Frequency Of Spontaneous Chromosomal Aberrations Research Articles

MEDICAL AND RADIOBIOLOGICAL ASPECTS OF RADIATION COMDPLICATIONS IN PATIENTS WITH ONCOGINECOLOGICAL PROFILE

Summary. Aim: to analyze the literature regarding the causes of the occurrence and features of distant complications of radiation therapy of cancer patients; investigation of the frequency and spectrum of spontaneous aberrations of chromosomes in the lymphocytes of peripheral blood of patients with oncogynecological profile (body cancer and cervix) before the onset of radiation therapy. Object and methods: peripheral blood lymphocyte test system with metaphase analysis of chromosome aberrations of 32 primary of cancer patients (follow-up group) and 30 conditionally healthy donors (comparison group). The examination of patients was performed before the onset of radiation therapy. Results: based on the analysis of literature data, the path to a personalized approach to the planning of radiation therapy for patients with oncogyne5 cological profile, the treatment of which is complicated by radiation lesions from the organs and tissues of the pelvis. The clinical and radiobiological aspects of the formation of radiation complications are considered in detail, the search for genetic indicators for the detection of patients with a high risk of developing radiation complications is justified. The results of cytogenetic examinations of patients with endometrial cancer and cervical cancer are close and indicate a 6-fold increase in the frequency of spontaneous chromosome aberrations compared to the population rate. In the spectrum of chromosomal restructures, complex restructures are recorded, which is uncharacteristic of the spontaneous level of aberration in healthy donors, as well as increased levels of chromatide type aberrations. Conclusions: the increased level of spontaneous chromosomal aberrations in T-lymphocytes of primary oncogynecological patients and the predominance of chromatid-type aberrations in the spectrum of registered chromosomal rearrangements indicate that genetic instability is formed in healthy cells before the start of radiation therapy, which predicts the risk of distant radiation complications, including the occurrence of secondary tumors radiation genesis. The examination of patients with the use of cytogenetic test will provide the most reasonable conclusion about the individual radio sensitivity of the patient to the onset of radiation therapy and will contribute to increasing its effectiveness, as well as improving the quality of life.

Frequency of Chromosomal Aberrations in Somatic Cells of Ukrainian Buffaloes (Bubalus bubalis L.)

Routine, GTG-, and Ag-staining techniques for the metaphase chromosome analysis were used to determine the spontaneous frequency of chromosomal aberrations and the level of chromosomal variability in the blood lymphocytes of Ukrainian buffaloes (Bubalus bubalis L.). It has been established that the diploid complement of chromosomes within the studied population of animals consists of 50 chromosomes (2n = 50, XX; 2n = 50, XY). Individual chromosomal variability was detected in some animals in the form of cells with aneuploidy and polyploidy sets of chromosomes as well as in the form of structural aberrations in autosomes. In particular, the chromosomal analysis based on differential staining technique has shown a duplicate in one chromosome of the second pair in individual females as well as an X monosomia and a chimerism over sex chromosomes. The total spontaneous level of chromosomal instability in the peripheral blood lymphocytes reached, on average, 14.35 ± 2.03% due to abnormalities of chromosomal and chromatid types. The Ag staining method was used to detect active NORs in six chromosome pairs (3p, 4p, 6q, 21q, 23q, and 24q).

Frequency of Spontaneous and Radiation-Induced Chromosomal Aberrations in Peripheral Blood Lymphocytes of Individuals of Different Ages

The frequency of spontaneous and X-ray-induced (0.25 Gy) in vitro chromosomal aberrations, as well as the level of chromosomal instability as a result of the bystander effect, in the blood lymphocytes of individuals aged from 12 to 102 years has been investigated using GTG staining of human metaphase chromosomes. The average group frequency of spontaneous chromosomal aberrations in adolescents (12–16 years), middle-aged people (33–52 years), and centenarians (90–102 years) was identical (p > 0.05), whereas it was higher in elderly people (60–70 years) due to increase of chromatid type aberrations rate (p 0.05); however, the total frequency of chromosomal aberrations in individuals aged 60–70 years exceeded the value of such indices in other age groups due to the chromosomal type damages (p < 0.05). In nonirradiated blood lymphocytes of adolescents, middle-aged, and elderly individuals cocultivated with lymphocytes irradiated in vitro (0.25 Gy), the induction of the bystander effect was detected. In lymphocytes of centenarians, the development of the bystander effect was not detected.

