The breeding of two polyploid rice lines with the characteristic of polyploid meiosis stability

Polyploidization is a basic feature of plant evolution. Nearly all of the main food, cotton and oil crops are polyploid. When ploidy levels increase, yields double; this phenomenon suggested a new strategy of rice breeding that utilizes wide crosses and polyploidization dual advantages to breed super rice. Because low seed set rates in polyploid rice usually makes it difficult to breed, the selection of Ph-liked gene lines was emphasized. After progenies of indica-japonica were identified and selected, two polyploid lines, PMeS-1 and PMeS-2 with Polyploid Meiosis Stability (PMeS) genes were bred. The procedure included seven steps: selecting parents, crossing or multiple crossing, back-crossing, doubling chromosomes, identifying the polyploid, and choosing plants with high seed set rates that can breed themselves into stable lines. The characteristics of PMeS were determined by observing meiotic behaviors and by cross-identification of seed sets. PMeS-1 and PMeS-2, (japonica rice), have several characteristics different from other polyploid rice lines, including a higher rate of seed set (more than 65%, increasing to more than 70% in their F1 offspring); and stable meiotic behaviors (pairing with bivalents and quarivalents nearly without over-quarivalent in prophase, nearly without lagging chromosomes in metaphase and without micronuclei in anaphase and telophase). The latter was obviously different from control polyploid line Dure-4X, which displayed abnormal meiotic behaviors including a higher rate of multivalents, univalents and trivalents in prophase, lagging chromosomes in metaphase and micronuclei in anaphase and telophase. There were also three differences of the breeding method between PMeS lines and normal diploid lines: chromosomes doubling, polyploidism identifying and higher seed set testing. The selection of PMeS lines is the first step in polyploid rice breeding; their use will advance the progress of polyploid rice breeding, which will in turn offer a new way to breed super rice. Supported by the National Natural Science Foundation of China (Grant Nos. 39970447, 30240090, 30471063 and 30650002), the High Technology Research and Development Program of China (Grant No. SZ-01-02-02), the Chenguang Youth Science and Technology Project of Wuhan City (Grant No. 20045006071-31), and the Educational Commission of Hubei Province of China (Grant No. 2004D004)     

Replication of euchromatin and heterochromatin in a mealybug

Asynchronous DNA replication of euchromatic (E) and heterochromatic (H) chromosomes and heterochromatic B chromosomes (B) were studied in the mealybug, Pseudococcus obscurus Essig (Homoptera: Coccoidea). The study was carried out on mycetocytes of adult females and on spermatocytes of mid-second instar males by employing tritiated thymidine labeling and autoradiography. In the mycetocytes the incorporation of the labeled thymidine began and ended later in the B's than in the E chromosomes. The S period was found to be about 21 hours. The DNA replication of the E chromosomes occupied about 86% of the S period and that of the B's 33%; during 18% of the mid-S period the replication of the two types of chromosomes overlapped. In the meiotic S period of the spermatocytes, the DNA of the E chromosomes started to replicate earlier than that of the H chromosomes and the B's, but the replication of the E chromosomes, the H chromosomes, and the B's overlapped. The H chromosomes completed their replication much later than the E chromosomes and slightly later than the B's.Supported by grants GB 1585 and GB 6745 to Dr. Uzi Nur from the National Science Foundation, Washington, D. C.Part of a thesis submitted to the University of Rochester in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

The band patterns of twelve D 98/AH-2 marker chromosomes and their use for identification of intraspecific cell hybrids

The chromosomal complement of the human cell line D98/AH-2 has been studied by quinacrine mustard and trypsin Giemsa banding techniques. The dispersion of chromosome counts has been shown to be due to non-random variation involving mainly a few particular chromosomes. — Twelve different marker chromosomes could be distinguished and the presumptive derivation of most of their chromosomal material from normal human chromosomes has been determined. Most cells in 6 different hybrid clones derived from fusion of D98/AH-2 cells with skin fibroblasts from a cystinotic patient contained a single copy of each marker chromosome.Supported by: united States Public Health Service Grant HD 04608, National Institute of General Medical Sciences Grant GM 17702 and American Heart Association Grant 71-981.Established Investigator of the American Heart Association.     

