Chromosome preparations of human blood lymphocytes—Evaluation of techniques

Several parameters of human lymphocyte culturing techniques and metaphase chromosome preparation procedures were studied and quantitatively evaluated in regard to their influence on the results of Q and G banding procedures. The culturing conditions were studied using³H thymidine incorporation as a parameter. A whole blood culturing technique using Ham's F12 medium was found to give optimal and consistent results. Colcemid concentration proved to be of no influence on chromosome contraction or on the number of metaphases obtained over the concentration range investigated. Prolonged exposure to colcemid was found to cause a decrease in the mean chromosome length but the absolute number of metaphases with a low degree of chromosomal contraction hardly decreased. Different spreading techniques were quantitatively analysed and factors important for the spreading of chromosomes were evaluated. Based on the results obtained, an optimal procedure is described which over a period of one year has given consistent results.     

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.     

Digoxigenin-labeled probes can detect single-copy genes in human metaphase chromosomes

A technique of in situ hybridization on metaphases of chromosomes by a digoxigenin-labeled probe is described. This technique was able to detect single DNA sequences of 2 and 7 kilobases. The results obtained were compared with those of a biotin streptavidin alkaline phosphatase-based detection system. The digoxigenin method was at least as efficient and sensitive as the biotin-streptavidin method.     

刺鳅X染色体DNA文库的构建

刺鳅（Mastacembelus aculeatus）是具有明显X和Y异形性染色体分化的淡水鱼。本实验室通过显微切割（Microdissection）和兼并引物PCR（DOP-PCR）方法,从雌性刺鳅中期染色体分裂相中分离获得X染色体并扩增其DNA,利用T载体和电转化方法,建立了刺鳅X染色体DNA质粒文库。该文库插入片段的平均长度约为500bp,理论上包含X染色体98％以上的序列。当用荧光原位杂交（FISH）来验证文库的专一性时发现,在无竞争性DNA杂交条件下,整个X和Y染色体上都表现出强烈的杂交信号,并且常染色体上也出现一些随机散布信号;当含有竞争性DNA时,常染色体上的信号消失,仅性染色体上异染色质区域保留有较强信号。就此,本文对刺鳅性染色体上的序列类型进行了探讨。     

Multi-locus (ML)-FISH is a reliable tool for nondisjunction studies in human oocytes

In the present study, we developed a fluorescence in situ hybridization (FISH) strategy, which allows a reliable determination of the chromatid number of specific chromosomes in mature human oocytes. 168 unfertilized oocytes were analyzed by dual-color FISH with two direct-labeled locus-specific DNA probes for chromosome 13 and 21. To exclude FISH failures, metaphases with abnormal signal patterns were reanalyzed by multi-locus-FISH (ML-FISH) for chromosome 13 and 21. Following dual-color FISH, abnormal signal patterns were detected in 21 out of 108 metaphases (19.4%). 17 of these metaphases were reanalyzed by ML-FISH. In contrast to the first FISH, seven metaphases showed normal signal patterns after rehybridization, whereas ten metaphases remained abnormal. Out of these real aneuploid metaphases, five showed gain or loss of a single signal (= chromatid), two showed missing double signals (= chromosome) and three showed both. In conclusion, locus-specific FISH probes facilitate differentiation between first meiotic nondisjunction of whole chromosomes and prematurely divided chromatids. Moreover, simultaneous hybridization with a second locus-specific probe on the same chromatid (ML-FISH) helps to differentiate between FISH failures and real meiotic division errors and therefore, allows a more reliable analysis of aneuploidies in human oocytes.     

No multistrandedness in mitotic chromosomes of Drosophila melanogaster

Feulgen cytophotometric measurements of neuroblasts in the first and third instar larvae of Drosophila melanogaster reveal the same DNA content for metaphases with chromosomes of different size. The total absorbance of all measured metaphases gives the four-fold value of that of the spermatids. Accordingly there seem to be no reasons to retain the assumption of a multistranded structure for the large chromosomes of metaphases in the third instar larvae.     

Fluorescence polarization and pulse width analysis of chromosomes by a flow system.

