doi:10.1093/nar/gkm688 DNA sequencing: bench to bedside and beyond y

Fifteen years elapsed between the discovery of the double helix (1953) and the first DNA sequencing (1968). Modern DNA sequencing began in 1977, with development of the chemical method of Maxam and Gilbert and the dideoxy method of Sanger, Nicklen and Coulson, and with the first complete DNA sequence (phage rX174), which demonstrated that sequence could give profound insights into genetic organization. Incremental improvements allowed sequencing of molecules>200 kb (human cytomegalovirus) leading to an avalanche of data that demanded computational analysis and spawned the field of bioinformatics. The US Human Genome Project spurred sequencing activity. By 1992 the first ‘sequencing factory ’ was established, and others soon followed. The first complete cellular genome sequences, from bacteria, appeared in 1995 and other eubacterial, archaebacterial and eukaryotic genomes were soon sequenced. Competition between the public Human Genome Project and Celera Genomics produced working drafts of the human genome sequence, published in 2001, but refinement and analysis of the human genome sequence will continue for the foreseeable future. New ‘massively parallel ’ sequencing methods are greatly increasing sequencing capacity, but further innovations are needed to achieve the ‘thousand dollar genome ’ that many feel is prerequisite to personalized genomic medicine. These advances will also allow new approaches to a variety of problems in biology, evolution and the environment

The structure and function of the replication terminator protein of Bacillus subtilis: identification of the 'winged helix' DNA-binding domain.

The replication terminator protein (RTP) of Bacillus subtilis impedes replication fork movement in a polar mode upon binding as two interacting dimers to each of the replication termini. The mode of interaction of RTP with the terminus DNA is of considerable mechanistic significance because the DNA-protein complex not only localizes the helicase-blocking activity to the terminus, but also generates functional asymmetry from structurally symmetric protein dimers. The functional asymmetry is manifested in the polar impedance of replication fork movement. Although the crystal structure of the apoprotein has been solved, hitherto there was no direct evidence as to which parts of RTP were in contact with the replication terminus. Here we have used a variety of approaches, including saturation mutagenesis, genetic selection for DNA-binding mutants, photo cross-linking, biochemical and functional characterizations of the mutant proteins, and X-ray crystallography, to identify the regions of RTP that are either in direct contact with or are located within 11 angstroms of the replication terminus. The data show that the unstructured N-terminal arm, the alpha3 helix and the beta2 strand are involved in DNA binding. The mapping of amino acids of RTP in contact with DNA, confirms a 'winged helix' DNA-binding motif

DNA sequencing: bench to bedside and beyond

ABSTRACT Fifteen years elapsed between the discovery of the double helix (1953) and the first DNA sequencing (1968). Modern DNA sequencing began in 1977, with development of the chemical method of Maxam and Gilbert and the dideoxy method of Sanger, Nicklen and Coulson, and with the first complete DNA sequence (phage rX174), which demonstrated that sequence could give profound insights into genetic organization. Incremental improvements allowed sequencing of molecules >200 kb (human cytomegalovirus) leading to an avalanche of data that demanded computational analysis and spawne

The Process of Infection with Bacteriophage ΦX174: X. Mutations in a ΦX Lysis Gene

The ability of conditional lethal mutants of phage ΦX174 to induce host cell lysis during infection under restrictive conditions has been studied. We have found amber (am) and temperature-sensitive (ts) mutants which present a variety of alterations in the normal lytic process. In particular, there is a class of am mutants which do not produce cell lysis but otherwise replicate normally in the restrictive host. These mutants constitute a single complementation group. The existence of these mutants implicates a phage-coded protein in the lytic process. This protein is not an essential structural component of the phage, since normal phage particles are produced in the absence of lysis

The Process of Infection with Bacteriophage ΦX174: X. Mutations in a ΦX Lysis Gene

The ability of conditional lethal mutants of phage ΦX174 to induce host cell lysis during infection under restrictive conditions has been studied. We have found amber (am) and temperature-sensitive (ts) mutants which present a variety of alterations in the normal lytic process. In particular, there is a class of am mutants which do not produce cell lysis but otherwise replicate normally in the restrictive host. These mutants constitute a single complementation group. The existence of these mutants implicates a phage-coded protein in the lytic process. This protein is not an essential structural component of the phage, since normal phage particles are produced in the absence of lysis

Kinetics of Bacteriophage Release by Single Cells of φX174-infected E. coli

The mechanism of release of φX174 by infected E. coli is of interest since its understanding must result in the discovery of either: (1) a phage-specific lytic enzyme (Streisinger, Mukai, Dreyer, Miller & Horiuchi, 1961; Jacob & Fuerst, 1958) which would be the first example of a φX-specific enzyme; or (2) a novel method of phage release. Recently some unsuccessful attempts to detect a lytic enzyme in the mature phage particle and in infected cells have been reported (Fujimura & Kaesberg, 1962; Eigner, Stouthamer, Van der Sluys & Cohen, 1963). It has been suggested (Eigner et al., 1963) that release of φX precedes cellular dissolution. Denhardt (1963, in preparation) has shown that even when the infection process is synchronized by infection in the presence of 0·003 M-KCN or by 'infection of starved cells phage release occurs over a period of time longer than the minimum latent period. These results suggested the possibility that ,PX might be released slowly from single infected complexes, as is the case with certain animal viruses (see, for example, Dulbecco & Vogt, 1954), rather than in bursts occurring at the time of lysis

Kinetics of Bacteriophage Release by Single Cells of φX174-infected E. coli

The mechanism of release of φX174 by infected E. coli is of interest since its understanding must result in the discovery of either: (1) a phage-specific lytic enzyme (Streisinger, Mukai, Dreyer, Miller & Horiuchi, 1961; Jacob & Fuerst, 1958) which would be the first example of a φX-specific enzyme; or (2) a novel method of phage release. Recently some unsuccessful attempts to detect a lytic enzyme in the mature phage particle and in infected cells have been reported (Fujimura & Kaesberg, 1962; Eigner, Stouthamer, Van der Sluys & Cohen, 1963). It has been suggested (Eigner et al., 1963) that release of φX precedes cellular dissolution. Denhardt (1963, in preparation) has shown that even when the infection process is synchronized by infection in the presence of 0·003 M-KCN or by 'infection of starved cells phage release occurs over a period of time longer than the minimum latent period. These results suggested the possibility that ,PX might be released slowly from single infected complexes, as is the case with certain animal viruses (see, for example, Dulbecco & Vogt, 1954), rather than in bursts occurring at the time of lysis