Sister chromatid exchanges in bromine incorporation into DNA cytosine nucleotides. III. 5-bromodeoxycytidine as a DNA thymine precursor. The characteristics of the exchanges]

Cell movement through the mitotic cycle and sister chromatid exchanges (SCE) were studied in human blood lymphocytes cultured in the presence of 5-bromodeoxycytidine (BrdC, 0.05 mM) plus thymidine (dT 0.4, 0.8, and 1.0 mM). In controls, lymphocytes were cultivated in the presence of 5-bromodeoxyuridine (BrdU, 0.05 mM) and deoxycytidine (0.1 mM), or BrdC alone. All nucleosides were added to the cultures 28 hours prior to fixation and were maintained in the medium for 16 hours. As determined from percentage of metaphases of 1st to 3rd divisions, BrdC did not release from thymidine block. This fact leads us to conclude that BrdC in contrast to deoxycytidine does not serve as a cytosine precursor. No significant differences in the frequency of SCE and their distribution among chromosomes were found between cultures treated with BrdC and with BrdU.     

Asynchronous Duplication of Chromosomes in Cultured Cells of Chinese Hamster

Chromosome duplication (DNA synthesis) was studied in cultured cells of Chinese hamsters by means of autoradiography following thymidine-H³ incorporation. The technique used was to expose an asynchronously dividing population of rapidly growing cells for a 10 minute interval to a medium with thymidine-H³. Cells were then transferred to a medium with excess unlabeled thymidine. The population was sampled at intervals thereafter and studies made of the frequency of labeled interphases and division figures, and the patterns of labeling of specific chromosomes. The average generation time during these experiments was about 14 hours. DNA synthesis occurred during an interval of about 6 hours and stopped 2 to 3 hours before metaphase. After metaphase the chromosomes usually begin duplication again within 5 to 6 hours. Grain counting, to estimate the amount of tritium incorporated after a short contact with thymidine-H³ and at intervals after transfer to a medium with excess unlabeled thymidine, indicated that the intracellular pool of labeled precursors was diluted within less than a minute so that further labeling would not be detected. The chromosomes labeled during the contact period retained their precise pattern of labeling through another duplication cycle and no turnover of DNA or loss of tritium was detectable. Five or 6 chromosomes of the complement have segments typically late in duplication. Two of these are the X and Y chromosomes. The long arm of the X chromosome and the whole Y chromosome are duplicated in the last half of the interval of DNA synthesis. The short arm of the X chromosome in a male strain is duplicated in the first half of the interval. In another strain (female), one X chromosome had the same timing, but the other one was all duplicated in the last half of the period of DNA synthesis. The DNA in the short arms of 2 medium sized chromosomes, as well as most of the DNA in 1 or 2 of the smallest chromosomes of the complement was replicated late. The study has led to the hypothesis that various chromosomes or parts of chromosomes have a genetically controlled sequence in duplication which may have some functional significance.     

Effects of toxic substances on natural bacterial assemblages determined by means of [h]thymidine incorporation

The effects of 3,5-dichlorophenol, 2,4-dinitrophenol, and potassium dichromate on natural bacterial assemblages were examined by means of [H]thymidine incorporation into trichloroacetic acid-insoluble material. Results from a large number of coastal marine and freshwater samples suggest the following. (i) The effects of the three toxicants included reductions in the bacterial cell number as well as changes in rates of [H]thymidine incorporation and in [H]thymidine incorporation per cell. The concentrations that inhibited [H]thymidine incorporation by 50% ranged from 3 to 11 mg liter for 3,5-dichlorophenol, 6 to 10 mg liter for 2,4-dinitrophenol, and 21 to 123 mg liter for potassium dichromate, with a tendency to higher values in bacterial assemblages from more eutrophic environments. (ii) The effects of 3,5-dichlorophenol and potassium dichromate determined by [H]leucine incorporation into bacterial protein were similar or larger than those obtained from [H]thymidine incorporation. (iii) Two to four hours of exposure to the toxicants was necessary before stable maximum effects were found in [H]thymidine incorporation. (iv) Storage of natural environmental samples should be avoided, since tests with water stored for 1 to 3 days sometimes produced results different from results obtained from in situ tests. (v) The effects of 3,5-dichlorophenol, 2,4-dinitrophenol, and potassium dichromate on natural bacterial assemblages were relatively constant during periods with different growth rates in the assemblages, during various periods of the year, and between samples from freshwater and marine localities. With some precautions, [H]thymidine incorporation can be used as a quick and sensitive method for determining the effects of toxicants on aquatic bacterial assemblages from natural environmental samples.     