Isolated Chinese hamster chromosomes have been analyzed using a multiparameter computer-controlled cell sorter to obtain information about unique properties of individual chromosomes. Parameters other than DNA content were sought that would further aid in distinguishing among chromosomes. The polarized emission of the DNA-specific bis-benzimidazole dye Hoechst 33342 was measured for each class of chromosomes identified by a distinct peak, i.e., differeing in DNA content. The emission anisotropy values for all chromosome classes was constant (emission anisotropy = 0.30), and the same value was obtained for purified DNA in solution. Pulse width was found to be a good parameter for resolving chromosomes as a function of total emission in the case of the smaller chromosomes and orientation (i.e., arm length) for large chromosomes. A simple theoretical model for predicting the pulse shapes generated by arbitrarily oriented, thin, rigid rods was developed and applied to the evaluation of the experimental data.     

Late replicating bands of human chromosomes demonstrated by fluorochrome and Giemsa staining.

The addition of thymidine (TdR) to cells growing in a medium containing 5-bromodeoxyuridine (BUdR) at the end of the first replication cycle results in the incorporation of TdR into the late replicating DNA regions. These sites can be visualized by staining the metaphase chromosomes with the fluorescent dye "33258 Hoechst" or a "33258 Hoechst" Giemsa procedure. A sequence of late replication patterns has been established in metaphase chromosomes of cultured human peripheral lymphocytes. The patterns are in agreement with those obtained by the standard autoradiographic procedures, but are more accurate. As is known from autoradiography, late replicating bands are in the position of G or Q bands. The "33258 Hoechst" Giemsa staining procedure of chromosomes which have replicated in the presence of BUdR first and in TdR for the last 2 hrs of the S phase is preferable to the currently used Giemsa banding techniques: the method yields very well banded metaphases in all preparations examined, as the chromosome structure is not disrupted by the pretreatment. The bands are very distinct, even in the "difficult" chromosomes (e.g. No. 4, 5, 8 and X). In female cells the late replicating X chromosome can be identified by its size and staining pattern. In addition to the replication asynchrony, the sequence of replication within both X chromosomes in female cells is not absolutely identical. The phenomenon of a phase difference in replication between the homologues is not a peculiarity of the X chromosome, but can be found in all autosomes as well as in homologous positions on the chromatids of individual chromosomes.     

A one-step efficient and specific non-radioactive non-fluorescent method for in situ hybridization of banded chromosomes

A non-radioactive method for in situ hybridization of cosmid probes to metaphase chromosomes is described. Two procedures are involved: (i) hybridization with a cosmid probe labelled by nick translation in the presence of digoxigenin dUTP. The signal is visualized by an alkaline phosphatase conjugated antibody. (ii) FPG banding of the chromosomes. The steps involved in these two procedures are combined in an order which allows simultaneous observation of the banding pattern and the hybridization signal. The metaphases can thus be analysed after a single photographic step. This technique is considerably simpler than the method used previously.     

Mitotic dynamics of micronuclei induced by amiprophos-methyl and prospects for chromosome-mediated gene transfer in plants

Summary Mitotic dynamics and the kinetics of mass induction of micronuclei after treatment of Nicotiana plumbaginifolia cell suspensions with the spindle toxin amiprophos-methyl (APM) are reported. The addition of APM to suspension cells resulted in the accumulation of a large number of metaphases. The course of mitosis was strikingly different from normal. Metaphase chromosomes showed neither centromere division nor separation of chromatids. Single chromosomes and groups of 2 or more chromosomes were scattered over the cytoplasm. After 5–6 h of APM treatment, chromosomes decondensed and formed micronuclei. When treatment duration was increased, the frequency of cells with micronuclei as well as those showing lobed micronuclei increased. Similarly, with an increase in APM concentration the frequency of cells with micronuclei increased. After removal of APM, chromosome grouping disappeared, cells showing lobed micronuclei further increased and mitoses with doubled chromosome numbers appeared in the next cell division. Cytological observations and DNA measurements revealed that several sub-diploid micronuclei containing 1 or a few chromosomes can be obtained, and that flow cytometry can detect and sort out these micronuclei. The applications of micronuclei for genetic manipulation of specific chromosomes and gene mapping are indicated.     

Preparation of monosomal lines containing individual human 15, 21, and X chromosomes]

Sorting of human--mouse or human--hamster hybrid cells with particular human chromosomes was performed by in situ hybridization. Total human genomic DNA was heavily labelled with. H and hybridized to metaphase spreads from hybrid clone cells. The method allowed us to not only identify human chromosomes in hybrid cells but also to detect terminal translocations and insertions from 1-2 bands in length to large ones. Biochemical markers of some human chromosomes were analysed using electrophoretic technique in the clones selected. Cytogenetic analysis (G staining) of these clones was made to visualize human chromosomes. Total 99 initial hybrid human--hamster and 26 human--mouse clones were obtained. 53 clones were analysed by in situ hybridization, only one of them being monochromosomal; the latter contained human X chromosome on the background of Chinese hamster chromosomes. Two other monochromosomal clones containing particular 15 and 21 chromosomes, respectively, were obtained by more complicated way from human--mouse hybrid clones using back selection, repeated hybridization and passing through a number of subsequent subclonings.     

