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.     

An alpha satellite DNA polymorphism specific for the centromeric region of chromosome 13

Alpha satellite DNA is composed of variants of a short consensus sequence that are repeated in tandem arrays in the centromeric heterochromatin of each human chromosome. To define centromeric markers for linkage studies, we screened human genomic DNA for restriction fragment length polymorphisms using a probe detecting alphoid sequences on chromosomes 13 and 21. We describe one such DNA polymorphism. Analysis of linkage of this DNA marker to other polymorphic markers in the CEPH pedigrees demonstrates linkage to markers on the proximal long arm of chromosome 13 and defines the centromeric end of the linkage map of this chromosome.     

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.     

Zonal Fractionation of Mammalian Metaphase Chromosomes and Determination of Their DNA Content

Chinese hamster cells in culture were synchronized, collected at metaphase, homogenized to release the chromosomes, and the chromosomes fractionated in a sucrose gradient using a zonal centrifuge with an A12 zonal rotor. Chromosomes in the separated fractions as well as in control metaphase spreads were quantitatively classified into five easily distinguished groups, according to individual measurements of length and centromeric index. For each zonal fraction, chemical determinations were made of the amount of DNA per average chromosome. Using the group compositional data for each fraction, the amount of DNA per average chromosome in each of the groups was then calculated to be: Group I (chromosomes 1, 2)= 1.00 ± 0.14 pgm/chromatid; Group II (chromosomes 4, X, 5) -0.39 ± 0.05 pgm/chromatid; Group III (chromosomes Y, 6, 7, 8)=0.24 ± 0.04 pgm/chromatid; Group IV (chromosomes 9, 10, 11)=0.13 ± 0.004 pgm/ chromatid; and Group V (a small marker in this cell line)=0.06 pgm/ chromatid. These values are in good agreement with the literature values for relative chromosomal DNA content derived from cytospectrophotometric measurements of fuelgen stained hamster metaphase spreads. They indicate that unlike the case for human chromatids the amount of DNA found in hamster chromatids is not directly proportional to the chromatid length.

The larger chromosomes contain more DNA per unit length than smaller chromosomes. The magnitude of this effect is considerably greater than that which may be ascribable to centromeric constriction.     

Linkage studies of polymorphic, repeated DNA sequences in centromeric regions of human chromosomes.

The DNA at human centromeric regions was characterized by using a repetitive sequence, 308, which localizes in situ exclusively to centromeres of all chromosomes. We previously noted that this sequence is enriched on chromosome 6 and has chromosome-specific organization on 6, 3, 7, 14, X, and Y. In addition to this basic organization, sequences homologous to 308 are polymorphic among normal individuals. The variants are transmitted in a Mendelian manner within a family. To determine the chromosome origin of the variants, we studied their linkage to markers of various chromosomes. Linkage analysis of one pedigree segregating two polymorphisms shows that the 2.6-kilobase (kb) BamHI and 2.6-kb TaqI fragments are linked to each other and to the HLA loci on chromosome 6. Data from another family shows that 2.8-kb TaqI, 4.0-kb TaqI, and 1.3-kb BamHI polymorphic fragments are linked and are probably near the Fy locus on chromosome 1. By dot blot analysis, we determined that the relative amount of these sequences in the genome is not measurably different between unrelated individuals. Thus, the polymorphisms represent changes in homologous 308 sequences on specific chromosomes and can be used as chromosome-specific markers. Linkage studies using polymorphisms of repeated sequences will be most useful within a kindred, especially from an inbred population, because polymorphic repeats of the same restriction size may be heterogeneous in origin.     

Alphoid DNA polymorphisms for chromosome 21 can be distinguished from those of chromosome 13 using probes homologous to both.

Although alphoid DNA sequences shared among acrocentric chromosomes have been identified, no human chromosome 21-specific sequence has been isolated from the centromeric region. To identify alphoid DNA restriction fragment length polymorphisms (RFLPs) specific for chromosome 21, we hybridized human genomic DNA with alphoid DNA probes [L1.26; aRI(680),21-208] shared by chromosomes 13 and 21. We detected RFLPs with restriction enzymes ECoRI, HaeIII, MboI,StuI, and TaqI. The segregation of these RFLPs was analyzed in the 40 CEPH families. Linkage analysis between these RFLPs and loci previously mapped to either chromosome 13 or 21 revealed RFLPs that appear to be specific to chromosome 21. These polymorphisms may be useful as genetic markers of the centromeric region of chromosome 21. Different alphoid loci within the centromeric region of chromosome 13 were identified.     

