Arbitrarily Primed Polymerase Chain Reaction Research Articles

Genetic instability and divergence of clonal populations in colon cancer cells in vitro

The accumulation of multiple chromosomal abnormalities is a characteristic of the majority of colorectal cancers and has been attributed to an underlying chromosomal instability. Genetic instability is considered to have a key role in the generation of genetic and phenotypic heterogeneity in cancer cells. To shed light on the dynamics of chromosomal instability in colon cancer cells, we have analyzed genetic divergence in clonal and subclonal derivates of chromosomally unstable (SW480) and stable (HCT116, LoVo) cell lines. Conventional G-banding karyotyping and arbitrarily primed PCR (AP-PCR) fingerprinting were used to calculate genetic distances among clones and parental cells, and to trace tree-type phylogenies among individual cells and clonal cell populations. SW480 cells showed enhanced karyotypic heterogeneity in clones as compared with parental cells. Moreover, genetic clonal divergence was also increased after two consecutive episodes of single-cell cloning, demonstrating that the homogeneity induced by the bottleneck of cloning is disrupted by genetic instability during clonal expansion and, as a consequence, heterogeneity is restored. These results demonstrate genetic drift in clonal populations originated from isolated cells. The generated cell heterogeneity coupled with selection provides the grounds for the reported feasibility of pre-neoplastic and neoplastic cells to generate new phenotypic variants with increased evolutionary potential.

Global DNA demethylation in gastrointestinal cancer is age dependent and precedes genomic damage

We studied the relationships between genetic and epigenetic alterations in gastrointestinal cancer by integrating DNA copy number changes determined by arbitrarily primed PCR (AP-PCR) with DNA methylation variations estimated by methylation-sensitive amplified fragment length polymorphism (MS-AFLP). We analyzed about 100 different chromosomal regions by AP-PCR and over 150 random CpG loci by MS-AFLP in human colon and gastric carcinomas. DNA hypomethylation and hypermethylation alterations distributed gradually and increased with cancer patient age, in contrast with the age-independent genomic alterations. Increased DNA hypomethylation and hypermethylation correlated with increased genomic damage, but only hypomethylation was highly significant in multivariate analyses. We conclude that age-dependent accumulation of DNA demethylation precedes diploidy loss in a significant subset of gastrointestinal cancers.

Molecular identification of Astragalus membranaceus at the species and locality levels

Astragalus membranaceus (Fisch.) Bunge, a commonly used Chinese medicinal material, from certain localities contains more favourable trace elements and fewer harmful trace elements than those from other localities. Therefore, there is a need to distinguish Astragalus membranaceus from different localities. Internal transcribed spacer 1 (ITS1) of the nuclear ribosomal RNA gene of 23 Astragalus membranaceus samples were sequenced to confirm the species of the samples. Arbitrarily primed polymerase chain reaction (APPCR) was then used to obtain unique fingerprints for each sample using several primers. The presence and absence of bands were used for calculating mean similarity indexes among the samples. It was found that the Heilongjiang samples were markedly distinguishable from samples of other localities. In addition, bands common for samples from the same locality were also identified and used to distinguish samples from Neimengu and Shanxi. Therefore, Astragalus membranaceus from these provinces, the major cultivation places in China, can be differentiated.

Detection of the frequency, antimicrobial susceptibility, and genotypic discrimination of Aeromonas strains isolated from municipally treated tap water samples by cultivation and AP-PCR

The frequency, antibiotic susceptibility, and genotypic discrimination of Aeromonas strains isolated from municipally treated drinking tap water distribution systems were investigated in this study. We have analyzed 148 tap water samples collected from 8 different locations by bacterial cultivation and arbitrarily primed polymerase chain reaction (AP-PCR). Gram negative, hemolytic, oxidase (+) and catalase (+) bacterial colonies were applied to the study. Identification of bacterial colonies was done by conventional biochemical method and API ID 20E panel (BioMerieux-France). Molecular epidemiological discrimination of the isolates was done by AP-PCR. Aeromonas spp. was detected in 6 of 148 (4%) tap water samples from 8 different locations. Five isolates were identified as Aeromonas hydrophila and one isolate was identified as Vibrio fluvialis by conventional biochemical method. These data were also confirmed by API 20E panel. One of 6 isolates was resistant to gentamicin, 2 of 6 isolates were resistant to trimethoprim/sulfamethoxazole, 4 of 6 isolates were resistant to ampicillin and ampicillin-sulbactam and all of 6 isolates were resistant to cephalothin. All isolates were found to be susceptible to amikacin, aztreonam, ceftazidime, ceftriaxone, ciprofloxacin. All 6 strains of Aeromonas were discriminated by AP-PCR and were determined that all isolates were from different genotypic sources. Although the frequency of the isolates was under the standard limits, the results indicate that hemolytic A. hydrophila are present in municipally treated tap water samples in Mersin City. While all strains were genotypically distinct, all of them were resistant to first generation beta lactam antibiotics tested in this study.

