Anti-kinetochore staining for single laser, bivariate flow sorting of Indian muntjac chromosomes.

Flow cytometric chromosome sorting typically relies upon dual-laser, bivariate analysis after staining with two different base pair-specific dyes for resolution of chromosomes with similar DNA content. The availability of FITC-conjugated antibodies offers the possibility of single-laser bivariate analysis when combined with propidium iodide (PI) DNA staining, but requires exploitable antigenic differences between chromosomes of interest. A technique was developed for indirect immunofluorescent anti-kinetochore staining of Indian muntjac chromosomes in suspension. Primary antibody binding within permeabilized whole cells minimized centrifugation-induced loss of chromosomal integrity. Subsequent FITC-conjugated second antibody binding was not affected by concurrent PI-counterstaining. Anti-kinetochore staining facilitated resolution of chromosomes No. 2 and X, which formed a doublet peak upon univariate DNA content analysis, as well as recognition of the Y2 peak which was indistinguishable from debris by univariate analysis. The technique allowed greater than 90% purification of each Indian muntjac chromosome.     

Spatial distribution of chromosomes 1 and Y in human spermatozoa.

The positions of chromosomes 1 and Y inside human spermatozoa were determined by differential staining techniques. In 85/100 cells the two chromosomes were in close contact and in association with a vacuole. This observation is in contrast to previous findings for chromosome No. 9 and the Y-chromosome whose positions do not appear to be correlated.     

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.     

Random position of human heterochromatin-bearing chromosomes in first and third mitoses of lymphocyte cultures

Summary The position of chromosomes 1, 9, and 16 in first and third mitoses of lymphocyte cultures was measured in BUdR-labelled air-dried chromosome preparations. No significant deviation from a random distance could be found between the two homologues of each chromosome, either in the first or in the third mitoses after phytohemagglutinin stimulation. Colchicine treatment also had no influence.     

Quantitative studies on the arrangement of human metaphase chromosomes. IX. Arrangement of chromosomes with and without spindle apparatus

Summary The spatial relationships in human male metaphase cells treated with and without colcemide were compared with each other. The following results were obtained: (1) In normal male metaphases the overall distributions of chromosomal distances regardless of chromosome identification numbers did not show normal distribution, neither in the colcemid-free sample nor in the colcemide-treated sample. (2) In both samples larger chromosomes showed a more peripheral position, and smaller chromosomes showed a more central position. This finding was statistically significant. (3) No differences between the two samples could be observed concerning the following parameters: overall distributions of the centromere-centromere distances, distributions of the distances between the homologous chromosomes (except the small acrocentric chromosomes), rank positions of the mean distances between homologous chromosomes, and rank positions of the mean distances of the different chromosomes from the center of the mitosis (except few chromosomes). (4) Visible, but not statistically accessible, differences appeared between the two samples in respect to rank positions of the mean distances of all possible acrocentric pairing groups, rank positions of the mean distances of the homologous acrocentric chromosomes from the center of the mitosis, and distances of the X chromosome from the center of the mitosis. (5) Statistically significant differences appeared between the two samples with respect to distance distributions of the small acrocentric chromosomes and positions of the chromosomes 1, 16, 18, Y, and 21, 22 in relation to the center of the mitosis.     

Characterization of human heterochromatin by in situ hybridization with satellite DNA clones

Biotinylated DNA from two satellite-related, repetitive DNA clones, pHuR 98 and pHuR 195 (specific for chromosomes 9 and 16, respectively), and from a Y-specific clone, pY-3.4A, were hybridized to human metaphase chromosomes using fluoresceinated avidin to detect binding. The chromosomes were simultaneously counterstained with distamycin-DAPI to identify the AT-rich heterochromatin of chromosomes 1, 9, 15, 16, and the Y chromosome. With this method, clear results were obtained under both normal and low stringency conditions, allowing hybridization between molecules sharing 80-85% and 60-65% identity, respectively. Thus, additional sites related to the probes could be identified. A close relationship was shown between the heterochromatin of chromosomes 1 and 16, both hybridizing with clone pHuR 195 under low stringency. Hybridization with clone pHuR 98 was highly specific for chromosome 9, even under low stringency. A relationship between chromosomes 9, 15, and the Y chromosome, however, was shown by hybridization with clone pY-3.4A. The chromosomal distribution of the three repetitive DNA clones used in this study, and data from the literature, are in accordance with the distribution of the heterochromatin types characterized by staining with different fluorescent dyes and dye combinations. Furthermore, our sequence data for clones pHuR 98 and pHuR 195 may explain the fluorescent properties on which the cytogenetic classification of the heterochromatin is based.     