Cytogenetic characterization of alpaca (Lama pacos, fam. Camelidae) prometaphase chromosomes

The present study provides specific cytogenetic information on prometaphase chromosomes of the alpaca (Lama pacos, fam. Camelidae, 2n = 74) that forms a basis for future work on karyotype standardization and gene mapping of the species, as well as for comparative studies and future genetic improvement programs within the family Camelidae. Based on the centromeric index (CI) measurements, alpaca chromosomes have been classified into four groups: group A, subtelocentrics, from pair 1 to 10; group B, telocentrics, from pair 11 to 20; group C, submetacentrics, from pair 21 to 29; group D, metacentrics, from pair 30 to 36 plus sex chromosomes. For each chromosome pair, the following data are provided: relative chromosome length, centromeric index, conventional Giemsa staining, sequential QFQ/C-banding, GTG- and RBG-banding patterns with corresponding ideograms, RBA-banding and sequential RBA/silver staining for NOR localization. The overall number of RBG-bands revealed was 391. Nucleolus organizer-bearing chromosomes were identified as pairs 6, 28, 31, 32, 33 and 34. Comparative ZOO-FISH analysis with camel (Camelus dromedarius) X and Y painting probes was also carried out to validate X-Y chromosome identification of alpaca and to confirm close homologies between the sex chromosomes of these two species.     

Chromosomal alterations in cell lines derived from mouse rhabdomyosarcomas induced by crystalline nickel sulfide

Summary Prior studies have shown a preferential decondensation (or fragmentation) of the heterochromatic long arm of the X chromosome of Chinese hamster ovary cells when treated with carcinogenic crystalline NiS particles (crNiS). In this report, we show that the heterochromatic regions of mouse chromosomes are also more frequently involved in aberrations than euchromatic regions, although the heterochromatin in mouse cells is restricted to centromeric regions. We also present the karyotypic analyses of four cell lines derived from tumors induced by leg muscle injections of crystalline nickel sulfide which have been analyzed to determine whether heterochromatic chromosomal regions are preferentially altered in the transformed genotypes. Common to all cell lines was the presence of minichromosomes, which are acrocentric chromosomes smaller than chromosome 19, normally the smallest chromosome of the mouse karyotype. The minichromosomes were present in a majority of cells of each line although the morphology of this extra chromosome varied significantly among the cell lines. C-banding revealed the presence of centromeric DNA and thus these minichromosomes may be the result of chromosome breaks at or near the centromere. In three of the four lines a marker chromosome could be identified as a rearrangement between two chromosomes. In the fourth cell line a rearranged chromosome was present in only 15% of the cells and was not studied in detail. One of the three major marker chromosomes resulted from a centromeric fusion of chromosome 4 while another appeared to be an interchange involving the centromere of chromosome 2 and possibly the telomeric region of chromosome 17. The third marker chromosome involves a rearrangement between chromosome 4 near the telomeric region and what appears to be the centromeric region of chromosome 19. Thus, in these three major marker chromosomes centromeric heterochromatic DNA is clearly implicated in two of the rearrangements and less clearly in the third. The involvement of centromeric DNA in the formation of even two of four markers is consistent with the previously observed preference in the site of action of crNiS for heterochromatic DNA during the early stages of carcinogenesis.     

Karyotype of mitotic metaphase and meiotic diakinesis in non-heading Chinese cabbage

Non-heading Chinese cabbage [Brassica rapa L. ssp. chinensis (L.) Hanelt] is one of the most popular leafy vegetables. Despite the economic importance of non-heading Chinese cabbage, little attention has been given to its cytogenetic profile. This study reveals the karyotype of non-heading Chinese cabbage. Fluorescence in situ hybridization (FISH) with 45S and 5S rDNA probes was performed on mitotic metaphase complementary regions. We located 45S rDNA on the centromeric or adjacent region of chromosomes A1 and A2, with the largest on the satellite of chromosome A5. Meanwhile, 5S rDNA co-localized with 45S rDNA on chromosomes A2 and A5, and on the telomeric region of chromosome A10. We performed DAPI fluorescence banding on the same metaphase chromosomes to identify homologous chromosomes. The DAPI fluorescence pattern was observed mainly on the centromeric heterochromatin regions of each chromosome. However, the lengths of chromosomes A2 and A6 were completely stained, except for their telomeric regions. Meiotic diakinesis chromosomes as new substrates in FISH-developed karyotype were revealed for the first time. The karyotype of non-heading Chinese cabbage reveals that it contains eight submetacentric chromosomes, one subtelocentric chromosome (bearing satellite), and one telocentric chromosome. Diakinetic chromosome pairing can overcome the difficulty of unlabeled chromosome identification. This study provided valuable information for cytogenetic research and molecular breeding of non-heading Chinese cabbage by using the combination of FISH and DAPI fluorescence patterns on mitotic and meiotic chromosomes.     