Molecular epidemiology of Vibrio nigripulchritudo, a pathogen of cultured penaeid shrimp ( Litopenaeus stylirostris) in New Caledonia

A collection of 57 isolates of Vibrio nigripulchritudo from either diseased or healthy shrimp and from shrimp farms environment was studied in order to gain a better understanding of the epidemiology of this pathogen, notably isolated from two distinct shrimp disease complexes. Molecular typing using two different techniques, arbitrarily primed PCR (AP-PCR) and multi-locus sequence typing (MLST), studied together with experimental pathology data allowed a relevant epidemiological insight into this possibly emerging pathogen. Additionally, results obtained with the two molecular typing techniques were congruent and allowed discriminating the strains associated with the “Summer Syndrome” from strains isolated from other contexts, especially the other shrimp vibriosis “Syndrome 93”. These results highlight that the “Summer Syndrome” is most probably caused by an emergent clonal pathogen that therefore deserves surveillance and that AP-PCR can satisfactorily be used for that purpose.

Genomic instabilities in squamous cell carcinoma of head and neck from the Indian population

Intrinsic genomic instability of an incipient tumor cell drives the development of cancer. In colorectal cancer, an inverse relationship between microsatellite instability (MIN) and chromosomal instability (CIN) has been reported. The relationship between MIN and CIN in head and neck squamous cell carcinoma (HNSCC) is uncertain. In the present study, we examined these two types of instabilities in HNSCC using microsatellite markers and arbitrary primed PCR (APPCR) technique. HNSCC tumors showed high frequency of MIN and loss of heterozygosity (LOH). We observed that, in contrast to colorectal tumors, the frequency of LOH increased with the increase in MIN. There was no significant difference between MIN- and MIN+ groups of HNSCC tumors in the extent of overall genomic alterations; rather higher MIN+ tumors exhibited higher incidence of deletion. Thus, in sporadic HNSCC, both MIN and CIN pathways operate simultaneously to drive tumor formation.

Increase in Probing Depth Is Correlated With a Higher Number of Prevotella intermedia Genotypes

The aims of this study were to determine the genotypic diversity of Prevotella intermedia in subgingival plaque samples by using two techniques, arbitrarily primed polymerase chain reaction (AP-PCR) and heteroduplex analysis, and to assess the relationship of this diversity with increase in probing depth. The subgingival plaque samples were obtained from 12 patients using paper points inserted into periodontal pockets (diseased sites) and healthy gingival sulci (healthy sites) of the same subjects. After isolation and identification, AP-PCR was performed for genotypic characterization of P. intermedia (80 isolates). The clinical samples with a positive result for P. intermedia were amplified by 16S rRNA-based PCR method, and the amplicons were subjected to heteroduplex analysis. The agreement between the two methods was very high; the AP-PCR and heteroduplex analysis showed that subjects harbored between one and five distinct genotypes of P. intermedia, with a positive association between numbers of genotypes by AP-PCR (P = 0.0042) or heteroduplex (P = 0.0099) and increase in probing depth. No matching of P. intermedia genotypes was observed between healthy and diseased sites of the same individual. Interindividual analyses demonstrated absence of identical clones and indicated a high level of genetic diversity in the species. A clear relationship was observed between a higher number of genotypes and increase in probing depth; these results suggest that environmental challenges in the periodontal pockets may modulate the microbiota by selecting genotypes best able to exploit the environment.