Human chromosome polymorphism and disordered reproductive function. I. Routine chromosome variants]

Routine polymorphic variants of chromosomes of 58 married couples with reproductive failure (two or more spontaneous abortions, stillbirths and malformed children) and 48 control couples, having two or more normal children and no spontaneous abortions and stillbirths, were investigated by conventional staining technique. Extreme variants of chromosomes 1, 9, 16, 17, 13--15, 21--22 and Y were found in 17.2% of subjects with reproductive loss and in 15.6% of control individuals. No significant differences in frequencies of scored routine variants were noted between married couples with reproductive failure and couples with normal reproduction.     

A quantitative study of the second meiotic metaphase in male mice (Mus musculus).

Over 11,000 second meiotic metaphase spreads stained for the pericentromeric region have been studied quantitatively in male mice of 14 strains. The sex-chromosome constitution of a cell could be judged objectively if X and Y chromosomes and ploidy were all scored. A bias arose if only Y chromosomes and ploidy were scored but could be corrected statistically. There was no sign of other forms of bias. The original contiguity of X and Y second metaphases in vivo was very occasionally evident in the preparations. Most of the subhaploid aneuploid counts were assumed to be artifactual. The incidence of truly aneuploid second metaphases in 13 strains was estimated as 0.38+/-0.12%. The estimated average rate per chromosome was 0.019+/-0.006%, with a comparable order of magnitude for the sex chromosomes alone. Simultaneous aneuploidy of two or more chromosomes of the haploid set was estimated to be very rare. Of the spreads from 13 strains, 9.6% were polyploid (2N, 3N, 4N) and showed most of the possible combinations of sex chromosomes. Nearly all the polyploid spreads were considered to arise by artifactual cell fusion at the time of second metaphase during the preparative technique, especially of the X and Y daughter-cell products of the first meiotic division. Other modes of origin (true polyploidy, accidental superposition of cells during preparation) were unlikely. The data could be accommodated by a statistical model with only four parameters. It allowed for artifactual fusion mainly between daughter cells but also between non-daughter cells, bias in one scoring method, and bias in the numbers of cells with given ploidy successfully mounted. Current techniques of chromosome preparation were thought to be wholly unsuitable for the recognition of true polyploidy. The artifactual origin of polyploid spreads was borne out by an absence of polyploid spermatozoa in 14 strains. There appeared to be a virtually constant transmission rate of paternal X and Y chromosomes from early meiosis to late blastocyst. The estimated rate of 49.05+/-0.67% with a Y chromosome also estimated the primary sex ratio. There was evidence of polymorphism in autosomal pericentromeric staining in 3 strains. No measure of the numbers of autosomes or sex chromosomes varied significantly between duplicate preparations or between duplicate males of a strain.     

Reliability of aneuploidy estimates in human sperm: Results of fluorescence in situ hybridization studies using two different scoring criteria

Aneuploidy estimates for chromosomes 1, 12, X, and Y were obtained in human sperm from five donors using multicolor fluorescence in situ hybridization (FISH) analysis. Disomy frequencies were obtained by scoring a minimum of 10,000 sperm for each chromosome probe per donor. This analysis was replicated for two scoring criteria: one used one half of a signal domain as the minimum distance between two signals to be counted as two and thus disomic; the other set one signal domain as the minimum distance between two signals. A total of 120,870 sperm were assessed using one half of a domain as the criterion, and 113,478 were scored using one domain as the criterion. The percentage of disomy for chromosomes 1, 12, X, Y, and XY was 0.18, 0.16, 0.15, 0.19, and 0.25, respectively, using the one-half-domain criterion, and 0.08, 0.17, 0.07, 0.12, and 0.16, respectively, using the one-domain criterion. The percentage of disomy decreased significantly with use of one domain as the minimum distance for signal separation for all chromosomes except for chromosome number 12. These lower disomy frequencies correlated well with frequencies derived from human sperm karyotypes analyzed in our laboratory. This suggests that the fluorescent signals for chromosomes 1, X, and Y split into more than one domain in decondensed interphase sperm, and that the use of the one-half-domain criterion would lead to an overestimate of aneuploidy frequencies. The factors known to affect aneuploidy estimates derived from FISH studies are discussed, and recommendations for stringent scoring criteria are proposed. © 1995 wiley-Liss, Inc.     