The effects of biological and life-style factors on baseline frequencies of chromosome aberrations in human lymphocytes

Objective: This study investigated the influence of sex and ageing on chromosomal damage and the role of life-style habits on the frequency of chromosomal aberrations (CAs) in peripheral blood lymphocytes (PBLs) of healthy Bosnian subjects. Materials and Methods: Peripheral blood samples were obtained from 100 healthy, unrelated individuals in Bosnia and Herzegovina during 2010 and 2011. Chromosome preparations were made by dropping and air drying and slides were stained with 10% Giemsa solution (pH 6.8). The cytogenetic analysis was carried out in a cytogenetic laboratory in the Department of Biology of the Faculty of Science in Sarajevo. The category of total structural CAs was sub classified as chromosome-type aberrations (CSAs) and chromatid-type aberrations (CTAs) while the category of total numerical CAs was sub classified as aneuploid and polyploid mitoses. All statistical analyses were carried out using Microsoft Excel 2010 (Microsoft Corporation) and the Windows Kwikstat Winks SDA 7.0.2 statistical software package (Texa Soft Cedar Hill, Texas). Results: Cytogenetic analysis revealed the average number of structural CAs was 2.84 and of numerical CAs was 9.56. There was a significant increase in the frequency of chromosome-type aberrations (1.92) compared with chromatid-type aberrations (CTAs) (0.92) and a significant increase in the frequency of aneuploid (8.83) compared with polyploid (0.73) mitoses. Significant positive correlations between age and CTAs in human PBLs were also demonstrated. Additional statistical analysis showed that ageing increase number of numerical CAs in lymphocytes of drinkers. The frequency of structural CAs of females exposed to radiation was significantly greater than in males. Analysis indicates the presence of a positive association between CAs and smoking in younger subjects but a negative correlation between aberrant cells frequencies and alcohol in older drinkers. Conclusion: The results of the study support the conclusion that sex and age, together with life-style habits of individuals as confounding factors can affect spontaneous frequency of CAs especially to CTAs in PBLs that are a biomarker of cancer risk.

Genotoxicity assessment in iron deficiency anemia patients using sister chromatid exchanges and chromosomal aberrations assays

Previous studies have shown that iron deficiency anemia is associated with oxidative stress produced by a decrease in antioxidant enzyme activities and/or a high level of oxidants. Because oxidative stress induces DNA damage, we investigated genotoxicity in lymphocytes from patients with iron deficiency anemia (IDA) using chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) assays. Eighteen IDA subjects and a similar number of age-matched healthy controls were included in the study. The results demonstrated that IDA was associated with a slight increase in the frequency of spontaneous CAs and a decrease in the frequency of SCEs (P<0.05). In addition, the level of SCEs was positively correlated with both the ferritin concentration (r=0.485, P<0.05) and hemoglobin content (r=0.514, P<0.05) in subjects. Moreover, vitamin E treatment reduced the frequency of SCEs in IDA patients and control subjects by the same percentage (∼30%) without affecting the magnitude of the difference in the levels of SCEs between the two groups. In conclusion, our results demonstrated that IDA has a differential effect on the frequency of spontaneous CAs and SCEs.

Physiologic Oxygen Enhances Human Embryonic Stem Cell Clonal Recovery and Reduces Chromosomal Abnormalities

Human embryonic stem cells (hESC) have great potential in regenerative medicine, provided that culture systems are established that maintain genomic integrity. Here we describe a comparison of the effects of culture in either physiologic oxygen (2%) or room oxygen (21%) on the hESC lines, H1, H9, and RH1. Physiologic oxygen enabled an average sixfold increase in clone recovery across the hESC lines tested (p < 0.001). FACS analysis showed that cells cultured in physiologic oxygen were significantly smaller and less granular. No significant changes had occurred in levels of SSEA4, SSEA1, TRA-1-60, or TRA-1-81. While karyotypic normalcy was maintained in both H1 and H9, the frequency of spontaneous chromosomal aberrations was significantly increased in room oxygen. This increase was not observed in physiologic oxygen. These results clearly demonstrate that physiologic oxygen culture conditions are indispensable for robust hES clone recovery and may enhance the isolation of novel hES lines and transgenic clones.