Changes in the cell cycle duration and karyotype of murine L cells with a shift in the mode of cultivation

A suspension subline LS of mouse fibroblasts L was adapted to the growth in monolayer (LSM subline). The duration of cell cycle and its phases was determined for both the sublines synchronized by a double thymidine block. The duration of cell cycle for LS cells is 17.0 hours, with G1, S and G2 + M being 8.8, 6.3 and 1.8 hours, resp., for LSM cells corresponding values being 31.6, 15.6, 11.0 and 4.8 hours. The karyotype of LSM cells differs from that of LS cells only slightly: there is a redistribution between the numbers of cells with 55 and 56 chromosomes, and a decrease in the number of polyploid cells from 2.0 till 0.2%. The data obtained indicate the heterogeneity of the L line culture which contains cells with long and short cycles. Depending on the mode of cultivation (suspension or monolayer), there is a certain predominance of population either with short or with long cell cycle.     

Thymidylate nucleotide supply for mitochondrial DNA synthesis in mouse L-cells. Effect of 5-fluorodeoxyuridine and methotrexate in thymidine kinase plus and thymidine kinase minus cells.

The effects of 5-fluorodeoxyuridine and methotrexate on [3H]thymidine and 32P labeling of mtDNA were studied in two lines of mouse L-cells. LMTK- cells, which lack the major cellular thymidine kinase (EC 2.7.1.21) but contain a genetically distinct mitochondrial enzyme, were compared to LA9 cells, which contain both thymidine kinase activities. LMTK- cells were resistant to 5-flurodeoxyuridine by a factor of 200 in comparison to LA9 cells. In both cells lines appropriate drug treatment increased utilization of exogenous thymidine for mtDNA synthesis. The maximum enhancement was 10- to 12-fold for LA9 cells and approximately 20-fold for LMTK- cells when treated with 10 muM methotrexate. The rates of mtDNA and nuclear DNA synthesis during drug treatment were analyzed with 32P labeling and 5-bromo-2'-deoxyuridine density labeling experiments. Synthesis of both mtDNA and nuclear DNA were strongly inhibited by drug treatment of either LA9 or LMTK- cells in the absence of exogenous thymidine. The rate of mtDNA synthesis substantially exceeded that of nuclear DNA in LA9 cells treated with 4 muM 5-fluorodeoxyuridine and less than 5 muM thymidine. Both synthetic rates approached those of untreated LA9 control cultures if 20 muM thymidine was present during 5-fluorodeoxyuridine treatment. In contrast, in LMTK- cells treated with 10 muM methotrexate and 20 muM thymidine, mtDNA synthesis continued at 50 to 60% of the control rate for at least 10 hours while nuclear DNA synthesis was 96% inhibited. Synthesis of mtDNA mass-labeled in both strands with 5-bromouracil occurred when LMTK- cells were incubated for 30 hours with 10 muM methotrexate and 20 muM 5-bromodeoxyuridine. These results indicate that mtDNA synthesis is resistant to a limitation of the thymidine triphosphate supply and is not strictly dependent upon concomitant nuclear DNA synthesis in these cells.     

