Chromosome DNA Content Research Articles

The DNA content of Chinese hamster meiotic metaphase chromosomes.

DNA values of Chinese hamster male meiotic metaphase chromosomes were measured by slide-based Feulgen cytometry. All the autosomes were distinguishable on the basis of their DNA content. No significant differences were found between the autosomes of the two male animals studied, but a significant difference was found in the DNA content of the sex-chromosome pair between these two animals.

A new experimental model for in vivo studies on differentiation and proliferation of leukemic cells using a mouse myeloid leukemia aneuploid line

We developed an experimental model to investigate in vivo differentiation and proliferation of leukemia cells using mouse myeloid leukemia aneuploid cells (LL-2) and syngeneic SL mice. The LL-2 cells were near-tetraploid cells isolated from mouse myeloid leukemia cell line M1 (clone D501). In suspension culture, the LL-2 cells were myeloblastic and grew well like parent D501 cells, but were distinct from the parental cells due to the large size of their nucleus, double chromosome number and DNA content. The LL-2 cells as well as D501 cells could be induced to differentiate in vitro into mature macrophage-like cells by a protein inducer of differentiation. After transplantation of 4 × 10 6 LL-2 cells into the intraperitoneal cavity of syngeneic SL mice, most of them died of leukemia within 10 weeks. On microscopic examination of the peritoneal cells of the mice, the transplanted LL-2 cells were clearly distinguishable from normal host cells by the size of their nucleus. We determined the increase in the LL-2 cells in the peritoneal cavity by morphological examination of the large-sized LL-2 cells. Survival times of the mice inoculated with the LL-2 cells were prolonged by administrations of an inducer of differentiation, poly(I)·poly(C). We found morphological changes in the peritoneal blastic LL-2 cells to mature macrophage-like cells after the serial administrations of poly(I)·poly(C) to the recipient mice. Thus the aneuploid LL-2 cells that grow in syngeneic mice may be useful to study in vivo differentiation and proliferation of leukemia cells, and to develop a therapeutic strategy of leukemia using various treatments including differentiation inducers.

In vivo effects of hyperdiploid Ly-1+ B cells of NZB origin.

Cells with increased chromosome number and DNA content have been found in the spleens of old NZB mice. These hyperdiploid cells are of clonal origin and demonstrate discrete IgH chain gene rearrangements by Southern blot analysis. In this report, hyperdiploid cells were analyzed by three-color flow cytometric techniques and found to be Ly-1+ B cells which were dull for Ly-1 and bright for surface IgM. These cells, unlike typical diploid Ly-1+ B cells, were negative for B220/6B2 and surface IgD. Hyperdiploid Ly-1+ B cells were found to be the predominant splenic subpopulation in animals receiving a spleen cell transfer from donors which possessed hyperdiploid Ly-1+ B cells. (NZB x DBA/2)F1 recipients of NZB spleen cells demonstrated a 10- to 1000-fold increase in Ly-1+ B cells in the spleen but showed no increased levels of Ly-1+ B cells in the peritoneum. Nearly all the splenic Ly-1+ B cells were hyperdiploid with the phenotype of the NZB parent. Cytogenetic analysis revealed that all the hyperdiploid cells were NZB donor cells. These findings suggest that the increase in splenic Ly-1+ B cells in the F1 recipients was due to expansion of injected splenic hyperdiploid Ly-1+ B cells of NZB origin. All of the F1 recipients of NZB hyperdiploid Ly-1+ B cells demonstrated a significant decrease in endogenous B cells as well as decreased serum IgM and anti-ssDNA autoantibodies. These studies suggest that hyperdiploid Ly-1+ B cells are different from typical peritoneal Ly-1+ B cells both in the lymphoid organs to which they home and in their proliferative capacity. NZB hyperdiploid Ly-1+ B cells, which may arise as a natural consequence of hyperactive Ly-1+ B cells, may play an immunoregulatory role in the spleen.

Chromosome numbers, genome sizes, cell volumes and evolution of snake‐head fish (family Channidae)

Chromosome number, C‐value and cell volume studies were carried out on three species of the genus Channa, viz., C. punctatus, C. striatus and C. gachua. The chromosome number, karyotypic structure and DNA content per cell along with cell volume are reported and described. A series of chromosomal rearrangements are established in three different karyotypes along with polyploidy. Both pericentric inversion and Robertsonian fusion played a major role in chromosome rearrangements. The nuclear DNA content of these three species is within 19‐29% of the present‐day placental mammals, and is thus lower than the median amount for fishes in general and teleosts in particular. Their lower DNA content suggests that the three species of the family Channidae are highly specialized, and this is supported by their known morphologic, reproductive, behavioural and ecological characteristics.The evolutionary significance of these chromosomal rearrangements, their origin and their mode of establishment are discussed. A probable phylogenetic model based on karyotype, C‐value and chromosomal rearrangements of the genus is presented.

