, 22, 15, 16, 13, 14, and 19 were most heteromorphic, and chromosomes 2-8 and X were least heteromorphic. The largest chromosome 21 was 45% larger than the smallest 21 chromosome observed. The base composition of the variable regions differed among chromosome types. DNA contents of chromosome variants determined from flow karyotypes were closely correlated to measurements of DNA content made of gallocyanin chrome alum-stained metaphase chromosomes on slides. Fluorescence in situ hybridization with chromosome-specific repetitive sequences indicated that variability in their copy number is partly responsible for peak-position variability in some chromosomes. Heteromorphic chromosomes are identified for which parental flow karyotype information will be essential if de novo rearrangements resulting in small DNA content changes are to be detected with flow karyotyping.