Chromosomal DNA content of sweet pepper determined by association of cytogenetic and cytometric tools

The nuclear DNA content of sweet pepper (Capsicum annuum L. var. annuum, 2n = 24) has been measured by flow and image cytometries but the DNA content of each chromosome of this species has not yet been regarded. DNA content of individual chromosomes has been quantified by the flow karyotyping technique, which requires a great quantity of intact metaphasic chromosomes and methods that allow the characterization of individual chromosomes; however, the obtainment of adequate number of metaphases can be difficult in some species like C. annuum. In order to estimate the DNA content of each C. annuum var. annuum cv. "New Mexican" chromosome, flow and image cytometries were associated with the cytogenetic methodology. First, the DNA amount (2C = 6.90 pg) was established by flow cytometry. Integrated optical density (IOD) values were calculated by image cytometry for each Feulgen stained metaphasic chromosome. Then, by distributing the correspondent metaphasic value (4C = 13.80 pg) proportionally to average IOD values, the following chromosomal DNA contents were obtained in pg: 0.74 (chromosome 1), 0.67 (2), 0.61 (3, 4), 0.60 (5), 0.59 (6, 7), 0.58 (8), 0.57 (9), 0.56 (10) and 0.39 (11, 12). This study reports an alternative and reproducible technique that makes quantifying the chromosomal DNA content possible.     

Flow cytometric DNA and cytogenetic studies in human tumors: a comparison and discussion of the differences in modal values obtained by the two methods

The numerical chromosome values in 53 human tumors were determined and compared with the modal DNA values as measured by flow cytometry. In tumors with chromosome counts in the diploid and tetraploid range, the modal DNA values were found to correspond to the modal values based on the chromosome counts. In tumors with chromosome counts in the triploid range, however, the modal DNA values were about 15% higher than expected. In order to explain this difference, the ratio between large and small chromosomes in the karyotyped metaphases was assessed. In addition, the DNA content of individual chromosomes, including markers and minutes, was calculated as a reflection of the DNA content of the whole cell. The ratio of large to small chromosomes did not deviate from the normal ratio found in cells with diploid, triploid, and tetraploid chromosome counts. Neither difficulties in karyotyping nor short-comings in the flow cytometric methodology could be used to explain the discrepancy between the expected and empirical modal DNA values. Some of the chromosomes in triploid tumors may, therefore, contain an increased amount of DNA.     

LBA technique in the detection of chromosome variants

Summary A method is described (LBA method) which uses DNA replication pattern in the detection of chromosome variants in man. In Part I, results on chromosomes with known sites of Q-variants, i.e., 5 pairs of acrocentrics, as well as 3 and 4, were presented.Fourty-one variants were detected in a total of 40 acrocentrics. Twentyeight of them were detected only by the LBA technique; 11 of them being in short arms and 17 of them in centromeres. Seven variants, including 4 of those in satellites, were detected only in QFQ-stained metaphases. Six short-arm variants were observed by both methods. It appears that a sequential QFQ-LBA technique is very useful in the detection of variants in D- or G-group chromosomes.     

Intra- and interspecific variations in nuclear parameters of two closely related species of Narcissus L.

Nuclear DNA amount, nuclear area, genome volume and karyotype length were analysed in different populations of two closely related species of Narcissus. There are intra- and interspecific variations in these parameters. 4C DNA amount and karyotype length, on one hand, and 2C DNA amount and telophase nuclear area, on the other, are not correlated. It seems that DNA content and chromosome length are independent parameters. However, 4C DNA content and karyotype volume are correlated, and are also correlated to different density estimations (4C DNA to Kar.length & 2C DNA to telophase area). These facts suggest that the relative length of the chromosomes is genetically controlled and that it is independent of the DNA that they contain. It seems that the interpopulational differences in DNA content are correlated with length changes of small segments in almost all chromosomes.     