Differences of cytosine methylation in parental lines and F1hybrids of Large White×Meishan crosses and their effects on F1performance

AbstractIn order to probe the effect of methylation on heterosis, the methylation-sensitive arbitrarily primed polymerase chain reaction (AP-PCR) technique was adopted to amplify pig genome DNA with 40 single arbitrary primers. The material involved parental lines and F1hybrids of Large White×Meishan crosses. Nineteen differentially methylated sites withRsaI+HpaII digestion and 14 differentially methylated sites withRsaI+MspII digestion between parental lines and the hybrid were found. All fragments detected in this study were grouped into four classes: (1) the same level of methylation in both parental lines and the hybrid; (2) the same level of methylation in one parent and the hybrid; (3) an increased level of methylation in the hybrid compared to the parents, and (4) a decreased level of methylation in the hybrid. Five sites had significant effects on seven traits (P<0.05). Sequence analysis showed that three sequences had a high-identity match in GenBank (greater than 87%) and two sequences had no match in the database. The percentage of G+C in three sequences was over 50, and the observed/expected CpG of all sequences was above 0.6. Furthermore, one sequence contained G/C boxes. This study demonstrated that the sites in CpG islands within a gene promoter were differentially methylated in the hybrid compared to parental lines; methylated sites contributed differentially to F1performance, showing that heterosis could benefit from either expression or repression of some genes.

Outbreaks of Serratia marcescens bacteriuria in a neurosurgical intensive care unit of a tertiary care teaching hospital: A clinical, epidemiologic, and laboratory perspective

Serratia marcescens is an aerobic gram-negative bacillus belonging to the family Enterobacteriacea. Infections caused by S marcescens may be difficult to treat because of their resistance to a variety of antibiotics, including beta-lactams and aminoglycosides. This study aimed to (1) identify the risk factors associated with the development of Serratia marcescens bacteriuria in neurosurgical intensive care units (NSICU); (2) genotype the pathogens to determine the source of infection; (3) compare these results with antibiograms; and (4) determine and implement appropriate control measures. A retrospective case-control study of the epidemiologic data, the surveillance of environmental cultures, and the genotyping of strains using arbitrarily primed polymerase chain reaction (AP-PCR) were performed at a 750-bed, tertiary care teaching hospital. Seventy-four bacteriuria patients were compared with 74 age/sex-matched control patients in the NSICU between March 2002 and March 2004. The factors assessed were patient demographics; duration of hospital stay; duration of indwelling catheter use before and during stay in the NSICU; chronic underlying illnesses (diabetes mellitus, cardiovascular disease, malignancy); other sites of infection; history of trauma; exposure to a nasogastric tube; mechanical ventilation; urinary catheterization; central venous catheterization; surgical drainage; tracheostomy; brain or spine surgery; and receipt of total parenteral nutrition (TPN), antimicrobials (beta-lactams, aminoglycosides, quinolones, carbapenems, vancomycins), or steroids. Patients with S marcescens bacteriuria were more likely to have a longer NSICU stay and other sites of infection. Environmental surveillance showed the handling of urine jugs to be the point source of contamination. Genotyping and antibiograms of 14 patients were the same except for those of 2 patients. The patient-related risk factors were identified, and a rapid identification of the organism was made. Heightened surveillance, infection control measures, and empiric therapy led to improved methods for handling urine jugs, which terminated the outbreak.

Novel DNA amplification on chromosomes 2p25.3 and 7q11.23 in cholangiocarcinoma identified by arbitrarily primed polymerase chain reaction

To detect and characterize amplified DNA sequences in cholangiocarcinoma (CCA). We extracted DNA from tumor and corresponding normal tissues of 30 patients with CCA and amplified with 30 random ten-mer arbitrary primers by the arbitrarily primed polymerase chain reaction (AP-PCR) technique. Our results showed gains of genomic sequences at high frequency. Using the AX-11 arbitrary primer, we determined an amplified DNA fragment occurred frequently in the tumors analyzed. The DNA fragment was isolated and identified as two sequences mapped to chromosomes 2p25.3 and 7q11.23. Specific primers were designed employing these sequences and used for detecting amplification by real-time quantitative PCR. The amplification of the DNA sequences on chromosomes 2p25.3 and 7q11.23 was detected in 10 (33%) and 6 (20%) cases, respectively. Thirteen (43%) cases showed amplification on both or one of the chromosomes. In addition, amplification of the DNA on chromosome 2p25.3 was predominantly observed in poorly differentiated tumors. Our findings suggest that the novel amplified DNA on chromosomal regions at 2p25.3 and 7q11.23 might be involved in the development and progression of CCA.