DNA amounts and chromatin compactness in Vicia

2C DNA amounts and areas of chromatin were determined with a M 86 Vickers microdensitometer in 56 species of Vicia (x=5, 6, 7), exhibiting large differences in chromosome size. There were significant differences between the species both in DNA content and chromatin area. The nuclear DNA amounts range from 3.85 to 27.07 pg. DNA distribution appears discontinuous; species cluster into distinct groups and the average nuclear DNA amount separating each successive pair is approximately the same (2.23 pg). The compaction of DNA in interphase nuclei increases with increasing DNA amount, which is, at least partly, due to a disproportionate increase in the heterochromatin relative to the euchromatin component of DNA. Comparisons of DNA readings at various stages of the cell cycle show that the DNA amounts are underestimated by microdensitometry in nuclei with high DNA density. Estimation of relative DNA content and area of individual chromosomes were made in twelve species. The results show that changes in DNA content within chromosomes affect the degree of metaphase coiling in an orderly fashion.

Karyotype ofCarapusacus(Brünnich, 1768) (Pisces, Carapidae)

SUMMARYFrom analysis of mitotic metaphase spreads the diploid number 2n = 36 (FN = 38) has been determined for Carapus acus from the Gulf of Palermo. It seems that, for this species, evolution is accompanied by a decrease in the chromosome number and DNA content.

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%.

Increased amplification of plasmids pBR322 and pBR327 by low concentrations of chloramphenicol.

The replication and amplification of plasmids pBR322 and pBR327 is maximal during partial inhibition of protein synthesis by low concentrations (10-20 micrograms/ml) of chloramphenicol in rich medium (LB). In this manner, 5- to 10-fold greater yields of plasmid DNA at 2- to 5-fold greater initial purity (less protein and chromosomal DNA content) can be obtained than by using the standard high concentrations of chloramphenicol. These results have been obtained with an improved method of plasmid quantitation in unfractionated bacterial lysates.

Cytogenetic studies of interspecific hybrids between diploid species of Festuca

Chromosome pairing in hybrids between diploid species of Festuca is described. The chromosome complements of the species from different taxonomic sections vary in chromosome size and DNA content. In interspecific hybrids involving species of the section Montanae there was a relationship between the difference in DNA content of the parental species and chromosome pairing in the F1 hybrids. The larger the difference between the DNA content of the parental species, the more pronounced the failure of chromosome pairing in the F1 hybrids. Factors other than divergence in genome size were also shown to have an effect on chromosome pairing in other hybrid combinations.Key words: chromosome pairing, DNA content, Festuca, hybrids (interspecific).

Recombination and genome size

Within complements the chiasma frequency per chromosome, which directly reflects the amount of recombination, is generally closely correlated with chromosome length, i.e. the chromosomal DNA content. The correlation does not apply when comparisons are made between the complements of different species. Analyses of results from three Angiosperm genera show a progressive decrease in the chiasma frequency per picogram of DNA with increase in nuclear DNA amount.

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.

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.

DNA content and base composition of human chromosomes

The relative DNA content and base composition of the 24 human chromosomes have been measured by flow cytometry. Isolated human metaphase chromosomes flowing through a finely focused laser beam were analyzed singly at a rate of 200 per s. The relative chromosomal fluorescence of the DNA specific dye propidium iodide correlated strongly ( r = 0.99) with the DNA-based human karyotype determined by image cytometry. The relative fluorescence of chromosomes stained with the dye combination Hoechst 33258 (AT binding specificity) and chromomycin A 3 (GC binding specificity) was compared with independent measurements of chromosomal base composition. The results correlated positively with quinacrine brightness ( r = 0.88) and autoradiographic determination of base-specific radioactive DNA precursor incorporation ( r = 0.55). The larger chromosomes were found to have a higher AT content than the average base composition of total human genomic DNA and the smaller chromosomes a higher GC content. Hoechst-chromomycin fluorescence depends primarily on chromosomal variation in base composition, but may also be influenced by chromosome-specific classes of repetitive DNA.

Flow cytometry and flow sorting of metaphase chromosomes from the dasyurid marsupial Dasyurus viverrinus.

Metaphase chromosomes (2n = 14) from D. viverrinus were analysed by flow cytometry and flow sorted into six homogeneous groups. Relative chromosomal DNA contents and distribution frequencies of the groups corresponded closely with values for the karyotype obtained by conventional methods.

Immunofluorescent detection of histone 2B on metaphase chromosomes using flow cytometry.

A monoclonal antibody against histone 2B (anti-H2B) was used as a reagent to stain isolated chromosomes for analysis using flow cytometry. Chromosome suspensions were treated with a mouse monoclonal antibody specific for the histone 2B (clone HBC-7) and then with a fluorescein-labeled goat anti-mouse-IgM antibody. The chromosomes were also stained for DNA content with either Hoechst 33258 or propidium iodide. The amount of antibody and the amount of DNA-specific stain bound to each chromosome were measured simultaneously using flow cytometry. The order of the steps in the staining protocol is important. Propidium iodide prevents anti-H2B from binding to chromosomes, and therefore must be added only after antibody labeling is completed. In contrast, the addition of Hoechst 33258 before antibody labeling reduces antibody binding by only 20%-30%. Binding of anti-H2B was proportional to the DNA content of both human and Chinese hamster chromosomes. Human chromosomes bind an average of three to four times more anti-H2B than do Chinese hamster or mouse chromosomes of the same DNA content. This was determined by analyzing mixtures of human and Chinese hamster chromosomes and human and mouse chromosomes. The results demonstrate that it is possible to label the proteins of chromosomes in suspension with fluorescent antibodies and to use these reagents for the analysis of chromosome structure by flow cytometry.