DNA polymerase theta is preferentially expressed in lymphoid tissues and upregulated in human cancers.

DNA polymerase theta (Pol theta) is a recently identified family A polymerase that contains an intrinsic helicase domain. Drosophila Pol theta mutants are hypersensitive to bifunctional DNA crosslinking agents and exhibit an elevated frequency of spontaneous chromosomal aberrations, suggesting a role for Pol theta in repair of DNA interstrand crosslinks and in the general maintenance of genome stability. To investigate a possible involvement of Pol theta in tumorigenesis, we have examined its expression in various normal and malignant tissues. Paired tumor and adjacent nontumorous tissues from patients with lung (n = 27), stomach (n = 28) and colon (n = 26) cancer, as well as 26 normal human tissues, were examined for Pol theta expression by RT-PCR, Northern or Western blot analysis. Pol theta was predominantly expressed in primary lymphoid organs including the fetal liver, thymus and bone marrow where lymphocyte progenitors undergo V(D)J rearrangements of their antigen receptor genes. In addition, Pol theta expression was upregulated in germinal center B cells, in which class switch recombination of the immunoglobulin genes occurs. Examination of Pol theta expression in matched cancer specimens revealed that Pol theta was barely detectable in the nontumorous tissues but was upregulated in 17 of 27 (63%) lung, 11 of 28 (39%) stomach and 20 of 26 (77%) colon cancers. Moreover, patients with high levels of Pol theta expression had a significantly poorer clinical outcome compared with those expressing low levels of Pol theta. These results implicate that Pol theta may have a specialized function in lymphocytes and that its overexpression may contribute to tumor progression.

A common link in the mechanism of the self-maintenance of malignant growth: The syndrome of the nonhealing wound

A general principle of the maintenance of malignant growth in all types of tumors has been formulated. According to this principle, stochastic but continuous death of some tumor cells due to the inherent genetic instability of their genome (fragility of chromosomes) is the main event stimulating tumor growth. The dead cells trigger a complex multicomponent process of wound healing expressed as further proliferation of living tumor cells, angiogenesis, stimulation of cell migration, and other events. Stimulation of the proliferation of living cells leads to further death of cells and, as a result, to further stimulation of the system of wound healing, etc. Thus, the tumor sacrifices a small amount of dying cells to stimulate proliferation of all its other cells. It is proposed that the nature of the genetic instability of malignant cells is related to the appearance of an uninemic structure in some regions of chromosomes, in whole chromosomes, or in whole genomes. The author bases his statements on the binemic structure of chromosomes, which has already been experimentally and theoretically substantiated. Uninemic regions have an exceedingly high frequency of spontaneous chromosome aberrations due to blockage of the mechanism of underlying repair of the DNA double break in the absence of a second DNA copy. Possible approaches to a search for more efficient methods of therapy are discussed.

Frequency of spontaneous chromosome aberrations in mice: effects of age

In this paper, we present data on chromosome aberration frequencies in mice which served as unexposed controls in a variety of radiation and chemical toxicology experiments conducted in our laboratory in recent years. All chromosome aberration data were obtained by chromosome painting. In peripheral blood lymphocytes from 102 animals, the frequencies of translocations and insertions increased significantly with age. No increase with age was seen for dicentrics or acentric fragments. When the data were analyzed by strain, the age-related increase in translocation frequencies was observed only in the 71 homozygous C57BL/6 mice and not in any of the three heterozygous strains. Very few aberrations of any type were observed in 62 bone marrow samples, and no effect of age was seen for any aberration type in this tissue. These results are similar to those observed in unexposed humans, and suggest that the increase in translocations is not the result of accumulated damage from chronic `background' environmental exposures but instead may be due to biological processes associated with aging.

In VitroChromosomal Radiosensitivity in Common Variable Immune Deficiency

Common variable immune deficiency (CVID) is characterized by low immunoglobulin levels and recurrent infections in patients with a period of normal immune function several years after birth. It is associated with diarrhea, malabsorption, bronchiectasis, and lymphoreticular malignancies. Radiation-induced chromosome instability may contribute to the high degree of susceptibility to neoplasia. Peripheral blood lymphocyte cultures were obtained from six patients with CVID and the healthy control group matched by age and sex. The groups did not differ in the frequency of spontaneous chromosome aberrations. After exposure to X-ray radiation, mitotic indices were found to be significantly low and incidence of chromosomal alterations were high in the CVID group. We conclude that chromosomes of cells from patients with CVID are significantly more radiosensitive than those of controls. Thus these patients must be protected from unnecessary X-ray examinations and in case of radiosensitive tumour, the dose of irradiation should be carefully monitored.