Genotyping of Streptococcus mutans by using arbitrarily primed polymerase chain reaction in children with Down Syndrome

The aim of this study was to compare the caries prevalence between Down Syndrome (DS) and non-DS children and to investigate the difference between the genotypes of Streptococcus mutans (S. mutans) colonized in both DS and non-DS groups. Sixty children with DS and 64 non-DS children aged between 7 and 12 years old were included to this study. All erupted teeth were evaluated according to the criteria recommended by the World Health Organization. Unstimulated saliva samples were carried out from the children and cultivated on S. mutans selective Tryptone-yeast-cystine (TYC) agar with 0.2 U/ml bacitracin and 15% sucrose. Molecular typing of S. mutans strains was performed by using arbitrarily primed polymerase chain reaction (AP-PCR) with OPA-05 primer. All data were analysed by using SPSS (SPSS Inc., Chicago, IL, USA) 11.0 software program for windows. The caries index scores were found significantly lower in DS individuals than the non-DS group (p < 0.05). The salivary S. mutans levels between DS and non-DS groups did not show significant difference (p > 0.05). The difference between dental caries and salivary S. mutans levels also was not statistically significant (p > 0.05). According to the results of the AP-PCR typing, all profiles of S. mutans which colonized in DS group were different from the control group. The relationship between these different profiles and dental caries prevalence was statistically significant (p < 0.05). The profiles of S. mutans colonized in DS group might be a reason of low caries prevalence.

Genotypic comparison of bacteria recovered from human bite marks and teeth using arbitrarily primed PCR

This study assessed, for forensic purposes, the feasibility of genotypically matching oral streptococci recovered from recent human bite marks with those from the teeth of the biter. Streptococci were isolated from the incisors of eight volunteers. Arbitrarily primed PCR (AP-PCR) distinguished 106 streptococcal genotypes among the participants, each harbouring at least eight distinct strains. In a crime simulation, a sample from an experimental bite mark was analysed by an experimenter unaware of its origin. The bacteria were unambiguously matched to the biter by comparing the amplicon profiles with those from the eight participants. In contrast, bacteria from an additional bite mark (not generated by one of the original participants) could not be matched to any of the eight participants. Between 20 and 78% of catalogued bacterial genotypes were recovered 12 months later from each participant. Throughout the study period, none of the bacterial genotypes were shared between participants. Streptococci isolated from recent bite marks can be catalogued by AP-PCR and matched to the teeth responsible for the bite. The study provides 'proof of concept' that genotypic analysis of streptococci from bite marks may provide valuable forensic evidence in situations where the perpetrator's DNA cannot be recovered.

Detection of MutA Genes in Transmitted Strains and Nontransmitted Strains of Mutans Streptococci

Our aim was to determine whether an isolate carrying one of the mutA genes was related to its ability to be transmitted from mother to her child. First, 200 mutans streptococci isolates were genotyped by arbitrarily primed polymerase chain reaction (AP-PCR) to demonstrate transmission between 20 mother-child pairs and to detect the transmitted and nontransmitted strains. Thenthemutacin structural genes mutA encoding mutacin types I, II, and III were screened by PCR. The results showed that all strains found to carry the mutAI gene were nontransmitted strains; PCR screening primers mutAII and mutAIII did not yield amplicons in any of the strains tested. Our data suggest that an isolate carrying the mutAI gene is related to reduced transmission. The low frequency of detection of mutAII, and mutAIII suggests that there is a high heterogeneity in the genetic determinants needed for the production of mutacin-like substances.

Caractérisation moléculaire des souches invasives d' Haemophilus influenzae isolées dans la région du Sahel tunisien

We reported a molecular characterization of 25 Haemophilus influenzae strains derived from cases of meningitis and sepsis in children aged less than five years hospitalized in pediatric wards from three hospitals in the Sahel area (Tunisia) during the period 1997–2002. These strains were biotyped and subjected to a capsular typing by Slide agglutination serotyping and Polymerase Chain Reaction (PCR). The genetic polymorphism of these strains was also studied in Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) with two sets of primers: RAP IV and 217 δ 2 as in Pulsed Field Gel Electrophoresis after digestion of the total DNA with the restriction enzyme SmaI (PFGE SmaI). Nineteen strains among 25 (76%) were of biotype I. The bexA gene was highlighted in 13 strains (52%) and in all the cases it was of the type b. Twelve strains (48%) were shown to be unencapsulated by PCR. AP-PCR RAP IV (23 genotypes/25 with a discrimination index ID =0.993) had shown nearly the same discriminatory power than PFGE (20 genotypes/21 strains with a discrimination index ID =0.995). We thus note, how capsular typing by PCR is more sensitive than slide agglutination serotyping. We also note the genetic diversity of the invasive strains isolated with a remarkable presence of non typable strains. AP PCR seems to be an alternative of choice for the epidemiologic follow-up of the Haemophilus influenzae invasive infections.