Variation in DNA content of 21 individual chromosomes among six subspecies in common wheat.

In order to consider at chromosomal level the phylogeny of wheat, DNA content of 21 individual chromosomes was measured cytophotometrically at M1 or A1 of meiosis in monosomic plants and compared among seven strains belonging to six subspecies of common wheat, Triticum aestivum.1) Nuclear DNA content at pollen tetrad did not differ at all among seven strains.2) DNA content of individual chromosomes varied from chromosome to chromosome in any strain.3) Significant difference was found among three genomes, A, B and D, the ratio being 1.14:1.21:1.00. This ratio apparently differed from those of two earlier reports based on nuclear DNA content of putative diploid ancestors (T. monococcum: Aegilops speltoides: Ae. squarrosa). The authors suggested diploid species of section Sitopsis, with a little higher DNA content than Ae. speltoides as B genome donor to polyploid wheat.4) There was not significant difference among seven homoeologous groups, but homologous chromosomes.5) Total DNA content of seven strains calculated from DNA content of individual chromosomes was very similar to one another, being clearly compatible with the results of nuclear DNA content at pollen tetrad.

Chromosome and DNA analyses of rat 13762NF mammary adenocarcinoma cell lines and clones of different metastatic potentials.

Chromosome morphologies revealed by Giemsa-banded karyotypes and chromosome numbers were compared between parental tumor-, lymph node- and lung metastasis-derived rat 13762NF mammary adenocarcinoma cell lines and clones having different spontaneous metastatic potentials. Although chromosome numbers in the cell lines and clones generally correlated with DNA content by flow cytometry, ploidy did not correlate with spontaneous metastatic potentials. Chromosome number and DNA content drifted during prolonged in vitro growth in each of the cell lines and clones. Common chromosome rearrangements were found, confirming a common origin for all the cell lines and clones, and the frequency and appearance of the individual marker chromosomes fluctuated during in vitro growth. Karyotypic analyses revealed that the markers coinciding with phenotypic drift in spontaneous metastatic potential and other biological properties of parental tumor-derived clones MTC and MTF7 and lung metastasis-derived clone MTLn3 involved chromosomes 3, 4, 5, 6, and 8. Clone MTC exhibited a shift in several markers and an increase in metastatic potential at passage T20, while clone MTF7 displayed a lesser spontaneous metastatic potential at high passage (T34) concomitant with an increase in the frequency of certain marker chromosomes. Lung metastasis-derived clone MTLn3 also exhibited a shift in some marker chromosomes, colonization preference and metastatic potential to lung and lymph nodes at high tissue culture passages. The changes in marker chromosomes during in vitro passage of clones MTC and MTLn3 suggested the presence of at least two cell subpopulations which could be responsible for the observed shift in spontaneous metastatic properties. Karyotypic features of the 13762NF cell lines and clones indicate that subtle cytogenetic changes, in contrast to gross chromosomal abnormalities, may be more important in determining metastatic phenotype.

Cytogenetics of Rutaceae. II. Nuclear DNA Content

SUMMARYNuclear DNA content was determined in 17 Rutaceae species. The results obtained suggest that both nuclear DNA content and mean chromosomal DNA content appear to be very sensitive indicators of the kin relationship between species or genera. The variation in nuclear DNA content among Rutaceae is mainly determined by high ploidy levels. Diploid and tetraploid species generally have a 4C of less than 5.5 pg, whereas those with higher ploidy have a 4C of more than 12 pg. The few exceptions in this tendency are encountered in species from temperate regions where the evolutionary strategies at the chromosome level seem to diverge from those developed by tropical plants.

The DNA content of the individual chromosomes of rye

The relative DNA content of individual chromosomes of Secale cereale L. was determined in 25 cells by microdensitometry of Feulgen stained preparations. The correlation value between relative DNA content and relative chromosome length was r=0.61 for all 328 chromosomes measured. However, the correlation coefficients calculated for individual cells as well as for mean values always approached 1. Taking into account the structure of rye chromosomes, this indicates that microdensitometric results may not be accurate when large quantities of heterochromatic DNA sequences are present in analyzed material.

Relative DNA content of individual chromosomes, karyotype and ploidy in crown gall tumor cells of Crepis capillaris.

Levels of ploidy, karyotype, mitosis and relative DNA content of chromosomes in crown gall tumor cells in stem and leaf of Crepis capillaris were studied. Except for about 7% tetraploid mitoses, dividing cells were found to be mainly diploid in both stem and leaf tumors. The karyotype and mitosis in tumor cells were not detectably different from normal cells. The relative DNA content of homologous pairs of chromosomes in the genome of tumor cells was similar to that of root tip cells. These observations indicate that no dramatic change in DNA content or DNA distribution occurred when tissue was transformed to crown gall tumor.