Karyotype and chromosomal banding pattern in Heteropeza pygmaea

The chromosomes of somatic and germ line cells of female embryos produced by paedogenesis were studied. The haploid set in somatic cells consists of one long submetacentric chromosome, one large acrocentric, one medium metacentric and two small acrocentrics. The length vs arm index karyogram makes it possible to distinguish all but the two pairs of small acrocentric chromosomes. — Attempts were made to develope a method for banding pattern visualization. The best result was obtained using trypsin which induced banding in the chromosomes of the somatic cells and occasionally also of the germ line cells. The resulting banding patterns were frequently not identical in members of a chromosome pair. There was also a variation between metaphases within an embryo as well as from different embryos. Some tentative explanations for these results are discussed.     

Chromosome abnormalities in tuberous sclerosis

Summary In fibroblasts cultured from biopsies of the skin lesions of six patients with tuberous sclerosis (TS) there was a variable but consistent degree of karyotypic variation. Premature centromere disjunction (PCD) of all or part of the chromosomes, micronuclei, an increased incidence of breaks, dicentric chromosomes and the presence of polyploid metaphases were found in all cultures. The PCD was of the type encountered in Roberts syndrome and its frequency varied from 8% to 30%. In metaphases with PCD of one and of two chromosomes, the chromosome involved were identified, and chromosome 3 was involved 21 times among 59 chromosomes with PCD. Chromosome 3 tends to be preferentially involved in dicentric formation. In lymphocyte cultures from the same patients there were no metaphases with PCD, but there was a slight increase of breaks and the presence of dicentric chromosomes, also involving chromosome 3. Polyploid metaphases were increased in some of the cases. Karyotypic variation can be considered a cellular phenotypic characteristic of TS in fibroblasts cultured from the skin lesions, and its type indicates disturbances in the mechanics of centromere division and of chromosome distribution at cell division.     

Photometric determination of the DNA distribution in the 24 human chromosomes

From five normal individuals the DNA content and the DNA arm ratios of the 24 metaphase chromosomes were determined by means of scanning densitometry of photographic negatives of Feulgen-stained metaphase preparations. The results showed high reproducibility of the measuring procedure. The obtained DNA values for the 24 chromosomes showed general correspondence between the individuals. No differences between males and females were found. The DNA arm ratios showed somewhat higher inter-individual variability, especially for the acrocentric chromosomes. Our data are in agreement with other data published so far, which were obtained with somewhat different techniques, indicating that the DNA content of the individual human chromosomes in general is highly constant. Attempts were made to distinguish chromosomes by their DNA content and DNA ratio. It appears that classification of chromosomes using these parameters cannot compete with classification according to the banding patterns. Determination of the total DNA content and DNA distribution along the metaphase chromosomes may, however, provide a frame of reference for cytochemical methods directed towards the localization and quantification of molecular properties of the chromosome.     

Cytogenetics of three breeds of river buffalo (Bubalus bubalis L.), with evidence of a fragile site on the X chromosome

The cytogenetic study of 182 river buffalo (Bubalus bubalis L., 2n = 50) of Murrah, Mediterranean and Jaffarabadi breeds, from the State of S?o Paulo, was carried out to characterize their chromosomes and to detect possible chromosomal abnormalities. The karyotypes were indistinguishable with conventional staining as well as with C and replication R banding techniques. In about 44% of the sample (8 males and 72 females), an X marker chromosome due to a fragile site was shown. The frequency of metaphases expressing the fragility site on the X was highly variable, from 2.86 to 41.03%. In females, the fragile site, rarely appeared on both X chromosomes. Most of the metaphases showed only 1 marker chromosome. In R-banded metaphases using 5-bromodeoxyuridine (BrdU) treatment, it corresponded in general to the late replicating X chromosome. No correlation between the X fragile site and altered phenotype was found. Structural and numerical chromosome rearrangements were ruled out in the present sample of buffalo.     

Non-histone proteins and the structure of metaphase chromosomes

Differential intensity of fluorescence corresponding to the banding patterns found in single metaphases can be obtained with isolated Chinese hamster chromosomes using the fluorochrome Hoechst 33258. Removal of histones from the chromosomes with 0.2 N HCl causes an approx. 50% increase in overall size, but does not abolish the gross metaphase morphology of the chromosomes or the ability to give their characteristic fluorescent banding patterns. In an attempt to study further the factors maintaining the characteristic metaphase structure, we have treated acid-extracted isolated chromosomes with DNase I, which was found to solubilize over 99% of the DNA content, while leaving stable ‘core’ structures which retain the basic features of metaphase chromosomes such as centromeric regions and defined chromatids. The cores appear to consist mainly of non-histone protein: they are destroyed by proteolytic action and unaffected by ribonuclease A. The structural implications of these findings are discussed.