The DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) removes alkylating adducts from the O(6) position of guanine and protects cells from cytotoxic and mutagenic effects. Expression of MGMT is decreased in some cancers, which may be the result of methylation of CpG islands of both the promoter and coding regions of the gene. We studied the methylation status of the MGMT promoter in a very large collection of brain tumors (85) using methylation-specific polymerase chain reaction (PCR). Aberrant methylation occurred in 48% of 85 human brain tumor samples. Quantitative real-time PCR showed that expression of MGMT mRNA levels was significantly decreased (P < 0.001) in those brain tumors that had methylation of the promoter region of their MGMT gene. MGMT can prevent G to A mutations by removing alkyl groups from the O(6) position of guanine. We found a significantly increased frequency of G:C to A:T mutations of the p53 gene in brain tumors having a methylated MGMT promoter compared with those having an unmethylated MGMT promoter (P < 0.05), and all the non-CpG dinucleotide G:C to A:T mutations of p53 were in samples with a methylated MGMT promoter.
Transposition mediated by the TnlOOO transposase was investigated by using transposon variants carrying synthetic or wild-type termini but no intact TnlOOO genes. Transposon Tnl001, whose only homologies to TnlOOO are in its 38-base-pair terminal inverted repeats, transposed at the same rate as Tn1005, an artificial construct carrying wild-type TnlOOO termini and approximately 1 kilobase of flanking Tn1000 DNA at each end, when transposase was supplied in trans. The majority of the transpositions into pOX38 gave rise to cointegrates, but approximately 10% of the products expressed phenotypes of direct transpositions. The expression and temperature dependence of the tnpA gene product were examined by studying transposition of TnlOOI to bacteriophage lambda. The temperature optimum for transposition was 37°C, and the transposase was stable for up to 2 h at this temperature.Transposon Tn1000 (-yb) was identified on the F plasmid of Escherichia coli K-12 (11, 15). Like other members of the Tn3 family, Tn1000 encodes a large transposase (Mr, 114,000), and it forms cointegrate intermediates which are later resolved by the tnpR gene product. The transposition properties of Tn3 and Tn1000 elements are expected to be similar, but there may be differences. The Tn1000 terminal inverted repeats, res site, and resolvase gene region are closely related to the corresponding elements of Tn3 (14,23,34). However, the inverted repeats of TnJOOO are only 35 base pairs (bp) long, and the Tn3 and TnlOOO tnpA gene products do not substitute for one another (20).The leftward end of Tn1000 (includes IRL) displays imperfect sequence homology to the inverted repeats of Tn3 over a 38-bp interval. In this paper we demonstrate that 38-bp terminal inverted repeats corresponding to the left end of TnlOOO were the only sequences required in cis for transposition sponsored by the Tn1000 transposase. We found a significant proportion of direct transpositions, and we describe the in vivo properties of the TnlOOO transposase. MATERIALS AND METHODSBacterial strains, bacteriophage, and plasmids. The bacterial strains (derivatives of E. coli K-12) and plasmids are listed and described in Table 1. Bacteriophage Xb2 is a deletion mutant of phage X with 12% of the parent phage genome, in the b region, deleted (31). Plasmid pMS421 (M. Susskind, unpublished results) is a derivative of pSC101 harboring the streptomycin and spectinomycin resistance genes, with the lacIq gene inserted on an EcoRI fragment. It was used to control the tnpA gene in pXRD4043 (Fig.1). Plasmid pOX38 is composed of largest HindIII fragment of F (17), which contains the entire tra operon.Small plasmid constructs are shown in Fig. 1 (Fig. 1D, region II). The adapter contains an artificial Shine-Dalgarno sequence and the first 7 bp at the amino-terminal end of the tnpA gene. In this construction, pACYC184 was digested with HindlIl and HincII, and the outer 12 bp of IRL (adjacent to tnpA) were removed at the HaeIII site. The sequence of the region shown in Fig. 1D was verified by the d...
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