Effect of hydroxyurea and normal plasma on DNA synthesis in lymphocytes from Fanconi anemia patients

Fanconi anemia (FA) is characterized at the cellular level by a high frequency of spontaneous chromosomal aberrations; crosslinking agents cause an abnormal increase in the frequency of chromosomal damage, and semiconservative DNA synthesis is severely inhibited. Deoxyribonucleotides are needed in both semiconservative and repair DNA synthesis. To investigate the involvement of deoxyribonucleotide pools in the inhibition of DNA synthesis in FA, we evaluated the effect on FA lymphocytes of hydroxyurea (HU), an inhibitor of ribonucleotide reductase which is known to alter the intracellular levels of deoxyribonucleotides. To achieve this goal, lymphocyte cultures of 4 FA patients and 4 normal individuals were used. Cultures were treated with HU and/or mitomycin C and normal human plasma. All cultures were processed to detect the number of DNA synthesizing nuclei by autoradiography. Scoring of 2000 nuclei for each kind of culture every 6 h in the last 24 h of incubation showed that, in long incubation periods, DNA synthesis in FA is largely inhibited by HU and this hypersensitivity may be partially decreased by addition of normal human plasma. It is known that recovery from damage induced by HU involves several enzymes such as flavin oxido-reductase, superoxide dismutase and catalase which are involved in the production or scavenging of O 2 radicals; FA cells are deficient in the detoxification of oxygen and this could explain the response of FA cells to HU.

A high frequency of induction of chromosome aberrations in the bone marrow cells of LEC strain rats by X-irradiation

LEC strain rats, which have been known to develop hereditarily spontaneous fulminant hepatitis 4 to 5 months after birth, are highly sensitive to whole-body X-irradiation when compared to WKAH strain rats. The present results showed that the frequencies of all types of chromosome aberrations induced by X-irradiation in the bone marrow cells of LEC rats were approximately 2- to 3-fold higher than those of WKAH rats, though no significant difference was observed in the frequency of spontaneous chromosome aberrations between LEC and WKAH rats.

The spontaneous frequency of chromosomal aberrations and sister-chromatid exchanges in cultured peripheral lymphocytes of a single blood donor sampled more than 200 times

Blood samples were taken from a single individual over 200 times during a 12-year period and the lymphocytes cultured with the intention of examining the frequencies of chromosomal aberrations and sister-chromatid exchanges (SCE). The data presented here are a compilation of the aberration frequencies in untreated control cultures from all experiments in which at least 100 metaphases were analysed for chromosomal aberrations (135 experiments), and at least 25 for the frequency of SCE (24 experiments). Since 52-h cultures displayed a high mitotic index and a low percentage of second-division metaphases (7%), this culture time was chosen for 70 experiments investigating the frequency of chromosomal aberrations. The inter-experimental variation in aberration frequency was found to be small, 0–4 aberrations per 100 cells, with a mean of 0.79±0.94. The fact that the spontaneous frequency of chromosomal aberrations was distributed according to the Poisson expectation indicates that the mean aberration frequency for a single subject is constant over time. On average, 80% of the aberrations were chromatid breaks (0.59 aberrations per 100 cells). Isochromatid breaks (or chromosome deletions) were not uncommon (0.11). Chromatid exchanges (0.03), as well as dicentric (0.014) and ring (0.014) chromosomes were rare. The incidence of SCE was examined in lymphocytes grown for 72 h in the presence of 20 μM 5-bromodeoxyuridine. The mean number of spontaneous SCE/cell was 6.41±0.69. The variation in SCE frequency between experiments was small, the coefficient of variation being only 0.11.

