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Biomedical subjects

B M Alberts

Publications and source records attributed to B M Alberts.

At least 19 recordsLinked to original sources
PubMed1992

Overexpression, purification, sequence analysis, and characterization of the T4 bacteriophage dda DNA helicase.

The bacteriophage T4 dda protein is a 5'-3' DNA helicase that stimulates DNA replication and recombination reactions in vitro and seems to play a role in the initiation of T4 DNA replication in vivo. Oligonucleotide probes based on NH2-terminal amino acid sequence were used to precisely map the location of the dda gene on the T4 chromosome. Using polymerase chain reaction techniques, the dda gene was then cloned into an expression vector, and the overproduced protein was purified in two chromatography steps. Both the genomic and cloned dda genes were sequenced and found to be identical, encoding a protein of 439 amino acids. The dda protein contains amino acid sequences resembling those of other known helicases, and is most homologous to the Escherichia coli recD protein. Protein affinity chromatography was used to show a direct interaction between the dda protein and the T4 uvsX protein (a rec A-type DNA recombinase).

Amino Acid Sequence↗
PubMed1992

Purification of a multiprotein complex containing centrosomal proteins from the Drosophila embryo by chromatography with low-affinity polyclonal antibodies.

A 190-kDa centrosomal protein interacts with microtubules when Drosophila embryo extracts are passed over microtubule-affinity columns. We have obtained a partial cDNA clone that encodes this protein. Using a fusion protein produced from the clone, we have developed a novel immunoaffinity chromatography procedure that allows both the 190-kDa protein and a complex of proteins that associates with it to be isolated in in a single step. For this procedure, the fusion protein is used as an antigen to prepare rabbit polyclonal antibodies, and those antibodies that recognize the 190-kDa protein with low affinity are selectively purified on a column containing immobilized antigen. These low-affinity antibodies are then used to construct an immunoaffinity column. When Drosophila embryo extracts are passed over this column, the 190-kDa protein is quantitatively retained and can be eluted in nearly pure form under nondenaturing conditions with 1.5 M MgCl2, pH 7.6. The immunoaffinity column is washed with 1.0 M KCl just before the elution with 1.5 M MgCl2. This wash elutes 10 major proteins, as well as a number of minor ones. We present evidence that these KCl-eluted proteins represent additional centrosomal components that interact with the 190-kDa protein to form a multiprotein complex within the cell.

Animals↗
PubMed1992

Reorganization of the cytoskeleton during Drosophila oogenesis: implications for axis specification and intercellular transport.

Inhibitor studies have implicated microtubules in at least three important developmental processes during Drosophila oogenesis: oocyte determination and growth during stages 1 through 6, positioning of the anterior determinant bicoid mRNA during stages 9 through 12, and ooplasmic streaming during stages 10b through 12. We have used fluorescence cytochemistry together with laser scanning confocal microscopy to identify distinct microtubule structures at each of the above three periods that are likely to be involved in these processes. During stages 1 through 7, maternal components synthesized in nurse cells are transported through cytoplasmic bridges to the oocyte. At this time, microtubules that appear to originate in the oocyte pass through these cytoplasmic bridges into the adjacent nurse cells; these microtubules are likely to serve as a polarized scaffold on which maternal RNAs and proteins are transported. During stages 7 and 8, microtubules in the oocyte cortex reorganize to form an anterior-to-posterior gradient, suggesting a role for microtubules in the localization of morphogenetic determinants. Finally, when ooplasmic streaming begins during stage 10 b, it is accompanied by the assembly of subsurface microtubule arrays that spiral around the oocyte; these arrays disassemble as the oocyte matures and streaming stops. During ooplasmic streaming, many vesicles are closely associated with the subsurface microtubules, suggesting that streaming is driven by vesicle translocation along microtubules. We believe that actin plays a secondary role in each of these morphogenetic events, based on our parallel studies of actin organization during each of the above stages of oogenesis.

Actins↗
PubMed1991

The T4 DNA polymerase accessory proteins form an ATP-dependent complex on a primer-template junction.

The DNA polymerase holoenzyme of bacteriophage T4 contains, besides the DNA polymerase itself (the gene 43 protein), a complex of the protein products of T4 genes 44 and 62 (a DNA-dependent ATPase) and of gene 45. Together, the 44/62 and 45 proteins form an ATP-dependent "sliding clamp" that holds a moving DNA polymerase molecule at the 3' terminus of a growing DNA chain. We have used a unique DNA fragment that forms a short hairpin helix with a single-stranded 5' tail (a "primer-template junction") to map the binding sites for these polymerase accessory proteins by DNA footprinting techniques. In the absence of the DNA polymerase, the accessory proteins protect from DNase I cleavage 19-20 nucleotides just behind the 3' end of the primer strand and 27-28 nucleotides on the complementary portion of the template strand. Detection of this DNA-protein complex requires the 44/62 and 45 proteins plus the nonhydrolyzable ATP analogue adenosine 5'-O-(thiotriphosphate). The complex is not detected in the presence of ATP. We suggest that ATP hydrolysis by the 44/62 protein normally activates the accessory proteins at a primer-template junction, permitting the DNA polymerase to bind and thus form the complete holoenzyme. However, when the polymerase is missing, as in these experiments, ATP hydrolysis is instead followed by a release (or loosening) of the accessory protein complex.

Adenosine Triphosphate↗
PubMed1991

DNA footprinting studies of the complex formed by the T4 DNA polymerase holoenzyme at a primer-template junction.

