Bruce Alberts

Rapid Bacteriophage Sedimentation in the Presence of Polyethylene Glycol and its Application to Large-Scale Virus Purification

Abstract Bacteriophages may be readily concentrated from crude lysates of infected bacteria after addition of polyethylene glycol (PEG). A general procedure is presented which can be used for lysate volumes of 17 liters or more with nearly quantitative recovery of infectivity over a wide range of phage titers (105 to 1013 PFU/ml). All the bacteriophages tested (λ, T4, T7, P22, fd, φX174, R17) are efficiently removed from solution by simple settling at concentrations of PEG 6000 between 2% and 10%. Bacteriophage pellets are redissolved in a small amount of buffer, allowing 100-fold concentration of the original lysate. While lower molecular weight polyethylene glycols are much less effective for concentrating bacteriophages than the PEG 6000 used, the efficiency is relatively insensitive to changes in pH and ionic strength. Asymmetric particles (tobacco mosaic virus and bacteriophage fd) are especially susceptible to PEG, and they can be purified from more symmetrical particles at low PEG 6000 concentrations (2% or less). Although the exact mechanism by which bacteriophages can be concentrated with PEG is unknown, a phase partition rather than a normal precipitation reaction seems to be involved, since the fraction of infective phages removed from solution by a fixed concentration of PEG is nearly invariant to changes in bacteriophage concentration over as much as a 108-fold range. Extension of this method to concentration and purification of other viruses and nucleic acids, as well as some preliminary mechanistic studies, are discussed.

The Cell as a Collection of Protein Machines: Preparing the Next Generation of Molecular Biologists

President, National Academy of Sciences2101 Constitution Avenue NWWashington, D.C. 20418Professor, Department of Biochemistry and BiophysicsUniversity of California, San Francisco tion A—just as it would in a machine of our commonSan Francisco, California 94143 experience (Alberts, 1984).Underlying this highly organized activity are ordered

Type II DNA topoisomerases: Enzymes that can unknot a topologically knotted DNA molecule via a reversible double-strand break

The T4 DNA topoisomerase is a recently discovered multisubunit protein that appears to have an essential role in the initiation of T4 bacteriophage DND replication. Treatment of double-stranded circular DNA with large amounts of this topoisomerase in the absence of ATP yields new DNA species which are knotted topological isomers of the double-stranded DNA circle. These knotted DNA circles, whether covalently closed or nicked, are converted to unknotted circles by treatment with trace amounts of the T4 topoisomerase in the presence of ATP. Very similar ATP-dependent enzyme activities capable of unknotting DNA are present in extracts of Drosophila eggs. Xenopus laevis eggs and mammalian tissue culture cells. The procaryotic enzyme, DNA gyrase, is also capable of unknotting DNA. We propose that these unknotting enzymes constitute a new general class of DNA topoisomerases (type II DNA topoisomerases). These enzymes must act via mechanisms that involve the concerted cleavage and rejoining of two opposite DNA strands, such that the DNA double helix is transiently broken. The passage of a second double-stranded DNA segment through this reversible double-strand break results in a variety of DNA topoisomerization reactions, including relaxation:super-coiling; knotting:unknotting and catenation:decatenation. In support of this type of mechanism, we demonstrate that the T4 DNA topoisomerase changes the linking number of a covalently closed double-stranded circular DNA molecule only by multiples of two. We discuss the possible roles of such enzymes in a variety of biological functions, along with their probable molecular mechanisms.

Rescuing US biomedical research from its systemic flaws

The long-held but erroneous assumption of never-ending rapid growth in biomedical science has created an unsustainable hypercompetitive system that is discouraging even the most outstanding prospective students from entering our profession—and making it difficult for seasoned investigators to produce their best work. This is a recipe for long-term decline, and the problems cannot be solved with simplistic approaches. Instead, it is time to confront the dangers at hand and rethink some fundamental features of the US biomedical research ecosystem.

Isolation and characterization of gene 5 protein of filamentous bacterial viruses

Abstract The product of gene 5 of filamentous bacteriophages is required for synthesis of the single-stranded progeny DNA. When extracts made from Escherichia coli infected with bacteriophages fd or M13 are chromatographed on single-stranded DNA/cellulose, a DNA-binding protein can be eluted which has a molecular weight of about 10,000 daltons and is made in at least 100,000 copies per cell. This protein is altered in amber mutant 5-H3 and temperature-sensitive mutant 5-HS1 of phage M13, which identifies it as the gene 5 product (Henry & Pratt, 1969). As judged by sucrose-gradient sedimentation at 4 °C, the pure protein binds tightly and co-operatively to single-stranded, but not to double-stranded, DNAs; at saturation, one protein monomer is bound per every 4 DNA nucleotides. This selective DNA-binding enables the gene 5 protein to denature double-stranded DNAs rapidly at physiological temperatures. In these respects, the gene 5 protein resembles the T4 bacteriophage gene 32 protein described previously, a protein which is required for both the replication and recombination of T4 bacteriophage DNA. However, electron microscopy reveals that the structure of the gene 5 protein complex with single-stranded DNA is quite different: whereas gene 32 protein forces the DNA into an extended linear conformation, the gene 5 protein coalesces two protein-covered DNA stands into a helical, rodlike structure. For this and other reasons, these two “DNA unwinding” proteins could have quite different roles in the replication process.

