Biochemistry - The Chemical Reactions of Living Cells, Volume 3 - D. Metzler 1980

Biochemical Genetics and the Synthesis of Nucleic Acids and Proteins
DNA Restriction and Modification
Restriction Enzymes in DNA Nucleotide Sequencing

Due to their ability to cleave DNA strands at strictly specific nucleotide sequences that may occur only a few times along the entire length of a DNA molecule, Restriction Endonucleases are widely used in DNA Sequencing [217]. Their use is largely analogous to the application of Trypsin for cleaving polypeptide chains into smaller fragments (ch. 2, sec. 3.2). A prime example of The Use of restriction Enzymes is The Study of The nucleotide sequence of SV40 virus DNA [218, 219]. This mammalian virus, capable of integrating into the host Cell genome and transforming The Cell into a tumor cell, contains a circular double-stranded DNA consisting of approximately 5,000 nucleotide pairs. One restriction enzyme from Hemophilus influenzae cleaves the SV40 DNA into 11 fragments, whereas the action of the Hemophilus parainfluenzae enzyme yields only four fragments. The Eco RI enzyme from E. coli breaks the circular DNA at a single unique site. Analysis of partial Hydrolysis products and overlapping fragments has made it possible to map the positions of all fragments on the circular map. Using pulse labeling with thymine, researchers were able to correlate the Location OF THE origin and the directions of Bidirectional Replication with the positions of fragments generated by restriction enzymes. Similarly, the direction of early and late METABOLISM/31.html">Transcription in productively infected Cells was established. The study of nucleotide sequences of individual fragments is progressing so successfully that it may soon be possible to decipher the complete nucleotide sequence of the SV40 virus molecule. Similar methodological approaches are already being used to study Mitochondrial and Chloroplast DNAs, as well as individual fragments of chromosomal DNA.