The Prevalence of Fecal Colonization of Enterococci, the Resistance of the Isolates to Ampicillin, Vancomycin, and High-Level Aminoglycosides, and the Clonal Relationship Among Isolates

The gastrointestinal tract carriage of enterococci was searched in 150 hospitalized patients and 100 outpatients, and clonal relatedness of the isolates and their resistance to ampicillin, vancomycin, and high-level streptomycin and gentamicin were investigated. A stool sample or rectal swab collected from each patient was inoculated into appropriate media within an hour. Enterococcus species were identified by using conventional biochemical tests, API-20 Strep assay, and BBL crystal kit. Antibiotic susceptibility tests were performed using Kirby-Bauer disk diffusion method. A polymerase chain reaction (PCR) was used to detect vanA and vanB genes. Pulsed-field gel electrophoresis (PFGE) and arbitrarily primed-polymerase chain reaction (AP-PCR) methods were used for molecular typing of the strains. Enterococci were isolated from 90 (60%) of the specimens collected from 150 inpatients. Of these 90 isolates, 37 (41%) had high-level gentamicin resistance, 36 (40%) had high-level streptomycin resistance, and 50 (55.6%) had ampicillin resistance. Fecal colonization was found in 30% of the outpatients. Resistances to ampicillin, high-level streptomycin, and gentamicin were 13%, 10%, and 3%, in these patients' isolates, respectively. No vancomycin-resistant enterococci were detected by both agar diffusion and PCR assays in our study. Both typing procedures were applied on 78 Enterococcus strains isolated from inpatients. AP-PCR typing showed that 30 (50.8%) of the 59 E. faecium and 5 (50%) of the 10 E. faecalis strains were clonally related. These values were found to be 12 (20.3%) and two (20%) by PFGE, respectively. The typing procedures did not find any clustered strains in the six E. durans and three E. avium isolates. Neither PFGE nor AP-PCR result was significantly different among the sensitive and resistant strains. Our results indicate that the high prevalence of colonization with ampicillin and highlevel aminoglycoside-resistant enterococci is an important problem in our medical center. The high clonal diversity among the isolates indicates limited spread of antibiotic-resistant strains between patients.

Anti-apoptotic Proteins Induce Non-random Genetic Alterations that Result in Selecting Breast Cancer Metastatic Cells

To shed light on the relationships between over-expression of anti-apoptotic proteins, genomic instability, and the metastatic ability of breast cancer cells, we analyzed genetic changes in tumors and metastases by orthotopically injecting MDA-MB 435 cells transfected with anti-apoptotic genes Bcl-xL or Bcl-2 into nude mice. Tumors and metastasis variants were extracted by primary culture from breast, bone, lung, and lymph node from mice with 435/Bcl-xL, 435/Bcl-2, and 435/Neo tumors. Using the Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), which permits the detection of allelic imbalances, we generated four different fingerprints utilizing four primers. We found that the genetic damage fraction (GDF) increased in 435/Bcl-2 (GDF=0.55) and 435/Bcl-xL cells (GDF=0.34), in regard to 435/Neo control cells (GDF=0.29), indicating that non-random genetic alterations occurred in cells secondary to Bcl-2 or Bcl-xL over-expression. Anti-apoptotic proteins render breast cancer cells susceptible to the in vivo acquisition of highly tumorigenic (Kruskal-Wallis, P=0.019) and metastatic (Kruskal-Wallis, P=0.004) activity. We therefore propose that genetic instability is a molecular mechanism favored by anti-apoptotic proteins involved in the selection of highly metastatic cells during tumorigenesis, a pathogenic event favoring the expansion of metastasis.