Evidence for chromosome number reduction and chromosomal homosequentiality in the 24-chromosome Korean frog Rana dybowskii and related species

The karyotype of the Korean frog Rana dybowskii with its pattern of C-band heterochromatin distribution was numerically analyzed. There are 2n = 24 chromosomes in the karyotype representing a reduction in number from the typical 2n = 26 chromosome karyotype of Rana. The karyotype shows other evidence of reorganization relative to 26-chromosome species. The chromosomes grade smoothly in size from largest to smallest without the two size classes that are characteristic for 26-chromosome species. In contrast to many 26-chromosome species, there are few centromeric C-bands but many interstitial ones. C-bands for each homologous chromosome pair are distinctive. A prominent secondary constriction is located on one of the smallest chromosomes, chromosome 11, in a position similar to that seen in most 26-chromosome species. The karyotype of R. dybowskii is compared to those of other species of Rana known to have 2n = 24 chromosomes; it is most similar to that of R. chensinensis, less so that of R. ornativentris and less still to that of R. arvalis in terms of the positions of centromeres and secondary constrictions. C-bands as well as secondary constrictions in the karyotypes of these frogs show evidence of chromosomal homosequentiality. The process and possible consequences of chromosome number reduction from an ancestral 26-chromosome karyotype is also evident in the karyotypes of these closely allied palearctic frogs. Pericentric inversions followed by fusion of two small elements apparently produced a new chromosome, chromosome 6, occurring originally among northeast Asian populations.     

High-resolution scanning-densitometry of photographic negatives of human metaphase chromosomes

Summary In photomicrographic negatives of cytochemically stained human metaphase preparations, images of individual chromosomes were scanned interactively with a Zeiss SMP interfaced to a PDP-12 computer.By means of the CHROSCAN computer program spot-scanning of selected chromosomes was performed in a direction perpendicular to the length axis, each measured value being corrected for background absorbance taken on both sides of the chromosome image. Plotting of the integrated absorbance values of each transversal scanline results in a graphic representation of the absorbance distribution over the chromsome length. Following this procedure, longitudinal curves were obtained which showed the characteristic patterns obtained after Q or G banding, and, in the case of Feulgen-staining, represented quantitative variations of DNA mass along the individual chromosomes. For Feulgen-stained chromosomes, the total integrated absorbance value and the ratio of integrated absorbance in the long arm over the total integrated absorbance, correspond with the DNA-absorbance and-arm ratio values per chromosome respectively.The results of investigations concerning the reproducibility and accuracy of cytochemical Feulgen staining and of the photographic procedures are presented, together with total integrated Feulgen-DNA absorbance and arm ratio values for a number of human chromosomes.For several chromosomes, Feulgen absorbance arm ratio measurements were found to result in values more constant over different metaphases when the long arm was considered to start at the lowest dip in the longitudinal absorbance curve, than when the microscopically observable primary constriction was taken to represent the centromere. The results indicate that the present method allows accurate photometry of naturally absorbing, or of stained or fluorescent objects, with measuring intervals of 0.16 . In addition it is shown that the arm ratio values and total DNA content can serve as very constant parameters for karyotype analysis.Supported by grant nr. 28-16915 of the Praeventiefonds, 's Gravenhage.     

