Prematurely condensed chromosome fragments in human lymphocytes induced by high doses of high-linear-energy-transfer irradiation

This study provides a useful biodosimetry protocol for radiation accidents that involve high doses of heavy particle radiation. Human peripheral blood lymphocytes (PBLs) were irradiated in vitro with high doses (5–50 Gy) of charged heavy-ion particles (carbon ions, at an effective linear-energy-transfer (LET) of 34.6 keV/ m), and were then stimulated to obtain dividing cells. PBLs were treated with 100nMcalyculin A to force chromosomes to condense prematurely, and chromosome spreads were obtained and stained with Giemsa. The G2 prematurely condensed chromosome (G2-PCC) index and the number of G2-PCC including fragments (G2-PCC-Fs) per cell for each radiation dose point were scored. Dose-effect relationships were obtained by plotting the G2-PCC indices or G2-PCC-Fs numbers against radiation doses. The G2-PCC index was greater than 5% up to doses of 15 Gy; even after a 30Gy radiation dose, the index was 1 to 2%. At doses higher than 30 Gy, however, the G2-PCC indices were close to zero. The number of G2-PCC-Fs increased steeply for radiation doses up to 30 Gy at a rate of 1.07 Gy−1. At doses higher than 30 Gy, the numbers of G2-PCC-Fs could not be accurately indexed because of the limited numbers of cells for analysis. Therefore, the number of G2-PCC-Fs could be used to estimate radiation doses up to 30 Gy. In addition, a G2-PCC index close to zero could be used as an indicator for radiation doses greater than 40 Gy.

A method on theoretical simulation of chromosome breaks in cells exposed to heavy ions

Background. The aim of this study is to assess an easy and quick method on simulating chromosome breaks in cells exposed to heavy charged particles. Methods. The theoretical value of chromosome break was calculated, and the validated comparison with the experimental value by using a premature chromosome condensation technique was done. Results. A good consistence was found to be appeared between the theoretical and experimental value. Conclusions. This suggested that a higher relative biological effectiveness of heavy ions was closely correlated with its physical characteristics and besides, a safe approach on predicting chromosome breaks in cells exposed to heavy ions at off-line environment come to be considered. Furthermore, three key factors influencing the theoretical simulation was investigated and discussed.

Comparison of clonogenic assay with premature chromosome condensation assay in prediction of human cell radiosensitivity

Aim: To determine whether the number of non-rejoining G2-chromatid breaks can predict the radiosensitivity of human cell lines. Methods: Cell lines of human ovary carcinoma cells (HO8910), human hepatoma cells (HepG2) and liver cells (L02) were irradiated with a range of doses and assessed both of cell survival and non-rejoining G2-chromatid breaks at 24 h after irradiation. Cell survival was documented by a colony assay. Non-rejoining G2-chromatid breaks were measured by counting the number of non-rejoining G2 chromatid breaks at 24 h after irradiation, detected by the prematurely chromosome condensed (PCC) technique. Results: A linear-quadratic survival curve was observed in three cell lines, and HepG2 was the most sensitive to gamma-radiation. A dose-dependent linear increase was observed in radiation-induced non-rejoining G2-PCC breaks measured at 24 h after irradiation in all cell lines, and HepG2 was the most susceptible to induction of non-rejoining G2-PCC breaks. A close correlation was found between the clonogenic radiosensitivity and the radiation-induced non-rejoining G2-PCC breaks (r=0.923). Furthermore, survival-aberration correlations for two or more than two doses lever were also significant. Conclusion: The number of non-rejoining G2 PCC breaks holds considerable promise for predicting the radiosensitivity of normal and tumor cells when two or more than two doses lever is tested.

Effect of gamma-ray radiation on physiological, morphological characters and chromosome aberrations of minitubers in Solanum tuberosum L.