Molecular characterization of Staphylococcus intermedius carriage by healthy dogs and comparison of antimicrobial susceptibility patterns to isolates from dogs with pyoderma

We conducted an epidemiological study of Staphylococcus intermedius using arbitrarily primed PCR (AP-PCR) and antibiograms. One hundred and twenty-five S. intermedius isolates were recovered from the oral cavity and/or cranial hair coat of healthy dogs enrolled in a pet therapy program. Commensal S. intermedius was cultured from 32% of the oral cavity cultures and 13% of the cranial hair coat cultures. We characterized the colonization of the dogs as transient, intermittent, or persistent. For dogs characterized as persistently colonized, 73% of the isolates came from the oral cavity. These isolates were also genotyped by AP-PCR. A single major AP-PCR type was observed in 91% of the dogs (n=22); minor variations were frequently observed in these major types. Antibiograms of these commensal isolates were compared to antibiograms from 97 historical clinical isolates (1988-1992) obtained from cases of canine pyoderma. Resistance was most often observed to penicillin (64% and 55%) and tetracycline (38% and 38%) among the commensal and clinical isolates, respectively. The commensal isolates were significantly less resistant to erythromycin, clarithromycin, clindamycin, and trimethoprim/sulfamethoxazole. Our data suggests that differences in both genotype and antimicrobial susceptibility phenotypes exist among S. intermedius strains isolated from different anatomic sites from the same dog and supports the opportunistic nature of S. intermedius in canine infections.

An improved arbitrary primed PCR method for rapid characterization of transposon insertion sites

Modifications were made to published arbitrary primed polymerase chain reaction (AP-PCR) procedures that resulted in increased specificity and sensitivity. Several arbitrary primer sequences were also evaluated, resulting in recommendations for primer design.

Pilot study of arbitrarily primed PCR-single stranded DNA conformation polymorphism analysis for screening genetic polymorphisms related to specific phenotypes

Background To investigate relationships between phenotypes and genotypes is not simple. We propose a phenotype-to-genotype screening strategy and pooled DNA system. As a pilot study of this strategy, we used arbitrarily primed polymerase chain reaction (AP-PCR) in combination with single-stranded DNA conformation polymorphism (SSCP) to screen for genetic polymorphisms associated with longevity. Methods Study subjects were separated into 3 age groups, individuals aged >100 years, 90–99 years and 60–69 years. Genomic DNAs were prepared from each individual, pooled to represent the 5 study groups, and then the pooled genomic DNAs were subjected to AP-PCR-SSCP analysis. Results We found 1 SNP more frequently in senior citizens with longevity. The genotype frequency of the 82133G>A polymorphism of human chromosome 3 clone RP11-61K12 (AC011199) differed significantly ( P=0.0189, Fisher's exact test) between older subjects (>90 years) and younger subjects (<70 years). It is noteworthy that the strategy we describe herein was useful for identifying an SNP that showed statistically significant differences in its distribution across the subject groups. Conclusions The pooled DNA strategy and quantitative genotype discrimination can also be applied to screening for the relationship between phenotype and genotype more effectively.

Germline genomic instability in PCNA mutants of Drosophila: DNA fingerprinting and microsatellite analysis

PCNA participates in multiple processes of DNA metabolism with an essential role in DNA replication and intervening in DNA repair. Temperature-sensitive PCNA mutants of Drosophila ( mus209) are sensitive to mutagens, impair developmental processes and suppress positional-effect variegation. To investigate the role of proliferating cell nuclear antigen (PCNA) in germline genomic stability, independent mus209-defective and mus209-normal lines were established and maintained over six generations. A time course study was carried out and general genomic alterations were analyzed in the progeny by using arbitrarily primed PCR (AP-PCR) and microsatellite analysis. The AP-PCR analysis has shown that a dysfunctional PCNA leads to germline genomic instability, being the amount of genomic alterations transmitted to the progeny directly related to the number of mus209 B1 mutant alleles. In addition, we have found that the frequency of genomic alterations tends to increase over successive generations. Surprisingly, the highest microsatellite instability was found in the heterozygous mus209-defective lines, suggesting a greater mutation rate in these individuals, in comparison with the homozygous mus209-defective lines. In conclusion, our results clearly indicate that PCNA is an important factor to maintain genomic stability in germinal cells, both in the overall genome and in simple repeated sequences. The implication of PCNA mutations in transgenerational genomic instability and related to cancer susceptibility is also discussed.