Flow cytogenetics of uncloned and cloned Chinese hamster cells

Flow cytometry has greatly facilitated the routine use of DNA content as a cellular indicator of the stages of the cell cycle and ploidy. DNA content can also be used to distinguish individual chromosomes. Fluorescent staining of chromosome DNA was done with a combination of ethidium bromide and mithramycin in hypotonic solution. Subsequent detergent treatment of the cells with Triton X-100 facilitated chromosome isolation. DNA flow cytometry of chromosomes of four established uncloned Chinese master cell lines showed 10 to 12 major subpopulations of chromosomes with varying degrees of overlap in the range of low and intermediate DNA content. Cloning of B14F28 cells, the line with the largest heterogeneity in chromosome number and DNA content, considerably reduced the dispersion in chromosome number and improved the resolution of DNA content distributions. Thus, cloned cells with a relatively homogeneous karyotype permit better discrimination of chromosome subpopulations by DNA content than uncloned cells and provide a more sensitive system to study mutagenic effects.     

Correlation between flow cytometric determination of nuclear DNA content and chromosome number in somatic hybrids within Brassicaceae

Summary Fourty-one somatic hybrids from two species combinations, Brassica oleracea + B. campestris and B. napus + Eruca sativa, were analysed for chromosome number and nuclear DNA content. The DNA content was measured in a flow cytometer using two internal standards as references and when related to the chromosome number a correlation of 0.91 was found. The chromosome number of the hybrids could be determined with an accuracy of ±10% by using flow cytometry, and the smallest statistically significant difference in DNA content between two individuals was 0.23 pg DNA/cell.     

HBsAg重组DNA转化中国地鼠卵巢细胞二氢叶酸还原酶阴性突变株的细胞染色体变化及致瘤性

整合了含乙型肝炎病毒表面抗原(HBsAg)基因和dhfr基因的CHO-dhfr~-细胞,其染色体的畸变率和畸变类型都比亲代CHO-dhfr~-细胞高。但转化前后两系细胞的重要特性都未发生变异,即两者的染色体总数无差别,都是20条。两系细胞株接种裸鼠,均未发现有致瘤性。     

Quantitative karyology of some species ofLuzula

The dimensions of metaphase chromosomes and nuclear DNA contents were measured in eight species ofLuzula. The 2 C DNA contents ranged from 8.51 pg inL. purpurea to 0.55 pg inL. pilosa. Total chromosome volume shows a linear relationship with DNA content; however, the total chromosome length of the complement of the different species is approximately constant. Nucleolar volume and the number of chromocentres in the different species also show a relationship with DNA content. Taken together, these data suggest that while chromosome fragmentation could have generated the present-day range of chromosome numbers in the genus, there have also been changes in the total quantity of DNA with the result that species with similar chromosome numbers have different DNA contents. The relationships of DNA content with chromosome volume inLuzula and other genera are compared and the differences discussed.     

Evidence for altered cell-cycle traverse of the non-modal cells of the heteroploid MCa-11 line

Classic stem cell theory states that the growth of heteroploid cell populations is due to the proliferation of 'main stemline'cells with modal DNA content and chromosome number. Cells with non-modal DNA content and chromosome number are thought to be blocked and/or destroyed at mitosis. To test this, we studied two chromo-somally stable cell populations (mouse bone marrow and WCHE-5 cells) and one heteroploid, chromosomally diverse cell line (MCa-11). The heteroploid MCa-11 cells showed significant [³H]dT labelling for cells with DNA contents below the modal G_o/G₁ peak and above the modal G₂ peaks ( P <0.001). This was consistent with the presence of cells with the non-modal DNA content that were engaged in replicative DNA synthesis. A percentage labelled mitosis analysis showed that MCa-11 cells with non-modal DNA content and chromosome number were able to complete mitosis, although with prolonged pre-karyokinetic time. These results suggest that many non-modal cells present in heteroploid cell populations are capable of continued proliferation.     

Discrepancies among the metaphase, telophase, and the G0/G1 and G2 DNA peaks of heteroploid cell lines

Heteroploid cell populations often show narrow peaks of G0/G1 and G2/M DNA content and broadly distributed chromosome numbers. This was originally explained by the selective metaphase arrest of the cells that have non-modal chromosome numbers. To test whether this explanation applies, we have measured the chromosome number distributions, as well as the G0/G1, G2, metaphase (M), and telophase (T) DNA distributions, of the cell lines WCHE-5, MCa-11, and HL-60. The WCHE-5 cells had narrowly distributed chromosome numbers and G0/G1 G2, M, and T DNA peaks. The MCa-11 and HL-60 cells also had narrowly distributed G0/G1 and G2 DNA peaks, but broadly distributed chromosome numbers and M and T DNA peaks. The widths of the MCa-11 and HL-60 M- and T-cell DNA peaks were similar to those of their chromosome number peaks, suggesting that all cells were completing mitosis, regardless of chromosome number or DNA content. Thus, selective metaphase arrest does not seem to be the cause of the narrow G0/G1 and G2 DNA peaks of heteroploid cell populations.     