Effect of inhibitor of poly (ADP-ribose) polymerase in blood lymphocyte cultures of untreated leprosy patients

Poly (ADP-ribose) polymerase is a cellular repair enzyme synthesised following damage to DNA. 3-Aminobenzamide (3-AB) is an inhibitor of this repair enzyme. To study repair efficiency in leprosy patients, who usually show a significantly higher frequency of spontaneous chromosome aberrations and sister-chromatid exchanges (SCEs), their blood lymphocyte cultures were treated with 3-AB. A marginal increase in the frequency of chromosome aberrations was observed following treatment with 3-AB in controls as well as in patient groups. There was also no significant difference in the frequency of SCEs in control cultures with or without 3-AB. A significant increase in the frequency SCEs was observed in lymphocyte cultures of paucibacillary (PB) and multibaacillary (MB) patients treated with 3-AB when compared with controls. Observation of a significant increase in the frequency of SCEs in 3-AB-treated cultures over the untreated value indicates that DNA damage caused in leprosy patients following mycobacterial infection is not repaired because of the presence of the inhibitor of repair enzyme.

Baseline Frequencies of Spontaneous Chromosome Aberrations, Sister Chromatid Exchanges and Micronuclei in Human Newborns.

A cytogenetic monitoring programme on human new borns carried out from 1983-1987 to find out the incidence of constitutional chromosome anomalies also included studies to obtain baseline data on spontaneous chromosomal aberrations (CA), sister chromatid exchanges (SCEs) and micronuclei (MN) from cord blood samples in limited number of human neonates. The results are compared with data obtained in our laboratory on these cytogenetic end points from adult peripheral blood lymphocytes. The micronuclei data was obtained from adult lymphocytes by the cytokinesis blocked lymphocyte assay, by Cytochalasin B. 120 cord blood samples analysed gave a mean frequency of 0.0052 chromatid breaks/metaphase and 0.0059 chromatid gaps/metaphase. Only one dicentric chromosome was noticed in the 12, 000 cells analysed. No statistically significant difference was noticed between male and female new borns in the spontaneous CA frequency. 20 adult blood samples analysed after 48 hr culture and 10 adult blood samples analysed after 72 hr culture gave mean frequency of 0.0085, 0.013 chromatid breaks/metaphase and 0.01414, 0.019 chromatid gaps/metaphase respectively. A large statistically significant elevation in SCE/cell was found when adult blood samples were compared to cord blood samples (the difference 1.94 SCE/cell, P<0.001 by simple ‘t’ test) the mean frequency of SCEs observed in cord blood samples and adult samples being 5.56±0.21 and 7.496±0.21 SCE/cell respectively. The frequency of micronuclei in cord blood lymphocytes was found to be 0-1.5/1000 cells, 1-3/1000 cells, 2-5/1000 cells in 48, 72 and 96 hr cultures respectively. Adult blood lymphocytes exhibited a micronuclei frequency of 0.0123±0.001 MN/CB cell. The low incidence reported here of all three cytogenetic end points may possibly reflect the influence of high folate levels in cord bloods on spontaneous chromosomal damage.

Louis-Bar syndrome: spontaneous and induced chromosomal aberrations in lymphocytes and micronuclei in lymphocytes, oral mucosa and hair root cells

Louis-Bar (L-B) syndrome, also called ataxia-telangiectasia, is cytogenetically characterized by an increased frequency of spontaneous and induced chromosomal aberrations (CA) in cultured lymphocytes and skin fibroblasts. However, it is not yet clear whether the chromosomal instability is also present in uncultured cells. The spontaneous and bleomycin-induced CA in peripheral lymphocytes of 8 L-B patients were evaluated. The micronucleus test was also performed, for the first time in lymphocytes by the cytokinesis-block method, and in uncultured cells of the oral cavity and hair root. The spontaneous frequency of CA and micronuclei in lymphocytes was about 3 times higher in L-B patients than in controls, these two cytogenetic parameters being highly correlated. Moreover, the induction by bleomycin of CA was higher in patients than in controls. The micronuclei in buccal and hair root cells of patients were normal. It remains to be determined whether the different responses obtained with cultured and uncultured cells are the result of the different L-B gene expression of chromosomal instability or whether they arise because of a particular cell sensitivity to culture conditions. The spontaneous and induced CA in lymphocytes of heterozygotes cultured in the presence of L-B serum were studied to evaluate a possible increased sensitivity of heterozygotes to a possible diffusible clastogenic factor present in the plasma of L-B patients. We could not demonstrate the presence of any factor that enhances CA in normal subjects or in heterozygote carriers.

