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B Alberts

Publications and source records attributed to B Alberts.

12 recordsLinked to original sources
PubMed1979

Purification of the gene 43, 44, 45, and 62 proteins of the bacteriophage T4 DNA replication apparatus.

A procedure has been developed which allows the T4 bacteriophage proteins corresponding to the products of genes 43, 44, 45, and 62 to be purified to near homogeneity from a single T4-infected cell lysate (greater than 90% single species as judged by sodium dodecyl sulfate polyacrylamide elctrophoresis). In these preparations, the major problem of removing all contaminating nucleases has been overcome. Each of the above proteins is known from genetic analysis to be essential for phage DNA replication. The protein product of gene 43 is T4 DNA polymerase, and its recovery can be monitored using a standard DNA polymerase assay. The other three gene products have been designated as "polymerase accessory proteins," since they directly enhance polymerase function on both single- and double-stranded DNA templates. Their activities were monitored by an "in vitro complementation assay," which measures the stimulation of DNA synthesis observed in a concentrated lysate of T4 mutant-infected Escherichia coli cells when the missing T4 wild type protein is added. Starting from 300 g of infected cell paste, we obtained 9.3 mg of gene 43 protein, 21 mg of gene 45 protein, and 70 mg of a tight complex made up of 44 and 62 proteins; final yields were estimated at 30%, 14%, and 28%, respectively, of the initial activity present in the lysate. When the above purified proteins are incubated with preparations of two other T4 DNA replication proteins (gene 41 and gene 32 proteins) plus deoxyribonucleoside and ribonucleoside triphosphates, extensive DNA synthesis occurs on both single- and double-stranded DNA templates. As reported elsewhere, this synthesis mimicks that catalyzed by the T4 DNA replication apparatus in vivo.

Coliphages↗
PubMed1977

Recent excitement in the DNA replication problem.

It is now possible to reproduce most of the reactions involved in DNA replication using prokaryotic enzymes in vitro. Such systems have revealed that DNA replication is a complex process depending on a relatively large number of proteins, and that nucleoside triphosphate hydrolysis energy is used at several discrete steps. Much of the complexity of DNA replication may arise from the need for extreme copying fidelity.

Adenosine Triphosphate↗
PubMed1977

Intracellular DNA-protein complexes from bacteriophage T4-infected cells isolated by a rapid two-step procedure. Characterization and identification of the protein components.

A simple technique has been developed for isolating intracellular DNA and its bound proteins from uninfected and phage-infected bacteria. This technique, which utilizes aqueous salt concentrations in the physiological range, is based upon the fact that DNA exists in normal cell lysates in a stiff random coil conformation, and has an unusually large excluded volume to mass ratio. Such stiff coils display a unique combination of low sedimentation coefficient and large Stokes radius, enabling them to be separated rapidly from all other cellular components by successive centrifugal and gel permeation steps. Analysis of this purified intracellular DNA fraction from bacteriophage T4-infected Escherichia coli reveals mainly DNA and protein, with a small amount of RNA also present. Among the major proteins obtained are the DNA-dependent RNA polymerase of the host and the products of T4 genes rIIA, rIIB, and 32 (DNA-"unwinding" protein). Small amounts of the proteins coded by T4 genes 43 (DNA polymerase) and 42 (dCMP hydroxymethylase) have also been identified, in addition to at least 13 other phage-coded proteins of unidentified genes. Much of the phage-coded protein in the complex, including the gene 32 protein, does not exchange readily with the same protein exogenously added in the lysate.

Chromatography, Gel↗
PubMed1976

Purification and physical characterization of nucleic acid helix-unwinding proteins from calf thymus.

We have devised a general protein fractionation procedure which selects for eukaryotic DNA-binding proteins, some of which resemble DNA-unwinding proteins from prokaryotes. Proteins were selected which (a) pass through a native DNA-cellulose column, (b) bind to a denatured DNA-cellulose column, and (c) remain bound to the latter column during a rinse with a dilute solution of the sodium salt of the polyanion dextran sulfate. When this fractionation was applied to the soluble proteins fo calf thymus, three major protein species were recovered. The predominant one has an apparent molecular weight of about 24,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is isoelectric near neutrality, and elutes as a monomer from denatured DNA-cellulose at moderate NaCl concentrations. This protein, designated calf-unwinding protein 1 (UP1), has been purified to homogeneity. However, isoelectric focusing reveals four or five subspecies (apparently separated by single-charge differences) which differ appreciably in their affinities for DNA. Two other major proteins are obtained which have apparent molecular weights in sodium dodecyl sulfate of 33,000: the first, which elutes with low salt from DNA-cellulose as a homogeneous preparation, appears to be a basic protein (although it is clearly not a histone); the other, which elutes from DNA-cellulose as the major component of a "high salt eluting fraction," is an acidic protein which co-purifies with less prominent species of higher molecular weights. Proteins similar to each of these three major calf thymus proteins have been observed by us and others in tissue culture cells of mouse, hamster, monkey, and humans, suggesting their wide occurrence among eukaryotes.

