Molecular Cloning and Preliminary Analysis of a Fragile Site Associated Gene

Objective To analyze the molecular coining of a fragile site-associated gene. Methods Genomic Chinese hamster ovary （CHO） DNA library was constructed using high molecular weight CHO DNA partially digested with MboI restriction enzyme from cultured CHO cells. Screening of genomic DNA library followed the established procedures. Genomic CHO in the positive clones was sequenced. Appropriate primers were designed for the reverse transcriptase-polymerase chain reactions （RT-PCR）. The RT-PCR products were cloned into a pCRII TOPO vector and confirmed by DNA sequencing. Antibodies were prepared using synthetic peptides as antigens by immunizing the rabbits. Immunohistochemical analyses were performed to evaluate the expression of the novel gene in different tissues. Results To investigate the molecular mechanism underlying the initial events of mdrla amplification, we cloned lq31 fragile site DNA. Strikingly, we found that this fragile site contained a novel gene which was designated as a fragile site-associated （FSA） gene. FSA encoded an unusually large mRNA of - 16 kb. Full-length human FSA eDNA was cloned. FSA mRNA was expressed in many cultured cells and tissue types. Immunohistochemical analyses also revealed an expression pattern of the encoded proteins in postmitotic, well-differentiated epithelial compartments of many organs, including colon, mammary glands, ovary, prostate, and bladder. Conclusion FSA plays an important role in regulating mammalian epithelial cell growth and differentiation.

CHROMOSOMAL FRAGILE SITES TO LYMPHOMA WITH OBSERVATION OF 40 PATIENTS

Chromosomal fragile sites studies were performed in 40 patients with lymphoma and 30 individuals as healthy controls. The results showed that there was a statistical difference of chromosomal aberration between the two groups; The patients carried 46 fragile sites totally; 21 out of 46 fragile sites in lymphoma corresponded with cancer breakpoints, and 9 fragile sites were located in the bands where oncogenes exist. These suggest a certain association between fragile sites, cancer breakpoints and oncogenes and thus indicate a possible important role of fragile sites in the pathogenesis of lymphoma.

Predominance of constitutional chromosomal rearrangements in human chromosomal fragile sites

Chromosomal fragile sites (CFSs) are loci or regions susceptible to spontaneous or induced occurrence of gaps, breaks and rearrangements. In this work, we studied the data of 4535 patients stored at DECIPHER (Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources). We mapped fragile sites to chromosomal bands and divided the 23 chromosomes into fragile and non-fragile sites. The frequency of rearrangements at the chromosomal location of clones found to be deleted or duplicated in the array/CGH analysis, provided by DECIPHER, was compared in Chromosomal Fragile Sites vs. non-Fragile Sites of the human genome. The POSSUM Web was used to complement this study. The results indicated 1) a predominance of rearrangements in CFSs, 2) the absence of statistically significant difference between the frequency of rearrangements in common CFSs vs. rare CFSs, 3) a predominance of deletions over duplications in CFSs. These results on constitutional chromosomal rearrangements are evocative of the findings previously reported by others relatively to cancer supporting the current line of evidence and suggesting that a common mechanism can underlie the generation of constitutional and somatic rearrangements. The combination of insights obtained from our results and their interrelationships can indicate strategies by which the mechanisms can be targeted with preventive medical interventions.

Loss expression of active fragile sites genes associated with the severity of breast epithelial abnormalities

Background WWOX and FHIT are two candidate tumor suppressor genes located in active fragile sites, the damage of which has been associated with the development of breast cancer. The association of the expression of these genes and the development of breast cancer has not been fully explored. We evaluated mRNA and protein expression of WWOX and FHIT in breast tissue with normal histological appearances, atypical ductal hyperplasia, ductal carcinoma in situ, and invasive cancer to see if a progressive decline in expression was present. Methods Reverse transcription-polymerase chain reaction and Western blotting were used to evaluate the specimens for mRNA and protein expression, including 28 specimens with normal tissue, 28 specimens with atypical ductal hyperplasia, 33 specimens with ductal carcinoma in situ, and 51 specimens with invasive ductal carcinoma. Results Compared with in situ and invasive cancer specimens, both normal and atypical hyperplasia specimens had greater rates of detectable mRNA （WWOX rate ratio=2.95, 95% CI 1.24-7.08; FHIT rate ratio=4.58, 95% CI 1.82-11.81） and Western blotting detectable protein （WWOX rate ratio=4.12, 95% CI 1.63-10.73; FHIT rate ratio=3.76, 95% CI 1.44-10.06）. For both proteins, differences between normal and atypical hyperplasia specimens and between in situ and invasive carcinoma specimens were explainable by chance （P 〉0.05 for each analysis）. Within each histological category, differences among fractions of specimens showed that FHIT and WWOX mRNA and protein expression were explainable by chance （P 〉0.05 for each analysis）. Conclusion Expression of FHIT and WWOX decreases along with breast tissue progress from a normal histological appearance to atypical ductal hyperplasia, in situ cancer, and the final invasive cancer.