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.     

人乳头状瘤病毒协同60钴照射促进食管上皮细胞恶性转化

为探明人乳头状瘤病毒(HPV)和促癌剂对食管上皮致癌作用,人胚食管上皮细胞转染HPV协同60钴(60Co)放射观察其恶性转化.用HPV18E6E7AAV转染的人胚食管上皮(SHEE),培养至13代,分为4组,实验组分别用60Co2、4、8Gy照射,每周1次共4周;SHEE未经照射为对照组.细胞形态用相差显微镜观察;细胞DNA合成和定量用3H-TdR掺入和用流式细胞仪分析;染色体众数用常规方法分析;致瘤性用软琼脂培养和裸小鼠接种;HPVDNA用PCR检测.经60Co照射后细胞呈凋亡和坏死(危象期).8周后SHEE 4Gy组细胞增殖,增殖指数(34%)和3H-TdR摄入增高,软琼脂培养和裸鼠接种出现致瘤性.对照组SHEE组细胞增殖指数24%,伴有少数3H-TdR掺入,裸鼠未成瘤.染色体众数:对照组,58～62;4Gy组,63～65;两组HPV18E6E7 PCR呈阳性条带.此结果表明,用HPV18E6E7协同60Coγ射线可以使人胚食管上皮恶性转化,60Co γ射线有加速食管上皮细胞恶性转化作用.     

Rapid detection of aneuploidy in <Emphasis Type="Italic">Musa</Emphasis> using flow cytometry

We report a procedure for the rapid and convenient detection of aneuploidy in triploid Musa using DNA flow cytometry. From a population of plants derived from gamma-irradiated shoot tips, plants were selected based on aberrant morphology and their chromosome numbers were counted. Aneuploids plants with chromosome numbers 2n=31 or 32 were found as well as the expected triploid plants (2n=3x=33). At the same time, the nuclear DNA content of all plants was measured using flow cytometry. The flow cytometric assay involved the use of nuclei isolated from chicken red blood cells (CRBC), which served as an internal reference standard. The relative DNA content of individual plants was expressed as a ratio of DNA content of CRBC and Musa (DNA index). In order to estimate the chromosome number using flow cytometry, the relative DNA content of plants with unknown ploidy was expressed as a percentage of the DNA content of triploid plants. The classification based on flow cytometry fully agreed with the results obtained by chromosome counting. The results indicated that flow cytometry is a convenient and rapid method for the detection of aneuploidy in Musa.     

麻竹花药诱导再生植株的染色体倍性分析

为阐明麻竹(Dendrocalamus latiflorus)花药培养再生植株的染色体倍性, 利用流式细胞仪和染色体标本制备方法对麻竹再生植株嫩叶DNA的含量和根尖染色体数目进行了研究。结果表明: 100株花药培养再生植株中有4株为六倍体, 96株为十二倍体。该结果进一步验证了麻竹花药培养体系, 对麻竹遗传改良和功能基因组学研究具有重要意义。     

Embryogenesis and genetic stability in long term megagametophyte-derived cultures of larch

Embryogenic cultures derived from megagametophytes of Larix decidua were maintained for up to 17 years. A few lines were divided into sub-lines, which were maintained in the same manner as the others. Embryogenic tissue was grown on 1/2 strength LM medium supplemented with glutamine and casein hydrolysate at constant temperature and light regimes. Chromosome counts were conducted at various times. DNA content was assessed by flow cytometry. Embryogenesis was monitored with each transfer and records of all appearances of green mature embryos were kept. Chromosome number was found to vary. DNA content and chromosome number, both of which had doubled a number of years after initiation, stabilized around 24 chromosomes for most cultures. A few lines showed substantial increases in chromosome number. One of these lines lost vigour and died. Another line showed a further doubling of DNA content. No lines were embryogenic over the entire period. Embryogenicity was lost completely in some lines, but in others the loss was temporary, as periodic restoration of embryogenesis was observed.     

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.     