Amino Acids↗
PubMed1976

Nucleic acid helix-coil transitions mediated by helix-unwinding proteins from calf thymus.

We have studied nucleic acid double helix destabilization mediated by purified calf helix-unwinding proteins, measuring ultraviolet hyperchromicity to detect helix melting. Both calf unwinding protein 1 (UP1) and a high salt eluting protein fraction are found to depress strongly the helix melting temperature (Tm) of the synthetic alternating copolymers poly[d(AT)] and poly[r(AU)], indicating that both DNA and RNA are recognized by these proteins. UP1 also destabilizes natural, GC-containing DNA helices, but to a smaller extent than observed with the above polymers. A simple model is presented to aid in the qualitative interpretation of the data, outlining the expected effect on the helix-coil transition of a protein ligand with differential affinity for the helix or coil form of nucleic acid. The observed helix-destabilizing effect of UP1 is dependent on the protein to nucleic acid ratio in an expected manner. Competition studies demonstrate a low, but appreciable affinity of UP1 for native DNA, opening the possibility that protein-mediated denaturation might be initiated by protein binding to the double helix. "Hairpin" helical regions of denatured DNA are strongly destabilized by UP1. Despite the fact that removal of these hairpin helices might greatly facilitate DNA renaturation, we failed to observe renaturation from the UP1-DNA complex after a switch to helix-stabilizing conditions. Thus, UP1 shows an important difference from its presumed prokaryotic analogue, T4 gene 32-protein. Possible in vivo functions of the calf proteins are discussed in light of these observations.

Animals↗
PubMed1976

Single-stranded DNA structure and DNA polymerase activity in the presence of nucleic acid helix-unwinding proteins from calf thymus.

In the preceding articles we have described the isolation and some of the properties of two calf thymus proteins which bind selectively to single-stranded DNA and which appear analogous to previously isolated prokaryotic DNA-unwinding proteins. In the present work we demonstrate two further points of analogy. First, both the calf UP1 and the high salt eluting proteins form protein-rich complexes with single-stranded DNA, and hold this DNA in a rigid, extended conformation. Second, these proteins stimulate the calf thymus DNA polymerase-alpha; phage T4 gene 32-protein does not. The stimulation of a homologous DNA polymerase is characteristic of several prokaryotic DNA-unwinding proteins and is assumed to reflect their in vivo role in DNA synthesis.

Animals↗
PubMed1976

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.

Chromatin↗
PubMed1975

The interaction of estradiol-receptor protein with the genome: an argument for the existence of undetected specific sites.

In extracts from rat and calf uterus, the steroid hormone 17 beta-estradiol stimulates the binding of its specific receptor protein to DNA. This interaction appears to be of low affinity (half of the estradiol-activated, 5S receptor bound at 300-400 mug/ml DNA) and nonspecific with respect to DNA base sequence. No binding to double-stranded RNA is observed. These findings are consistent with several in vivo observations. In particular, when the cytoplasmic receptor protein binds hormone, it migrates to the cell nucleus to an extent consistent with its affinity for DNA in vitro, and this in vivo nuclear binding is uniform and nonsaturable in the testable range (to greater than 3 times 10-4 sites per cell). The level of biological response appears to parallel the hormone dose up to these high levels of receptor binding. How are these observations to be reconciled with the prevalent view of steroid receptors as gene control proteins regulating transcription at specific loci on the genome? Our model is based on an analogy with the DNA binding properties of the E. coli lac repressor protein. We believe that the estradiol receptor exerts its effect by binding to a small number of high affinity sites on the genome, while also having a finite low affinity for nonspecific DNA sequences. These nonspecific loci, because of their vast number, completely mask the presence of the high affinity sites. We estimate that up to 10-3 specific sites, with affinities in the range 10- minus 8 minus 10- minus 10 M, could exist without being detected by bulk binding assays currently in use. However, alternative approaches should allow detection of these sites, and some of these are suggested.

Animals↗