Chromosome polymorphism involving heterochromatic blocks in Chinese hamster chromosome 9

A chromosome polymorphism was detected between two early passage euploid Chinese hamster cell strains when a fluorescence shift of the small metacentric No. 9 chromosome was resolved by flow cytometry. The characteristics of the polymorphism were studied using cultures established from ear clippings taken from 16 additional hamsters from our breeding colony. Additional variants of chromosome 9 were detected using flow cytometry, and a subset of these variants were analyzed by G- and C-banding. An increase of fluorescence recorded by flow cytometry correlated with an increase of centromeric heterochromatin. Autosomal normalization of the flow karyotype from 18 different animals indicated three distinct peak positions for chromosome 9. The results indicate that a discrete block of constitutive heterochromatin may be present in one or two extra copies within the small inbred colony of hamsters studied. To determine the inheritance patterns, hamsters with known polymorphic No. 9 chromosomes were bred. The flow karyotypes derived from the offspring of these matings provide strong evidence that chromosomal polymorphisms are inherited in Mendelian fashion.     

Detection of novel centromeric polymorphisms associated with alpha satellite DNA from human chromosome 11

Summary The pericentromeric region of human chromosomes is composed of diverse classes of repetitive DNAs, which provide a rich source of genetic variability. Here, we describe two novel centromeric polymorphisms associated with a subset of alpha satellite repetitive DNA, D11Z1, which is specific for human chromosome 11. Segregation and inheritance of the polymorphisms are demonstrated and their relative frequencies are determined. These polymorphisms may be useful genetic tools for distinguishing between individual chromosome 11 centromeres. In addition, these polymorphisms may be applied to the development of a centromerebased genetic linkage map of chromosome 11. Molecular models for the generation of these polymorphisms are discussed.     

Structure of the major block of alphoid satellite DNA on the human Y chromosome

Alphoid DNA is a family of tandemly repeated simple sequences found mainly at the centromeres of the chromosomes of many primates. This paper describes the structure of the alphoid DNA at the centromere of the human Y chromosome. We have used pulsedfield gradient gel electrophoresis, cosmid cloning and DNA sequencing to determine the organization of the alphoid DNA on each of the Y chromosomes present in two somatic cell hybrids. In each case there is a single major block of alphoid DNA. This is approximately 470,000 bases (475 kb) long on one chromosome and approximately 575 kb long on the other. Apart from the size difference, the structures of the two blocks and the surrounding sequences are very similar. However, one restriction enzyme, AvaII, detects two clusters of sites within one block but does not cleave the other. The alphoid DNA within each block is organized into tandemly repeating units, most of which are about 5.7 kb long. A few variant units present on one chromosome are about 6.0 kb long. These variants, like the AvaII site variants, are clustered. The 5.7 kb and 6.0 kb units themselves consist of tandemly repeating 170 base-pair subunits. The 6.0 kb unit has two more of these subunits than the 5.7 kb unit. Our results provide a basis for further structural analysis of the human Y chromosome centromeric region, and suggest that long-range structural polymorphisms of tandemly repeated sequence families may be frequent.     

Giemsa banding,quinacrine fluorescence and DNA-replication in chromosomes of cattle (Bos taurus)

Almost all the 30 chromosome pairs of cattle can be identified by their banding patterns made be visible by a Giemsa staining technique described previously. The banding pattern of the X chromosome shows striking similarities with the banding pattern of the human X chromosome. — The centromeric region of the acrocentric autosomes contains a highly condensed DNA. This DNA is removed by the Giemsa staining procedure as can be shown by interference microscopic studies. If the chromosomes are stained with quinacrine dihydrochloride these centromeric regions are only slightly fluorescent. — Autoradiographic studies with ³H-thymidine show that the DNA at the centromeric regions starts and finishes its replication later than in the other parts of the chromosomes.     

三种姬鼠的染色体比较研究

本文采用染色体分带技术（Ｇ－,Ｃ－带和银染色）,对中华姬鼠（Ａｐｏｄｅｍｕｓｄｒａｃｏ）、大林姬鼠（Ａ．ｐｅｎｉｎｓｕｌａｅ）和大耳姬鼠（Ａ．ｌａｔｒｏｎｕｍ）的核型进行了观察分析。结果表明：３种姬鼠的２ｎ均为４８。中华姬鼠的染色体均为端着丝点染色体。大林姬鼠的常规核型中,除１对中着丝点染色体（Ｎｏ．２３）外,其余均为端着丝点染色体。大耳姬鼠的核型中,有１３对端着丝点染色体,２对亚端着丝点染色体,１对亚中着丝点染色体和７对中着丝点染色体。中华姬鼠Ｃ－带核型中,所有染色体着丝点Ｃ－带都呈强阳性,异染色质非常丰富,Ｙ染色体整条深染。在大林姬鼠Ｃ－带核型中,Ｎｏｓ．７,１１,１５,２１,２２着丝点Ｃ－带弱化甚至近阴性,其余染色体着丝点异染色质Ｃ－带都呈现程度不同的阳性。且Ｎｏｓ．２,４,７有强弱不同的端位异染色质带。Ｘ染色体着丝点区有大块的异染色质斑带出现,Ｙ染色体整条深染。大耳姬鼠除Ｎｏｓ．３,４,１０,１２,１３染色体着丝点Ｃ－带很弱外,其余染色体着丝点Ｃ－带均呈阳性,并有８对（Ｎｏｓ．１６－２３）染色体出现异染色质短臂。从总体上看,大林姬鼠和大耳姬鼠的着丝点异染色质明显比中华姬鼠的少。中华姬鼠的Ａｇ－ＮＯＲ     

