A spot cloning method for restriction landmark genomic scanning

Ohsumi, Tatsuya; Okazaki, Yasushi; Hirotsune, Shinji; Shibata, Hideo; Muramatsu, Masami; Suzuki, Harukazu; Taga, Chiharu; Watanabe, Sachihiko; Hayashizaki, Yoshihide

doi:10.1002/elps.1150160135

Cited by 22 publications

(7 citation statements)

References 8 publications

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“…Four spots in the epididymis of the DES-treated mice were cloned using a modified Not I trapper method [21]. In this study, Easy Anchor Not I (Nippon Gene) was used instead of the Not I trapper.…”

Section: Spot Cloningmentioning

confidence: 99%

Neonatal Exposure to Diethylstilbestrol Alters the Expression of DNA Methyltransferases and Methylation of Genomic DNA in the Epididymis of Mice

et al. 2006

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Abstract. Fetal and neonatal exposure to diethylstilbestrol (DES) is known to cause many abnormalities, such as cancer, in the male and female reproductive tracts later in life, and epigenetic mechanisms, such as DNA methylation, may be involved in these processes. In the present study, newborn C57BL/6 male mice were exposed to 3 µg of DES from postnatal days 1 to 5. Subsequently, the expression levels of the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b and the transcription factors Sp1 and Sp3, which have been reported to regulate the expression of Dnmts, were examined at days 5, 14 and 30. Furthermore, restriction landmark genomic scanning (RLGS), which can analyze genome-wide DNA methylation, was performed to clarify whether or not aberrant DNA methylation was present in the epididymis of the DES-treated mice at day 30. Increased expression of Dnmt3b was observed at days 5 and 14, followed by increased expression of Dnmt1 and Dnmt3a at day 30, as evaluated by real-time RT-PCR. The expression of Sp1 was also increased at day 30. The RLGS analysis revealed that 7 loci of the genomic DNA were demethylated and 1 locus was methylated in the epididymis of the DES-treated mice. Four of these loci specifically demethylated in DES-treated mice were cloned, and all were found to be located within CpG islands near genes. In conclusion, our results indicated the possibility that DES-induced abnormalities of reproductive organs are associated with altered expression levels of DNA methyltransferases and DNA methylation. THE synthetic estrogen diethylstilbestrol (DES) was prescribed to pregnant women to prevent miscarriage from the 1930s through to the 1970s. However, in 1971, a correlation between DES exposure of mothers and the occurrence of vaginal clear cell carcinoma in their daughters was reported [1]. Subsequently, many other problems of reproductive organs, such as malformations and dysfunctions, were reported to be associated with exposure to DES [2]. Studies using murine models revealed that DES induces similar reproductive abnormalities in mice [3][4][5], and DES is now widely used in a model for estrogenic endocrine disruptors. Our previous studies revealed that mice treated neonatally with DES or other endocrine disruptors show increased or decreased expression levels of various genes in the epididymis or testes, even in adulthood [6][7][8][9]. Since these changes in gene expression last for a long

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Section: Spot Cloningmentioning

confidence: 99%

Neonatal Exposure to Diethylstilbestrol Alters the Expression of DNA Methyltransferases and Methylation of Genomic DNA in the Epididymis of Mice

et al. 2006

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“…To further characterize the nature of the genomic, we cloned RLGS spots by the restriction trapper-based RLGS spotcloning method (Ohsumi et al, 1995). To facilitate cloning of the RLGS fragments, NotI fragments were enriched from a 100 mg DNA sample using a restriction trapper (AGENE, Kamakura, Japan) that consisted of latex beads with covalently attached double-stranded DNA adapters having a free NotI restriction sequence at the end.…”

Section: Cloning Of the Dna Fragment From The Rlgs Spot Of Interestmentioning

confidence: 99%

Correlation of postoperative recurrence in hepatocellular carcinoma with demethylation of repetitive sequences

Itano

Ueda

Kikuchi³

et al. 2002

Oncogene

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Restriction landmark genomic scanning (RLGS) was utilized to identify novel genomic alterations in hepatocellular carcinoma (HCC). Thirty-one HCC samples were examined by RLGS. Two high intensity spots were common to several RLGS pro®les of di erent HCCs. Nucleotide sequencing and homology search analysis showed that these spots represented repetitive sequences, Human tandem repeat sequence (Genbank, L09552) and centromeric NotI cluster (Genbank, Y10752). These intensi®ed signals were attributable to the occurrence of demethylated areas in the recognition sequence of the NotI site of the corresponding fragments. The intensity of these spots in the RLGS pro®le re¯ects their degree of demethylation, which was signi®cantly correlated with postoperative recurrence, even in patients regarded as belonging to the good prognosis group by conventional prognostic factors. Multivariate analysis showed that the intensities of the two spots retained independent prognostic value. This is a new type of predictive factor for HCC based on epigenetic changes in hepatocarcinogenesis, and in the future it is expected to be of great value in making preoperative diagnosis and selecting postoperative therapy.

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“…In particular, spot A was common to 64.5%. One of the advantages of RLGS is that DNA fragments can be cloned from the spots (Miwa et al, 1995(Miwa et al, , 1996Costello et al, 1997;Hirotsune et al, 1993;Ohsumi et al, 1995a). In an eort to characterize the genomic changes corresponding to spots associated with HCC, cloning studies are now in progress.…”

Section: Discussionmentioning

confidence: 99%

A new predictive factor for hepatocellular carcinoma based on two-dimensional electrophoresis of genomic DNA

Itano

Ueda

Kikuchi³

et al. 2000

Oncogene

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Molecular genetic analyses have clari®ed that accumulation of genomic changes provides important steps in carcinogenesis and have identi®ed a number of valuable genetic markers for certain cancers. To date, however, no prognostic markers have been identi®ed for hepatocellular carcinoma (HCC). In this study, we used restriction landmark genomic scanning (RLGS), a new high-speed screening method for multiple genomic changes, to detect unknown genetic alterations in HCC. Thirty-one HCC samples and their normal counterparts were examined by RLGS. Eight spot changes were common in several cases, and all were seen only on the HCC pro®le. Five of these spots were detected in more than 12 of 31 cases (38.7%). Viral infection had no in¯uence on changes in the RLGS spots. The disease-free survival rate for patients with 516 changed RLGS spots was signi®cantly lower than that for patients with fewer changed RLGS spots (415 spots) (P50.001). In multivariate analysis, the number of changed spots was proven to retain an independent prognostic value (relative risk 1.095: P=0.0031). These results suggest that the number of changed RLGS spots may be a useful biological marker for recurrence of HCC.

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A spot cloning method for restriction landmark genomic scanning

Cited by 22 publications

References 8 publications

Neonatal Exposure to Diethylstilbestrol Alters the Expression of DNA Methyltransferases and Methylation of Genomic DNA in the Epididymis of Mice

Neonatal Exposure to Diethylstilbestrol Alters the Expression of DNA Methyltransferases and Methylation of Genomic DNA in the Epididymis of Mice

Correlation of postoperative recurrence in hepatocellular carcinoma with demethylation of repetitive sequences

A new predictive factor for hepatocellular carcinoma based on two-dimensional electrophoresis of genomic DNA

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