Purpose: To investigate the effects of gamma-ray radiation on the physiological, morphological characters and chromosome aberrations of minitubers. Materials and methods: Minitubers of one potato cultivar, 'Shepody', were irradiated with 8 doses of gamma-rays (0, 10, 20, 30, 40, 50, 60, 70 and 80 Gy [Gray]) to investigate the effects of radiation on emergence ability, plant height and root length, morphological variations, chromosome aberrations, M-1 (first generation mutants) tuber number and size of minituber plants. Results: Compared with the non-irradiated controls, the whole period of emergence was prolonged by 10-15 days for minitubers treated with gamma-ray radiation, but low doses of radiation (10, 20 and 30 Gy) promoted the emergence percentage of minitubers. With an increase in radiation dose, the emergence percentage, plant height and root length of minituber plants were significantly inhibited at 40 and 50 Gy. No emergence occurred at 60 Gy and higher doses. After radiation, a series of morphological variations and chromosome aberrations appeared in minituber plants. Radiation with 20 Gy promoted tuber formation, and the average number and diameter of M-1 tubers per plant were significantly increased over the control by 71% and 34%, respectively. Conclusion: Low doses of radiation (10-30 Gy) might be used as a valuable parameter to study the improvement of minitubers by gamma-ray radiation treatment.

Differences in the rDNA-bearing chromosome divide the Asian-Pacific and Atlantic species of Crassostrea (Bivalvia, Mollusca)

Karyotype and chromosomal location of the major ribosomal RNA genes (rDNA) were studied using fluorescence in situ hybridization (FISH) in five species of Crassostrea: three Asian-Pacific species (C. gigas, C. plicatula, and C. ariakensis) and two Atlantic species (C. virginica and C. rhizophorae). FISH probes were made by PCR amplification of the intergenic transcribed spacer between the 18S and 5.8S rRNA genes, and labeled with digoxigenin-11-dUTP. All five species had a haploid number of 10 chromosomes. The Atlantic species had 1-2 submetacentric chromosomes, while the three Pacific species had none. FISH with metaphase chromosomes detected a single telomeric locus for rDNA in all five species without any variation. In all three Pacific species, rDNA was located on the long arm of Chromosome 10 (10q)-the smallest chromosome. In the two Atlantic species, rDNA was located on the short arm of Chromosome 2 (2p)-the second longest chromosome. A review of other studies reveals the same distribution of NOR sites (putative rDNA loci) in three other species: on 10q in C. sikamea and C. angulata from the Pacific Ocean and on 2p in C. gasar from the western Atlantic. All data support the conclusion that differences in size and shape of the rDNA-bearing chromosome represent a major divide between Asian-Pacific and Atlantic species of Crassostrea. This finding suggests that chromosomal divergence can occur under seemingly conserved karyotypes and may play a role in reproductive isolation and speciation.

Construction and characterization of a bacterial artificial chromosome library of marine macroalga Porphyra yezoensis (Rhodophyta)

A large-DNA-fragment library is necessary for research into the Porphyra genome. In this study, a bacterial artificial chromosome (BAC) library of Porphyra yezoensis was constructed and characterized. The library contains 54,144 BAC clones with an average insert size of about 65 kb and fewer than 0.7% of clones without large inserts. Therefore, its capacity is more than 6.6 P. yezoensis genome equivalents, and the probability of recovering any nuclear DNA sequence from the library is higher than 99%. The library shows good fidelity and stability. A putative trehalose-6-phosphate synthase (TPS) gene was successfully screened out from the library. The above results show that the library is useful for gene cloning and genomic research in P. yezoensis.

