Bøger af Saluz

A Safety Considerations Genomie sequencing involves a number oj hazard­ ous steps, such as high eurrent, high voltage, radioaetive and highly toxie chemieals. It is, the- jore, absolutely essential that the instruetions oj equipment manu/aeturers bejollowed and that par­ tieular attention is paid to the loeal and jederal safety regulations. I Introduction 13 B Introduction Hypomethylation ofDNA has been positively correlated with thc activation of many eucaryotic genes. During the transition from inactive to active genes changes in the protein/DNA interaction pattern occur. Tran­ scriptional activation of eucaryotic genes is mediated by specific interac­ tions oftransacting factors with their respective DNA binding sites in Lhe control regions (promoters, enhancers) ofthe genes. This process is ofLen accompanied by changes in local chromatin strucLure, witnessed by the appearance of nuclease hypersensitive sites, as weil as by changes in protein-DNA interactions and, in the case of higher eucaryotes, alterations ofthe cytosine methylation pattern. The sole available experimental tech­ nique that permits the study ofthe latter phenomena at single nucleotide resolution is direct genomic sequencing/footprinting, pioneered by Church and Gilbert (1984). This method combines the chemical DNA­ sequencing procedure of Maxam amI Gilbert (1980) with thc detection 01' DNA sequences by electroblotting and indirect end-Iabeling by hybridiza­ tl0n. An alternative possibility is the novel procedure (Saluz and . lost, 1989), using Taq polymerase. The first steps 01' both meLhods are essen­ tially the same: total genomic DNA is digested wiLh a suilable restriction enzyme and the resulting DNA fragments are chemically sequeneed.

More recently, the widely expressed intuition that discrete sequences within these elements constitute binding sites for sequence-specific binding proteins has been confirmed, especially through the use of "footprinting" assays (for examples, Galas and Schmitz, 1978).

A Safety Considerations Genomic sequencing involves a number of hazardous steps, such as high current, high voltage, radioactive and highly toxic chemicals. It is, therefore, absolutelyessen­ tial that the instructions of equipment manufacturers be followed and that particular attention is paid to the local and federal safety regulations. INTRODUCTION 9 B Introduction During the cloning of genomic DNA many of its characteristics are perma­ nently lost. It was therefore necessary to develop a new technique that would give us a closer look at a gene in its normal environment. The powerful technique of genomic sequencing, first described by Church and Gilbert (1984) now makes it possible to have a precise view of a given DNA sequence in a chromosome. This method combines the chemical DNA-sequencing procedure of Maxam and Gilbert (1980) with the detection of DNA sequences by electroblotting and indirect end-labeling by hybridization. Besides studies on the methylation state of single bases in a given gene (Nick et al. , 1986; Saluz and Jost, 1986; Saluz et al. , 1986), genomic sequencing can also be used to study specific DNA-protein interactions in vivo (Church et al. , 1985; Giniger et al. , 1985; Becker et al. , 1986; Ephrussi et al. , 1985; Martin et al. , 1986; Nick et al. , 1986; Zinn and Maniatis, 1986).