麻竹花药诱导再生植株的染色体倍性分析

为阐明麻竹（Dendrocalamus latiflorus）花药培养再生植株的染色体倍性,利用流式细胞仪和染色体标本制备方法对麻竹再生植株嫩叶DNA的含量和根尖染色体数目进行了研究。结果表明：100株花药培养再生植株中有4株为六倍体,96株为十二倍体。该结果进一步验证了麻竹花药培养体系,对麻竹遗传改良和功能基因组学研究具有重要意义。     

Egg maturation and parthenogenetic recovery of diploidy in the scorpion Liocheles australasiae (Fabricius) (Scorpions, ischnuridae)

In the scorpion Liocheles australasiae, egg maturation and parthenogenetic recoveries of chromosome number and nuclear DNA content were examined by histological, karyological observations and quantitative measurements of DNA. The primary oocyte becomes mature through two successive maturation divisions. The first maturation division takes place in the primary oocyte to produce a secondary oocyte and a first polar body. The second maturation division soon occurs in the secondary oocyte, in which the nucleus is divided into a mature egg nucleus and a second polar body nucleus, not followed by cytoplasmic fission. The first polar body, in one case, was successively divided into two second polar bodies; in the other case it was not divided. In either case, these polar bodies remained attached to the early embryo. The fate of these polar bodies during further embryogenesis were studied. In the karyological analysis, the chromosome number was divided into two groups, one from 27-32, the other was 54-64. The former was presumably the metaphase chromosome number at the meiotic division; the latter was presumably the metaphase chromosome number at the mitotic division. DNA content in the diploid nucleus of the primary oocyte, doubled before the maturation divisions, was reduced through the maturation divisions by one-half in the nuclei of the secondary oocyte and the first polar body and by one-fourth in the nuclei of the egg and the second polar bodies. The first reduction of DNA content corresponded to halving the number of the chromosomes in the first maturation division and the second to the nuclear division in the secondary oocyte. These reductions represent a common process of egg maturation, except the final production of the mature egg with two haploid nuclei, an egg nucleus, and a second polar body nucleus. These two nuclei, which were formed apart in the mature egg, drew near to fuse into a zygote nucleus. The chromosome number and nuclear DNA content were doubled in the zygote and each blastomere in embryos, supporting the hypothesis that the egg nucleus fuses with the second polar body nucleus and this conjugation initiates subsequent embryonic development.     

Establishment and characterization of human uterine cervical epidermoid carcinoma cell line HHUS containing HPV 59 DNA

The cell line designated HHUS was established from human uterine cervical keratinizing squamous cell carcinoma. The HHUS cell line was subcultivated more than 70 times within 3 years. The cultured cells, polygonal or spindle, with neoplastic and pleomorphic features, appeared epithelial in shape, with a pavement-like arrangement and grew in multi-layers without contact inhibition. The chromosome number was varied from 40 to 88, and the modal number was stable in diploid range. The cultured cells produced keratinizing squamous cell carcinomas by heterotransplantation into the subcutis of nude mice. The HHUS cells were characterized as producing large amounts of SCC, in vitro and possessing HPV-59 DNA genomes.     

宽体沙鳅的染色体核型与DNA含量分析

为了解宽体沙鳅(Sinibotia reevesae)的种质特征,以野生宽体沙鳅为材料,采用腹腔注射植物血球凝集素(PHA)和秋水仙素,肾组织细胞短期培养、常规空气干燥法制备染色体标本,并对其核型进行分析。以鸡(Gallus gallus)血细胞DNA含量(2.50 pg/2c,2c指二倍体)为标准,用流式细胞仪测定宽体沙鳅外周血细胞的DNA含量。结果表明:(1)宽体沙鳅的染色体数目为2n=96,核型组成公式为2n=36m+14sm+20st+26t,染色体总臂数NF=146;未发现与性别相关的异型染色体。(2)宽体沙鳅的DNA含量为(2.60±0.36)pg/2c。通过与其他26种鳅科鱼类核型进行比较,发现宽体沙鳅属于鳅科鱼类中的特化类群,其染色体核型经历了罗伯逊易位和染色体多倍化等过程。本研究结果可为宽体沙鳅种质资源保护和细胞遗传学研究提供基础资料。     

Size and physical map of the chromosome of Haemophilus influenzae.

A variation of pulse-field electrophoresis, field-inversion gel electrophoresis, was used to determine the size and physical map of the chromosome of Haemophilus influenzae. The DNA of H. influenzae had a low G + C content (39%) and no restriction sites for the enzymes NotI or SfiI. However, a number of restriction enzymes (SmaI, ApaI, NaeI, and SacII) that recognized 6-base-pair sequences containing only G and C nucleotides were found to generate a reasonable number of DNA fragments that were separable in agarose gels by field-inversion gel electrophoresis. The sizes of the DNA fragments were calibrated with a lambda DNA ladder and lambda DNA restriction fragments. The sum of fragment sizes obtained with restriction digests yielded a value for the chromosome of 1,980 kilobase pairs. Hybridization of a labeled fragment with two or more fragments from a digest with a different restriction enzyme provided the information needed to construct a circular map of the H. influenzae chromosome.     