Characterization of hybrids between bovine (MDBK) and mouse (L-cell) cell lines

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient mutants of a bovine kidney cell line (MDBK) were selected following mutagenesis with ethylmethane sulfonate or ICR-170G. MDBK mutants were hybridized to thymidine kinase-deficient L cells and selected in HAT medium. Parental and hybrid cells were characterized for isozyme patterns of lactic dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, and glutamate oxalate transaminase. Chromosomes of MDBK can be distinguished from mouse L cells by configuration and by fluorescent staining with Hoechst 33–258 stain. Hybrid cells contained both MDBK and L-cell chromosomes and had elevated DNA content. MDBK cells are normally restrictive for mengovirus replication. Both permissive and restrictive hybrids were found. Our data indicate that there was preferential loss of MDBK chromosomes in the hybrid cell lines.This work was supported by a grant from the Institute for General Medicine [GM18924 (M. W. T.)]. V. G. C. was a predoctoral fellow on Genetic Training Grant No. GM12 from the National Institute for General Medicine.     

Computer image analysis of variance between human chromosome replication sequences and G-bands.

A computer image analysis system was applied to the quantitative study of chromosomal early- and late-replication patterns from the leukocytes of several normal human donors, and these patterns were compared with the chromosomal G-banding patterns. The first and last few hours of replication were discriminated by selective bromodeoxyuridine vs. thymidine incorporation in DNA and a Hoechst-blacklight-Giemsa stain technique. Image analysis with Tufts Piquant system involved automatic determination of chromosome boundaries, centromeres and telomeres, linear chromatid axes, chromatid density measurements along each axis, and comparative length normalized density profiles for each chromatid and the chromosome. Consistent complementary early- and late-replication patterns were determined for autosomes 1-6 and the X chromosomes. Limited intracellular or interindividual variability occurred in the intensity of a few active replication peaks but not in their location. However, there were very distinct regions of noncorrespondence between the late-replication patterns and the G-band patterns, in contrast with previous observations, although many similarities were also evident. These differences are interpreted with reference to a general model of replication sequence control of cell differentiation.     

Autoradiographic studies of the human Y chromosome

An autoradiographic analysis (using continuous labeling with tritiated thymidine) was made on 317 cells from four normal males. The labeling pattern of the Y chromosome was compared to the first and the last chromosomes to complete replication as well as to G21–22. The Y chromosome was never found to be the last chromosome in the cell to complete replication. Instead, it completed DNA synthesis relatively early (usually among the first 10 chromosomes) but had a distinctively heavy label during the earliest stages of late-S. In 51% of those cells with one labeled G+Y chromosome, a G21–22 was labeled and the Y was not.—It was concluded, therefore, that the human Y chromosome is not a late-replicating chromosome but terminates replication earlier than most of the autosomes. In addition, the Y chromosome cannot be distinguished from the G chromosomes on the basis of a consistent and differential labeling pattern.Supported by USPHS Grant GM 15361.     

Thymidine kinase activity and human chromosome No. 18

Somatic cell hybridization studies have suggested that the locus for human thymidine kinase is on a No. 17 or No. 18 chromosome. To evaluate further this possibility, fibroblast cell cultures from patients with partial monosomy 18 and trisomy 18 were assayed for thymidine kinase activity; the enzyme levels in these cell extracts were normal. If the enzyme activities reflect a simple gene dose effect, these results suggest that the human thymidine kinase locus is not situated on the No. 18 chromosome.Supported in part by Grant # MR 0504A-69 from the Division of Mental Retardation, Social and Rehabilitation Service, Department of Health, Education and Welfare, and by the Institute of Child Health and Human Development, Grant # DO 4612, Mental Retardation Research Center, UCLA/NPI.     

Complementation in heterokaryons derived from a thymidine kinase deficient cell line and senescent human diploid cells.

Incorporation of [3H]thymidine was observed in both parental nuclei in heterokaryons resulting from the fusion of post-mitotic, "senescent" human diploid cells and a thymidine kinase-deficient murine cell line (3T3der-4E). The senescent nuclei displayed a sudden increase of activity approximately 4--6 hours after fusion. A moderate increase of thymidine incorporation was observed in 3T3der-4E cells cocultivated with but not fused to senescent cells, consistent with metabolic cooperation. Chromosome preparations of cultures fixed approximately 24 hr after fusion revealed the presence of hybrid metaphase cells containing almost the entire human complement. All of the identifiable human chromosomes were bi-armed, suggesting that the senescent nuclei were stimulated to reinitiate replicative DNA synthesis rather than repair synthesis in these heterokaryons.     