Flow cytometric analysis and sorting of plant chromosomes from Petunia hybrida protoplasts

A method to obtain a high metaphase index and thereafter a plant chromosome suspension is described for Petunia hybrida (2n = 14). Mesophyll protoplast cultures have been used, giving easily disrupted cell walls and a high percentage of dividing cells after 42 h. On 2.5 mM colchicine-treated cells, metaphase indexes reaching 10% were routinely obtained. The lysis medium in which the protoplast-derived cells were disrupted was a simplified culture medium. After chromosome release, samples were stained with Hoechst 33342 dye and analysed by flow cytofluorometry. The histogram of fluorescence intensities included three peaks of metaphase chromosomes and a duplication of this flow karyotype provoked by "monochromatid chromosome." This interpretation was established after flow sorting; micronuclei could also be observed and sorted. Of the 7 chromosomes, only the largest formed a distinct peak while the others were incompletely resolved, due to the similar DNA content of various chromosomes. Model distributions of Petunia hybrida chromosomes have been computed according to the relative chromosome length. The theoretical histograms indicated that low variability is indispensable for resolving distinctive chromosome peaks. The experimental flow karyotype was consistent with one of the models having CV of 2.5%.     

Karyotype of Norway spruce by multicolor FISH

The chromosomes (2n = 2x = 24) of Norway spruce are very large since their size reflects the huge amount of genomic DNA (2C = 30 × 10⁹ bp). However, the identification of homologous pairs is hampered by their high degree of similarity at the morphological level. Data so far presented in the literature were not sufficient to solve all the ambiguities in chromosome identification. Several genomic Norway spruce DNA clones containing highly repetitive sequences have been identified and characterised in our laboratory. Three of them were selected for fluorescent in situ hybridization (FISH) experiments because of their strong signals and suitability for chromosome identification: PATR140 hybridized at the centromeric site of three chromosome pairs; PAF1 hybridized in six subtelomeric and two centromeric sites; 1PABCD6 co-localized with the subtelomeric sites identified by PAF1. The statistical analysis of microscopic measurements of chromosomes in combination with the FISH signals of these probes allowed the unambigous construction of Norway spruce karyotype. We also compared the karyotype of Norway spruce with that of other spruce species to infer the number and kind of rearrangements that have occurred during the evolution of these species.Communicated by D.B. Neale     

Centromeric index measurement by slit-scan flow cytometry

We report here the application of slit-scan flow cytometry (SSFCM) in the classification of muntjac, Chinese hamster, and human chromosomes according to centromeric index (CI) and total fluorescence. Chromosomes were isolated from mitotic cells, stained with propidium iodide and processed through the SSFCM where fluorescence profiles were measured. The centromere for each profile was taken as the point of maximum difference between the measured profile and a standard profile having no centromeric dip. The areas under the profile on either side of the centromere were then calculated and the CI was calculated as the ratio of the larger area to the total area under the profile. Relative DNA contents for each chromosome were taken to be proportional to the total fluorescence. Mean CI's for muntjac chromosomes 1, 2, and X + 3 were 0.52, 0.88, and 0.73, respectively; CI's for Chinese hamster M3-1 chromosomes 1, 2, 5, 8, and M2 were 0.53, 0.55, 0.57, 0.77, and 0.86, respectively; and average CI's for chromosome groups 4 + t (X;5), 6 + 7 + Y, 9 + M1, and 10 + 11 were 0.56, 0.82, 0.58, and 0.60, respectively. These results were, on average, within 4.4% of CI measurements made by image cytometry. CI's measured for human chromosomes 9 through 12, were, on average, within 2.0% of those made by image cytometry.     

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.