Nuclear DNA content and chromosome number in some diploid and tetraploid Centaurea (Asteraceae: Cardueae) from the Dalmatia region

Given the paucity of information about genome size in the genus Centaurea, nuclear DNA content of 15 Centaurea taxa, belonging to four subgenera and six different sections, has been investigated for the first time. The sample concerns 21 populations from the Dalmatia region of Croatia. The 2C DNA content and GC percentage were assessed by flow cytometry and chromosome number was determined using standard methods. Genome size of studied Centaurea ranged from 2C=1.67 to 3.72 pg. These results were in accordance with chromosome number and especially with ploidy level that varies throughout this group; 2C DNA values ranged from 1.67 to 3.43 pg for diploid, and from 3.19 to 3.72 for polyploid taxa. No significant intraspecific variations of DNA amount were found between two subspecies of C. visiani and C. ragusina, nor between two varieties of C. gloriosa. However, some populations of C. glaberrima and C. cuspidata showed a significant difference in DNA amount. Three different basic chromosome numbers were observed in studied species (x=9, 10, and 11). The most frequent basic number was x=9. C. rupestris, C. ragusina ssp. ragusina, and C. r. ssp. lungensis possessed x=10 and C. tuberosa x=11. The species with a basic chromosome number of x=9 had a small genome size and the smallest chromosomes (on average 0.09 to 0.12 pg/chromosome) but frequently present polyploidy. Centaurea ragusina ssp. ragusina and C. r. ssp. lungensis had a mean base composition 41.3% GC.     

禾本科植物基因组大小与种子特性的相关性分析

在检索植物C值数据库和种子数据信息库的基础上,对禾本科282种植物的基因组参数（倍性、染色体数、C值、GS值和平均每条染色体DNA含量）和种子特性（千粒重、含油量和蛋白含量）进行了统计分析。分析结果表明,禾本科植物C值在0.35～19.7 pg,大多位于1.6～3.2 pg之间,呈偏正态分布,种子千粒重在0.05～252 g,绝大多数位于0.05～20.0 g,呈偏态分布,二者平均值分别为4.14 pg和7.1 g。随着染色体倍性增加,C值在二倍体到八倍体之间显著增加,而GS值和平均每染色体DNA含量在二倍体到六倍体之间显著下降（p<0.05)。雀麦属和羊茅属随着倍性增加,C值显著增加,表现与禾本科相似的变化规律,GS值下降却不明显。相关性分析表明,禾本科植物C值与倍性、染色体数、GS值及平均每条染色体DNA含量均呈极显著正相关（p<0.01),与种子千粒重无相关性。GS值与染色体数、倍性呈极显著负相关,而与千粒重呈极显著正相关。C值与种子含油量呈显著负相关,但与种子蛋白含量之间无相关性。以上结果表明,禾本科植物在系统演化和进化过程中,主要通过倍性和染色体的增加来增大C值,可能通过某种删除或丢失机制来降低GS值,从而保持较高的适应环境能力和进化速率。     

恶性横纹肌样瘤（MRT）的起源研究——高变异率HeLa细胞致裸鼠产生MRT的实验研究

HeLa细胞KB株、X株、NM20／X株、H株的染色体众数依次为60±3（超二倍体）、62±3（超二倍体）、68±3（超二倍体和亚四倍体）和78±2（亚四倍体）,所占比率分别为72%～76%,69%,52%和40%。在纯化3代的肿瘤阴性对照二倍体猫肾（染色体众数38所占比率80%）和犬肾原代细胞皮下接种裸鼠的致癌／致瘤率分别为0%（0／22）和0%（0／10）,X株HeLa细胞冻融裂解物皮下接种裸鼠产生进行性缩小肿瘤的比率为20%（1／5）的前提下,HeLa细胞KB株、X株、NM20／X株皮下接种裸鼠产生进行性生长恶性肿瘤的比率分别为100%（10／ 10）,100%（25／25）和100%（5／5）,H株细胞皮下接种裸鼠产生恶性肿瘤的比率为50%（5／10）。其中,只有HeLa细胞KB株10～11代（染色体结构畸变率高达20%,出现18%双着丝点和2%断片）以超高数量接种的1组4只裸鼠（0.17ml12.75×10