C,Q and H-banding in the analysis of Y chromosome rearrangements in Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae)

In Lucilia cuprina C-banding produces procentric bands on all autosomes and deep staining over most of the X and Y chromosomes which conciderably facilitates the analysis of complex Y chromosome rearrangements. The Y chromosome is generally darkly C-banded throughout while in the X chromosome a pale staining segment is found in the distal portion of the long arm. Modulation of the banding reaction results in grey areas in both X and Y. When C-banding is compared with allocycly it is clear that not all heteropycnotic regions in the sex chromosomes C-band to the same extent. Secondary constrictions in the short arms of both X and Y chromosomes are clearly revealed by C-banding, the X satellite being polymorphic for size.— Q-banding results in a brightly fluorescing band in the short arm of structurally normal Y chromosomes. This band loses its fluorescence in some translocations, probably through a position effect. Hoechst 33258 staining does not produce any brightly fluorescing bands.     

鼷鹿云南亚种（Tragulus javanicus williamsoni）的核型分析

本文以染色体分带技术,发现鼷鹿云南亚种的染色体数目为2n=32。全部为双胃染色体,NF=64。所有染色体着丝点区分布有C带,多数染色体的端部或两端也有C带。某些染色体还有插入C带。Y染色体C带阳性,有一Ag-NORs。文章对鼷鹿云南亚种染色体独特的C带分布以及和核型进化的关系进行了讨论。     

石貂的染色体研究

本文对分布在我国的石貂北方亚种染色体进行了较详细的研究。结果表明２ｎ＝３８,核型为１４（Ｍ）＋４（ＳＭ）＋１８（ＳＴ）,ＸＹ（Ｍ,Ａ）。Ｃ－带显示该亚种的一些染色体着丝粒区域结构异染色质弱化或消失。Ｎｏ,９染色体的短臂完全异染色质化;Ｘ染色体长臂丰出现插入杂色质带;Ｙ为完全结构异染色质组成。     

Centromere localization at meiosis and the position of chiasmata in the male and female mouse

Techniques for obtaining differential Giemsa staining of the paracentromeric (p.c.) regions of male and female mouse meiotic chromosomes (centromeric heterochromatin) were explored and standard procedures developed for the different meiotic cells in the two sexes. The best result followed the use of heat at controlled pH in Sörensen's phosphate buffer or in Standard Saline Citrate (SSC) solutions. With these techniques, morphological features of the p.c. regions and their variation were studied in normal animals (CFLP strain) and in a strain (AKR) homozygous for a centric fusion [T(11; ?)-1 Ald] between chromosomes No. 6 and No. 15 (Miller et al., 1971). The Y chromosome was often found to show distinct p. c. staining at first and apparently at second meiotic metaphase, and the X and Y chromosomes were found to associate as bivalents by their long arms. Autosomal p.c. regions showed variation in size which might indicate differences between non-homologous chromosomes but a tendency to similarity between homologues. Differences were found between males and females in respect to proportions and variation of bivalents with single and double chiasmata. The relative positions of chiasmata were different in the two sexes. The presence of the centric fusion in the males did not seem to affect the pairing behaviour of the remaining autosomes or of those taking part in the centric fusion. The possibility is discussed that the p.c. regions, to which also other functions would seem to appertain, may be important for chromosome recognition and pairing, possibly on a quantitative basis.     

Isolabeling of the long arm of the human Y chromosome demonstrated by the FPG technique

Isolabeling segments were found in the distal region of the long arm of Y chromosomes derived from human leukocytes grown through two replication cycles in medium containing BrdU and stained by the FPG technique. Three main types of Y chromosome staining patterns were demonstrated: I-Y chromosome with typical SCD, II-Y chromosome with weakly stained distal regions of long arms (isolabeling segments), III-Y chromosome with both terminal regions displaying SCD interrupted by one isolabeled segment. The existence of different types of Y chromosome staining patterns was explained on the basis of the previously described hypothesis of unequal distribution of thymine residues between two DNA polynucleotide chains in the distal part of the long arms of human Y chromosomes.     

Distribution of C-band heterochromatin in the ZW sex chromosomes of European and American eels (Anguillidae, Teleostomi)

The ZW sex chromosomes of the European eel, Anguilla anguilla, and the American eel, A. rostrata, were examined with C-band and fluorescent staining to demonstrate the C-band heterochromatin. The W as well as Z chromosomes in both species are C-band negative except for a small amount of C-band heterochromatin in the centromeric region, in contrast to the W or Y elements of most other vertebrates. No fluorescing W-associated body is evident either in interphase nuclei or in metaphase plates. The ZW chromosomes of the two species have substantially similar size, morphology, and patterns of C-band heterochromatin. Karyologic and evolutionary implications are discussed.     