Construction and characterization of two bacterial artificial chromosome libraries of Zhikong scallop, Chlamys farreri Jones et Preston, and identification of BAC clones containing the genes involved in its innate immune system

Large-insert bacterial artificial chromosome (BAC) libraries are necessary for advanced genetics and genomics research. To facilitate gene cloning and characterization, genome analysis, and physical mapping of scallop, two BAC libraries were constructed from nuclear DNA of Zhikong scallop, Chlamys farreri Jones et Preston. The libraries were constructed in the BamHI and MboI sites of the vector pECBAC1, respectively. The BamHI library consists of 73,728 clones, and approximately 99% of the clones contain scallop nuclear DNA inserts with an average size of 110 kb, covering 8.0x haploid genome equivalents. Similarly, the MboI library consists of 7680 clones, with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1x. The combined libraries collectively contain a total of 81,408 BAC clones arrayed in 212 384-well microtiter plates, representing 9.1x haploid genome equivalents and having a probability of greater than 99% of discovering at least one positive clone with a single-copy sequence. High-density clone filters prepared from a subset of the two libraries were screened with nine pairs of Overgos designed from the cDNA or DNA sequences of six genes involved in the innate immune system of mollusks. Positive clones were identified for every gene, with an average of 5.3 BAC clones per gene probe. These results suggest that the two scallop BAC libraries provide useful tools for gene cloning, genome physical mapping, and large-scale sequencing in the species.

Construction of bacterial artificial chromosome libraries for Zhikong Scallop Chlamys farreri

Two Large-insert genomic bacterial artificial chromosome (BAC) libraries of Zhikong scallop Chlamys farreri were constructed to promote our genetic and genomic research. High-quality megabase-sized DNA was isolated from the adductor muscle of the scallop and partially digested by BamH I and Mbo I, respectively. The BamH I library consisted of 53 760 clones while the Mbo I library consisted of 7 680clones. Approximately 96 % of the clones in BamH I library contained nuclear DNA inserts in average size of 100 kb, providing a coverage of 5.3 haploid genome equivalents. Similarly, the Mbo I library with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1 haploid genome equivalents.

Chromosome inheritance in triploid Pacific oyster Crassostrea gigas thunberg

Reproduction and chromosome inheritance in triploid Pacific oyster (Crassostrea gigas Thunberg) were studied in diploid female x triploid male (DT) and reciprocal (TD) crosses. Relative fecundity of triploid females was 13.4% of normal diploids. Cumulative survival from fertilized eggs to spat stage was 0.007% for DT crosses and 0.314% for TD crosses. Chromosome number analysis was conducted on surviving progeny from DT and TD crosses at 1 and 4 years of age. At Year 1, oysters from DT crosses consisted of 15% diploids (2n = 20) and 85% aneuploids. In contrast, oysters from TD crosses consisted of 57.2% diploids, 30.9% triploids (3n = 30) and only 11.9% aneuploids, suggesting that triploid females produced more euploid gametes and viable progeny than triploid males. Viable aneuploid chromosome numbers included 2n + 1, 2n + 2, 2n + 3, 3n - 2 and 3n - 1. There was little change over time in the overall frequency of diploids, triploids and aneuploids. Among aneuploids, oysters with 2n + 3 and 3n-2 chromosomes were observed at Year 1, but absent at Year 4. Triploid progeny were significantly larger than diploids by 79% in whole body weight and 98% in meat weight at 4 years of age. Aneuploids were significantly smaller than normal diploids. This study suggests that triploid Pacific oyster is not completely sterile and cannot offer complete containment of cultured populations.

Chromosome segregation in fertilized eggs from triploid Pacific oysters, Crassostrea gigas (Thunberg), following inhibition of polar body 1

Chromosome segregation in fertilized eggs from triploid Pacific oysters, following inhibition of the first polar body (PB1), was studied with acetic orcein staining techniques. To block the release of PB1, fertilized eggs were treated with 0.5 mg/l of cytochalasin B (CB). Four types of segregation were observed, namely, ''tripolar segregation'' (54.5%), ''united bipolar segregation'' (12%), ''separated bipolar segregation'' (2.5%), and ''incomplete united bipolar segregation'' (4%). The remaining 23% could not be classified because of chromosome disorganization, but appeared to be variants of the above. It seemed clear that the predominant pattern that gave rise to tetraploids was united bipolar segregation, although certain separated bipolar segregations might also lead to the formation of tetraploids. The sequential events of meioses observed in CB-treated eggs are described. The asynchrony of meiotic events and possible mechanisms for the various types of chromosome segregation are discussed.