Genetic recombination: The nature of a crossed strand-exchange between two homologous DNA molecules

Abstract Molecular models have been used to demonstrate that a crossed strand-exchange between two homologous DNA molecules, a likely intermediate in genetic recombination, can be formed with all of the bases in the two double-helices remaining paired. There are two “outside” and two connecting, or “bridging”, strands in this structure. Since the positions of these two pairs of strands can be interchanged in the model by rotation, all four strands participating in the exchange must be regarded as equivalent. As a consequence, when pairing is terminated by strand-scissions, there is an equal probability of generating two kinds of DNA molecules: those with genes flanking the region of strand-exchange in parental configuration and those with flanking genes recombined.

Head-on collision between a DNA replication apparatus and RNA polymerase transcription complex

An in vitro system reconstituted from purified proteins has been used to examine what happens when the DNA replication apparatus of bacteriophage T4 collides with an Escherichia coli RNA polymerase ternary transcription complex that is poised to move in the direction opposite to that of the moving replication fork. In the absence of a DNA helicase, the replication fork stalls for many minutes after its encounter with the RNA polymerase. However, when the T4 gene 41 DNA helicase is present, the replication fork passes the RNA polymerase after a pause of a few seconds. This brief pause is longer than the pause observed for a codirectional collision between the same two polymerases, suggesting that there is an inherent disadvantage to having replication and transcription directions oriented head to head. As for a codirectional collision, the RNA polymerase remains competent to resume faithful RNA chain elongation after the DNA replication fork passes; most strikingly, the RNA polymerase has switched from its original template strand to use the newly synthesized daughter DNA strand as the template.

A model for chromatin based upon two symmetrically paired half-nucleosomes

We propose that the basic unit of chromatin is constructed of two isologously paired heterotypic protein tetramers each containing one molecule of H2A, H2B, H3, and H4 histone. These proteins form a core that holds 140 base pairs (bp) of DNA in a single left-handed, non-interwound DNA super-coil approximately 95 bp in circumference, creating a nucleosome particle (DNA and protein) organized about a dyad axis of symmetry. Such a nucleosome can open up into its separate half-nucleosomes to allow genetic readout without requiring histone displacement.

Characterization by electron microscopy of the complex formed between T4 bacteriophage gene 32-protein and DNA☆

Abstract Techniques have been developed for visualization of gene 32-protein/DNA complexes by electron microscopy. With DNA in excess, gene 32-protein can be seen to bind co-operatively to single-stranded DNA. With protein in excess, all single-strands appear to be uniformly coated with protein. The saturated complex has a flexible rod-like conformation, with a diameter of about 60 A and a length of about 4.6 A per nucleotide. This structure is unchanged in the presence of 1 m m -spermine, a condition which induces extensive folding in the free single-strands. Using glutaraldehyde fixation, gene 32-protein induced denaturation of double-stranded DNAs can be visualized at physiological temperatures. The denaturation map derived with bacteriophage lambda DNA closely resembles that obtained earlier with heat and alkaline denaturation in the absence of protein (Inman, 1967) , indicating that the protein preferentially invades A + T rich double-helical regions, without a requirement for a physical discontinuity such as a cleaved phosphodiester bond or a double-stranded end. With supercoiled DNA, only a single short region of double-helix is opened by gene 32-protein, reflecting the difficulty of denaturation where unwinding is geometrically restricted.

The Drosophila GAGA transcription factor is associated with specific regions of heterochromatin throughout the cell cycle.

In virtually all eukaryotes the centromeric regions of chromosomes are composed of heterochromatin, a specialized form of chromatin that is rich in repetitive DNA sequences and is transcriptionally relatively silent. The Drosophila GAGA transcription factor binds to GA/CT‐rich sequences in many Drosophila promoters, where it activates transcription, apparently by locally altering chromatin structure and allowing other transcription factors access to the DNA. Here we report the paradoxical finding that GAGA factor is associated with specific regions of heterochromatin at all stages of the cell cycle. A subset of the highly repetitive DNA sequences that make up the bulk of heterochromatin in D. melanogaster are GA/CT‐rich and we find a striking correlation between the distribution of GAGA factor and this class of repeat. We propose that GAGA factor binds directly to these repeats and may thereby play a role in modifying heterochromatin structure in these regions. Our observations demonstrate for the first time that a transcriptional regulator can associate with specific DNA sequences in a fully condensed mitotic chromosome. This may help explain how the distinctive character of a committed or differentiated cell can be maintained during cell proliferation.