Uptake of radioactive thymidine and cytidine by Ehrlich ascites tumor cells in different stages of growth

Summary In Strong A female mice, the Ehrlich ascites tumor inoculated into the peritoneal cavity grows exponentially for the first 7 days with a doubling time of about 36 hours. The tumor enters then into a late stage during which the number of tumor cells in the peritoneal cavity does not increase. The uptake of intraperitoneally injected thymidine decreases from the exponential to the late stage, mostly because of a decrease in the fraction of cells in DNA synthesis. During the exponential phase, the uptake of thymidine is a function of the amount of radioactive thymidine injected per tumor cell, the utilization decreasing with increasing cell dose. The uptake of intraperitoneally injected cytidine decreases slightly with time after inoculation although the fraction of tumor cells in RNA synthesis remains constant.Dedicated to Professor Friedrich Wassermann with admiration and affection on the occasion of his 80th birthday.This investigation was supported by U.S.P.H.S. Grant CA-05667. The author is a U.S.P.H.S. Research Career Development Awardee.     

Selective lethal effect of thymidine on human and mouse tumor cells

Human cell lines derived from a melanoma and a colon carcinoma, and cultures of human melanocytes and intestinal epithelial cells, as well as a mouse mesenchymal non-neoplastic cell line and a malignant subline of the same have been quantitatively studied in tissue culture for their sensitivity to thymidine. All three tumor lines produced solid tumors when injected into nude thymus-deficient mice. No tumors were obtained by injecting cells of the human normal long-term cultures or of the non-neoplastic mouse line. The tumor-producing lines showed a greater sensitivity to the lethal effects of high concentrations of thymidine than their non-tumor-producing counterparts. Less than 23% of the tumor cells survived 72 hours in the presence of 1 mg/ml of thymidine, in contrast to 60% or more of the non-tumor cells. Colony formation was much more inhibited by thymidine and the differential between normal and tumor cells was even more pronounced. Tumor cells which also were treated for 72 hours with 1 mg/ml of thymidine and then plated in fresh medium formed very few colonies. If the plating efficiency of the untreated controls is considered as 100%, 4.3% or less of the treated tumor cells formed colonies, in contrast to 33% or more of the non-tumor cells.     

Further studies on pachytene pairing failure in maize

Summary Pairing failure in pachytene chromosomes was studied against a genetically constant background. Very significant negative correlations were found between total chromosome length per cell on the one hand and number of terminal pairing failures and their total extent (per cent length) on the other, and between number of terminal failures and their average extent (per cent length). No significant correlation was found, however, between total chromosome length per cell and the average extent (per cent length) of pairing failure. If the pairing failures are dissociations increasing in extent and number during the pachytene stage studied, the simplest reconciliation of the results requires that the average rate of their extension be roughly proportional to total chromosome length or that certain constants be related to each other as described. An alternate interpretation, that the pairing failures were present before pachytene and that chromosomes are shortening faster in cells containing them, is favored by the findings that heterogeneity is low between anthers in number of failures per cell and that no significant correlation was found between anthers in total chromosome length and number of pairing failures. The possibility is also discussed that the pairing failures are a complex combination of both initial synaptic failure and later dissociation.Distributions of chromosomes containing pairing failure at both ends and of those containing both terminal and intercalary pairing failures generally follow expectations of randomness.This work was supported by National Institute of Health Grant Number RA-6492 and by National Science Foundation Grant Number G 7068.     

