Fast-BAC with Cassette Player

Generating transgenic mice to study gene function requires highly efficient strategies for cloning and manipulating large genomic sequences. The large cloning capacity (up to 300-kb insert) of bacterial artificial chromosome (BAC)-based vectors makes them a popular tool for this purpose. Chromosome engineering, or recombineering, using BACs can, however, be hampered when using large targeting constructs containing multiple repeated elements because of undesirable recombination events. To overcome this problem, L. Blaas and colleagues at Ludwig Boltzmann Institute for Cancer Research (Vienna, Austria) describe in this issue the use of ΦC31-mediated cassette exchange technique to introduce a large construct (16.5 kb) containing several repeated elements into a BAC. The ΦC31 integrase catalyzes recombination between a pair of heterologous sites, namely attP and attB. The authors first introduced an attP-flanked ampicillin (Amp) cassette, with 200 bp homology arms on either side, into a Rosa26 BAC to form a docking site, followed by targeting a large attB-flanked construct (16.5 kb) consisting of repetitive modules to the docking site. The ΦC31-mediated recombination resulted in the substitution of the Amp cassette by the second construct containing the repeats. The authors show that their new technology is efficient, straightforward, and unidirectional. In addition, this technique is useful where the presence of similar sequences between the BAC and the targeting construct may make it rather difficult to obtain a correctly targeted BAC via homologous recombination. Therefore, the technique developed in this study can complement homologous recombination technology and will be a potent tool to facilitate the modification of BACs with highly complex targeting constructs.

(See “ΦC31-mediated cassette exchange into a bacterial artificial chromosome” on page 659.)

Heavy Methyl: Tracking Histone Modifications

Post-translational modification of histones—through methylation, acetylation, and ubiquitination—is an important means of chromatin regulation. One type of histone methylation is mediated by the histone lysine methyltransferases (HKMTs), which methylate histone tails using S-adenosyl-L-methionine (AdoMet) as the methyl donor at specific lysine residues. A better understanding of the mechanisms of these enzymes is important, since they represent potential targets for anticancer drugs. The current assays for HKMT activity share the disadvantage of being discontinuous. P. Rathert and his colleagues at Jacobs University School of Medicine in Bremen, Germany, have developed an alternate approach that allows for the continuous measurement of HKMT (and other protein methyltransferases) activity in a convenient, accurate, inexpensive, and high-throughput manner by modifying their previous biotin/avidin microplate peptide methylation assay. In this improved assay, biotinylated target peptide is bound to the streptavidin-coated wells of special 96-well microplates, which are also coated with a thin layer of polystyrene-based scintillant. Enzyme and AdoMet with a tritium-labeled methyl group are then added to the wells, after which no additional washing or pipetting steps are required. Since the weak β-particles from the labeled AdoMet in solution are easily quenched by solvent, there is no scintillation signal until the tritiated methyl group is enzymatically transferred to target peptide bound on the wall of the microplate well; the resulting proximity of the tritium to the scintillant results in a strong signal. The results obtained are highly accurate, since the continuous data generated allows for the averaging of many data points. This assay is therefore ideal to produce accurate data for enzyme activity with many different substrates under multiple conditions or with different inhibitors.

(See “Continuous enzymatic assay for histone lysine methyltransferases” on page 602.)

Hybrid Vehicle Runs on Methyl

With increasing evidence for transcriptional regulation of developmentally important or cancer-related genes by DNA methylation of their upstream regions, accurate assessment of the methylation status of these genes has become increasingly important from both a molecular biological and clinical viewpoint. While methylation of specific residues is critical for the regulation of some genes, in most others it appears that the overall methylation density of upstream DNA regions correlates strongly with transcriptional repression. Two main types of methods have been devised for assaying the methylation of DNA. The first relies on digestion of the target DNA with methylation-sensitive restriction enzymes (MSREs), which are blocked by methylation of the recognition site, followed by measurement of undigested DNA by Southern blotting or quantitative PCR. The second approach employs the bisulfite conversion of unmethylated cytosines into uracil, which is then assayed by direct DNA sequencing of clones or by methylation-specific PCR (MSP) using primers specific for methylated or unmethylated cytosine residues. Both of these techniques suffer from various limitations, however. Incomplete digestion with MSRE can lead to false positive results. For the bisulfite-based methods, the conversion reaction is time-consuming and laborious, as is the cloning and direct sequencing of bisulfite-treated DNA, while MSP is complicated by the difficulty of choosing appropriate primers and probes. To surmount these problems, H. Holemon and colleagues at Orion Genomics (St. Louis, Missouri) have devised a new method, MethylScreen, that uses a combination of a methylation-dependent restriction enzyme (MDRE) and an MSRE in single and double digests, which, coupled with quantitative PCR, allows for the sensitive quantitation of cytosine methylation. The addition of the MDRE not only eliminates the false positives associated with an MSRE-only assay, but also permits the assessment of the methylation density of the region being examined, allowing intermediately methylated input DNA to be distinguished from sparsely or densely methylated input DNA using the appropriate calculations. Employing the MethylScreen assay, methylation of the 5′ region of the GSTPI gene, which is the most frequently hypermethylated gene in prostate cancer, was examined in DNA derived from several different sources, and the results were validated by clone-based bisulfite sequencing. Since MethylScreen is fast, efficient, and suitable for high-throughput application, it appears to be well suited for cancer detection based on the analysis of the regional methylation status of selected cancer-related genes.