褐家鼠精母细胞中联会复合体的研究 Ⅱ.性染色体配对形态和行为的研究

用表面铺展-AgNO_3和PTA染色技术,对雄性褐家鼠性染色体配对形态和行为进行研究表明:X和Y轴在减数分裂前期Ⅰ的不同阶段固缩速度不同;性染色体在配对之前轴心增粗、配对延迟到早粗线期;性染色体首次配对起始区发生在X和Y短臂端粒区,其次配对起始区发生在X与Y长臂的端粒区或长臂的中间区;在中粗线期几乎整条Y与约1/3 X配对形成X-YSC;配对区的侧生组分分为两股,其中一股发生泡状变形,不配对片段发生多种变形。本文对X和Y配对起始位点,配对的同源性及XY轴心增厚与变形机制作了讨论.     

Lateral asymmetry in human constitutive heterochromatin

Human lymphocytes were grown for one replication cycle in BrdU, stained with 33258 Hoechst, exposed to UV light and subsequently treated with 2 x SSC and stained with Giemsa. This technique differentially stains the constitutive heterochromatin of chromosomes 1, 9, 15, 16, and the Y. In the heterochromatin of chromosome 9 both sister chromatids stained darkly and symmetrically but in the other four chromosomes the heterochromatin showed lateral asymmetry, one chromatid being darkly stained while its sister chromatid was as pale or paler than the rest of the chromosome. The lateral asymmetry is presumed to reflect an underlying asymmetry in distribution of thymine between the two strands of the DNA duplex in the satellite DNA component of the chromosomes. In some number 1 chromosomes compound lateral asymmetry was seen; darkly staining material was present on both sister chromatids although at any given point lateral asymmetry was maintained so that if one chromatid stained darkly the corresponding point on the sister chromatid was very pale. The pattern of compound lateral asymmetry varied among the number 1 chromosomes studied but was constant for any one homologue from one individual. This technique reveals a previously unsuspected type of polymorphism within the constitutive heterochromatin of man.     

Human chromosome variation: the discriminatory power of Q-band heteromorphism (variant) analysis in distinguishing between individuals, with specific application to cases of questionable paternity.

The chromosomes from 57 persons were analyzed by means of quinacrine fluorescent staining in order to assess the amount of variation and the discriminatory power of Q-band heteromorphism analysis. Chromosomes 3, 4, 13, 14, 15, 21, 22, and Y of each person were visually compared to those of 56 others, for a total of 1,596 comparisons. No two persons were found to have the same set of variants. The number of differences between chromosomes for each comparison ranged from 2 to 12 out of a possible total of 14 for females and 15 for males. Relatives were also distinguishable, and differences ranged from two to seven. We used the frequency with which each chromosome was useful for telling two people apart, and estimated the probability of finding two persons with the same set of quinacrine variants as .0003. Distinctly different heteromorphisms were found in the 39 unrelated persons for each of the chromosomes examined. In this small population, the number of different sets of variants observed for chromosomes 3, 4, 13, 14, 15, 21, 22, and Y were six, seven, 27, 16, 20, 15, 24, and five, respectively, for a total number of possible combinations of 1.14 X 10(15). As a test of the usefulness of chromosome heteromorphisms in paternity cases, 12 father-mother-child trios of virtually certain paternity, owing to the father-child segregation of a rare structural rearrangement, were coded and recombined at random to produce 120 cases of uncertain paternity. When the code was broken, 108 "alleged fathers" had been excluded correctly and the 12 biological fathers had been included correctly.     

Satellite DNA and speciation: A species specific satellite DNA of Drosophila guanchel

The heterochromatin of the chromosomes of Drosophila gunche consists mainly of a satellite DNA composed of multiple, tandemly arranged copies of a 290 b p basic sequence. Five clones containing one or two copies of the basic unit were sequenced. As expected from CsCl density centrifugation and AT specific staining of mitotic chromosomes the sequence is AT rich. The average nucleotid variability between the cloned sequences is 11.6%. In situ hybridization on the mitotic chromosomes revealed, that this satellite DNA is present in the centromeric regions of all chromosomes but the Y. The nucleotide variability between copies of different tandem clusters seems to be higher than between members of the same cluster. The copy number of the sequence in the haploid genome was estimated to be approximately 80000. The sequence is species specific and is not present in the genome of sibling species D. subobscura and D. madeiren-sis. The evolutionary origin of the satellite DNA and its possible role in species formation is discussed.