Cytogenetic Effects of Pulsing Electromagnetic Field on Domestic Pig Lymphocytes in Vitro

The effects of pulsing electromagnetic fields(PEMFs)on cells are very important subjects in the field of bioelectromagnetics.In this experiment,the cytogenetic effects of PEMF on domestic pig lymphocytes were tested in vitro.Pig lymphocytes in RPMI 1640 medium were exposed to PEMFs of 100 kHz and 200 kHz for 12,24 and 48 hours.Chromosomal aberrations(aneuploidy,breaks,gaps,et al)were significantly increased in exposed cultures,and of these aberrations,56%chromosomal or chromatid breaks and 42%gaps induced by PEMFs were the points of pig chromosomal fragile sites.The baseline frequency of sister chromatid exchange(SCE)increased after exposing lymphocytes continuously to PEMFs of 100 kHz and 200 kHz for 48 hours.These results suggested that the exposure to PEMFs might induce a type of DNA lesion and chromosomal aberrations.

DEtail-seq is an ultra-efficient and convenient method for meiotic DNA break profiling in multiple organisms

Programmed DNA double-strand break(DSB)formation is a crucial step in meiotic recombination,yet techniques for highefficiency and precise mapping of the 3’ends of DSBs are still in their infancy.Here,we report a novel technique,named DNA End tailing and sequencing(DEtail-seq),which can directly and ultra-efficiently characterize the 3’ends of meiotic DSBs with near single-nucleotide resolution in a variety of species,including yeast,mouse,and human.We find that the 3’ends of meiotic DSBs are stable without significant resection in budding yeast.Meiotic DSBs are strongly enriched in de novo H3K4me3 peaks in the mouse genome at leptotene stage.We also profile meiotic DSBs in human and find DSB hotspots are enriched near the common fragile sites during human meiosis,especially at CCCTC-binding factor(CTCF)-associated enhancers.Therefore,DEtail-seq provides a powerful method to detect DSB ends in various species,and our results provide new insights into the distribution and regulation of meiotic DSB hotspots.

Hot spots of DNA double-strand breaks and genomic contacts of human rDNA units are involved in epigenetic regulation

DNA double-strand breaks(DSBs)are involved in many cellular mechanisms,including replication,transcription,and genome rearrangements.The recent observation that hot spots of DSBs in human chromosomes delimit DNA domains that possess coordinately expressed genes suggests a strong relationship between the organization of transcription patterns and hot spots of DSBs.In this study,we performed mapping of hot spots of DSBs in a human 43-kb ribosomal DNA(rDNA)repeated unit.We observed that rDNA units corresponded to the most fragile sites in human chromosomes and that these units possessed at least nine specific regions containing clusters of extremely frequently occurring DSBs,which were located exclusively in non-coding intergenic spacer(IGS)regions.The hot spots of DSBs corresponded to only a specific subset of DNase-hypersensitive sites,and coincided with CTCF,PARP1,and HNRNPA2B1 binding sites,and H3K4me3 marks.Our rDNA-4C data indicate that the regions of IGS containing the hot spots of DSBs often form contacts with specific regions in different chromosomes,including the pericentromeric regions,as well as regions that are characterized by H3K27ac and H3K4me3 marks,CTCF binding sites,ChIA-PET and RIP signals,and high levels of DSBs.The data suggest a strong link between chromosome breakage and several different mechanisms of epigenetic regulation of gene expression.