Increased bleomycin-induced chromosome damage in lymphocytes of patients with common variable immunodeficiency indicates an involvement of chromosomal instability in their cancer predisposition.

To study mutagen-induced chromosome instability in cancer disposition, late S and G2 lymphocytes of 15 patients with common variable immunodeficiency and 14 healthy controls were exposed to bleomycin in vitro. The groups did not differ in the frequency of spontaneous chromosome aberrations. In bleomycin-treated samples we found higher numbers of break events per cell and increased frequency of cells with aberrations compared to the control group. A slightly reduced breakage of chromosome group D was noted in patients. These results support the hypothesis that a higher incidence of cancer in patients with genetically determined immunodeficiencies may be explained by an increased mutagen-induced chromosome instability in at least some of them.

Cytogenetical characterisation of Chinese hamster 43-3B transferants with the amplified or non-amplified human DNA repair gene ERCC-1

A comparative study on the biological responses to different mutagens (UV, 4NQO, MMC, MMS and EMS) was made on CHO wild-type cells (CHO-9), its UV-hypersensitive mutant 43-3B, and 2 types of its transferants, i.e., one containing a few copies of the human repair gene ERCC-1 and the other having more than 100 copies of ERCC-1 (due to gene amplification). Cell survival, chromosomal aberrations and SCEs were used as biological end-points. The spontaneous frequency of chromosomal aberrations in the transferants was less than found in 43-3B mutant cells, but still 2–3 times higher than in wild-type CHO cells. The spontaneous frequency of SCEs in the transferants was less than in 43-3B and similar to that of wild-type cells. The induction of SCEs by all tested agents in transferants was similar to that found in CHO-9 cells, while the mutant is known to respond with higher frequencies. ERCC-1 also bestowed resistance to MMS and EMS on the mutant to induction of chromosomal aberrations and cell killing to levels comparable with those of the wild-type strain. On the other hand ERCC-1 could not completely regain the repair proficiency against cell killing and induction of chromosomal aberrations by UV or MMC to the wild-type level. These results suggest that the ERCC-1 corrects the repair defect in CHO mutant cells, but it is unable to rectify fully the defect; probable reasons for this are discussed. However, amplified transferants (having more than 100 copies of the ERCC-1 gene) restored the impaired repair function in 43-3B to UV-, MMC- or 4NQO-induced DNA damage better than non-amplified transferants with a few copies of the ERCC-1. This difference may be due to the high amount of gene product involved in the excision repair process in the amplified cells.

Chromosome abnormalities in peripheral blood lymphocytes from untreated Hodgkin's patients. A possible evidence for chromosome instability.

We describe the presence of a high frequency of spontaneous chromosome aberrations in lymphocytes from six untreated patients with Hodgkin's disease. The characteristics of the chromosome abnormalities observed suggest the existence of a certain degree of chromosome instability in these cases, that could be a predisposing factor for the development of malignancies.

Characterization of a Simian virus 40-transformed Fanconi anemia fibroblast cell line

We have characterized a SV40-transformed human fibroblast cell line (GM6914) derived from a patient with Fanconi anemia (FA) in order to establish its usefulness for biochemical and genetic experiments, including DNA-mediated gene transfer.. GM6914 cells have a growth rate similar to that of SV40-transformed normal human fibroblasts and an indefinite lifespan in culture. As has been established for other FA cell types, GM6914 cells have an increased sensitivity to DNA-crosslinking agents such as mitomycin C (MMC). The D 10 for GM6914 cells is 8 times lower than for equivalent controls. GM6914 cells also have an elevated frequency of spontaneous chromosome aberrations and this frequency can be increased by MMC concentrations which show no effect on control cells. Genetic complementation studies with lymphoblasts derived from two affected sibs of the donor of GM6914 cells show that GM6914 belongs to FA complementation group A. In DNA-transfection studies using plasmid pRSVneo, colonies of GM6914 cells resistant to the drug G-418 were observed at frequencies ranging from 1.7 to 16 × 10 −4, values similar to those observed with several other SV40-transformed human cell lines. GM6914 should be a useful recipient cell line in experiments using DNA-mediated gene transfer to clone the normal allele of the gene which is defective in FA complementation group A. GM6914 would also be an excellent cell line for studies on mutagenesis, recombination and repair using plasmid vectors.