The contribution of cell death to medium-released fractions of cell cultures

Summary Cell death was estimated by prelabeling primary chick embryo skeletal muscle cell cultures with [³H]thymidine and by subsequently measuring the release of label into complete culture medium or serum-and embryo-extract-free medium for a 6 h period. Cultures of the established muscle cell line L6 and the fibroblastic cell line 3T3 were used for comparative purposes. Comparison of the nigrosin exclusion test with the thymidine release test shows that the former underestimates cell death because it measures only the instantaneously occurring cell death. The [³H]thymidine release test estimates the cumulative amount of cell death. From cumulative cell death estimates it is calculated that 12.0 and 17.8% of the³H-fucosylated medium-released fractions from primary cell cultures are the result of cell death contamination when release occurs in complete or macromolecule-free media, respectively. High speed centrifugation is shown to eliminate most contamination from cell death. Evidence is presented that the absence of macromolecules in the culture medium has little effect on the release process. Contamination of the released fraction resulting from cell death is much less in the established cell lines than in the primary cells. It is concluded that the release process can be studied in primary muscle cell cultures and especially in established cell lines if adequate precautions are taken and if corrections for cell death contamination are taken into account. This research benefited from use of the Cell Culture Facility supported by National Cancer Institute Grant CA 14733. This research was supported by a Muscular Dystrophy Association Grant to Dr. Heinz Herrmann and by American Cancer Society Grant RDP 8 and was submitted in partial fulfillmant of a Ph.D. degree at the University of Connecticut (T. C. Doetschman).     

Effects of Toxic Substances on Natural Bacterial Assemblages Determined by Means of [3H]Thymidine Incorporation

The effects of 3,5-dichlorophenol, 2,4-dinitrophenol, and potassium dichromate on natural bacterial assemblages were examined by means of [³H]thymidine incorporation into trichloroacetic acid-insoluble material. Results from a large number of coastal marine and freshwater samples suggest the following. (i) The effects of the three toxicants included reductions in the bacterial cell number as well as changes in rates of [³H]thymidine incorporation and in [³H]thymidine incorporation per cell. The concentrations that inhibited [³H]thymidine incorporation by 50% ranged from 3 to 11 mg liter⁻¹ for 3,5-dichlorophenol, 6 to 10 mg liter⁻¹ for 2,4-dinitrophenol, and 21 to 123 mg liter⁻¹ for potassium dichromate, with a tendency to higher values in bacterial assemblages from more eutrophic environments. (ii) The effects of 3,5-dichlorophenol and potassium dichromate determined by [³H]leucine incorporation into bacterial protein were similar or larger than those obtained from [³H]thymidine incorporation. (iii) Two to four hours of exposure to the toxicants was necessary before stable maximum effects were found in [³H]thymidine incorporation. (iv) Storage of natural environmental samples should be avoided, since tests with water stored for 1 to 3 days sometimes produced results different from results obtained from in situ tests. (v) The effects of 3,5-dichlorophenol, 2,4-dinitrophenol, and potassium dichromate on natural bacterial assemblages were relatively constant during periods with different growth rates in the assemblages, during various periods of the year, and between samples from freshwater and marine localities. With some precautions, [³H]thymidine incorporation can be used as a quick and sensitive method for determining the effects of toxicants on aquatic bacterial assemblages from natural environmental samples.     

Radioautography of incorporated 3H-thymidine and its metabolism during meiotic prophase in microsporocytes of Lilium

Zygotene cells of Lilium were cultured in the presence of ³H-thymidine for 24 hours and the culture continued in isotope-free medium. Radioautographs of the cells at subsequent stages of meiosis showed the zygotene label to be chromosomal and to be more or less generally distributed over all the chromosomes. Exposure of cells to ³H-thymidine for periods longer than 24 hours resulted in widespread incorporation of thymidine catabolites into a variety of acid-insoluble compounds. Such catabolism is characteristic of meiotic prophase and is virtually absent at premeiotic interphase.This work was supported by a grant from the National Science Foundation, GB 29562 and the Ministry of Education of Japan.     

Consequences of Accounting for Isotopic Dilution in Thymidine Incorporation Assays

Rates of thymidine incorporation into DNA were corrected for isotope dilution by internal nucleotide pools and were compared with rates obtained from uncorrected data. Differences as large as 109% were observed between corrected and uncorrected estimates of thymidine incorporation. The degree of underestimation varied seasonally and, to a lesser extent, spatially.