We have used DNA footprinting techniques to analyze the interactions of five DNA replication proteins at a primer-template junction: the bacteriophage T4 DNA polymerase (the gene 43 protein), its three accessory proteins (the gene 44/62 and 45 proteins), and the gene 32 protein, which is the T4 helix-destabilizing (or single-stranded DNA-binding) protein. The 177-nucleotide-long DNA substrate consisted of a perfect 52-base pair hairpin helix with a protruding single-stranded 5' tail. As expected, the DNA polymerase binds near the 3' end of this molecule (at the primer-template junction) and protects the adjacent double-stranded region from cleavage. When the gene 32 protein binds to the single-stranded tail, it reduces the concentration of the DNA polymerase required to observe the polymerase footprint by 10-30-fold. Periodic ATP hydrolysis by the 44/62 protein is required to maintain the activity of the DNA polymerase holoenzyme (a complex of the 43, 44/62, and 45 proteins). Footprinting experiments demonstrate the formation of a weak complex between the DNA polymerase and the gene 45 protein, but there is no effect of the 44/62 protein or ATP on this enlarged footprint. We propose a model for holoenzyme function in which the complex of the three accessory proteins uses ATP hydrolysis to keep a moving polymerase tightly bound to the growing 3' end, providing a "clock" to measure polymerase stalling.

Autoradiography↗
PubMed1991

Amplification of snap-back DNA synthesis reactions by the uvsX recombinase of bacteriophage T4.

The uvsX protein of bacteriophage T4 is a recA-type recombinase. This protein has previously been shown to help initiate DNA replication on a double-stranded DNA template by catalyzing synapsis between the template and a homologous DNA single strand that serves as primer. Here, we demonstrate that this replication-initiating activity of the uvsX protein greatly amplifies the snap-back (hairpin-primed) DNA synthesis that is catalyzed by the T4 DNA polymerase holoenzyme on linear, single-stranded DNA templates. Amplification requires the presence of uvsX protein, the DNA polymerase holoenzyme, T4 gene 32 protein, and a T4 DNA helicase, in a reaction that is modulated by the T4 uvsY protein (an accessory protein to the uvsX recombinase). The reaction products consist primarily of large networks of double-stranded and single-stranded DNA. With alkali or heat treatment, these networks resolve into dimer-length single-stranded DNA chains that renature instantaneously to reform a monomer-length double helix. A simple model can explain this uvsX protein-dependent amplification of snap-back DNA synthesis; the mechanism proposed makes several predictions that are confirmed by our experiments.

Adenosine Triphosphatases↗
PubMed1990

The UvsY protein of bacteriophage T4 modulates recombination-dependent DNA synthesis in vitro.

An in vitro system containing the T4 gene 43, 45, 44/62, 32, dda, and uvsX proteins catalyzes DNA synthesis that is dependent on the synapsis step of homologous genetic recombination. The rate of DNA synthesis in this system is highly dependent on the concentration of the uvsX recombinase (a recA-like protein). Here we report the effect of the T4 uvsY protein, a recombination accessory protein, on this reaction. Low concentrations of uvsY protein greatly stimulate DNA synthesis at low concentrations of uvsX protein, but these same concentrations inhibit DNA synthesis at high concentrations of uvsX protein. As a result, the addition of small amounts of uvsY protein lowers the minimum concentration of uvsX protein needed for the reaction 8-fold, and it lowers the uvsX protein concentration for maximum activity 4-fold. The uvsY protein can affect either the initiation or elongation phase of DNA synthesis, depending on the concentration of uvsX protein present. The implications of these results for the function of the uvsY protein in T4 DNA replication in vivo are discussed.

Adenosine Triphosphatases↗
PubMed1990

In vitro replication through nucleosomes without histone displacement.

A well-characterized set of proteins encoded by bacteriophage T4 replicates DNA in vitro and generates replication forks that can pass nucleosomes. The histone octamers remain associated with newly replicated DNA even in the presence of excess DNA competitor, and intact nucleosomes re-form on the two daughter DNA helices. It is concluded that nucleosomes are designed to open up transiently to allow the passage of a replication fork without histone displacement.

DNA Replication↗
PubMed1990

Effects of the bacteriophage T4 gene 41 and gene 32 proteins on RNA primer synthesis: coupling of leading- and lagging-strand DNA synthesis at a replication fork.

We have demonstrated previously that the template sequences 5'-GTT-3' and 5'-GCT-3' serve as necessary and sufficient signals for the initiation of new DNA chains that start with pentaribonucleotide primers of sequence pppApCpNpNpN or pppGpCpNpNpN, respectively. Normally, the complete T4 primosome, consisting of the T4 gene 41 (DNA helicase) and gene 61 (primase) proteins, is required to produce RNA primers. However, a high concentration of the 61 protein alone can prime DNA chain starts from the GCT sites [Cha, T.-A., & Alberts, B. M. (1986) J. Biol. Chem. 261, 7001-7010]. We show here that the 61 protein can catalyze a single-stranded DNA template-dependent reaction in which the dimers pppApC and pppGpC are the major products and much longer oligomers of various lengths are minor ones. Further addition of the 41 protein is needed to form a primosome that catalyzes efficient synthesis of the physiologically relevant pentaribonucleotides that are responsible for the de novo DNA chain starts on the lagging strand of a replication fork. The helicase activity of the 41 protein is necessary and sufficient to ensure a high rate and processivity of DNA synthesis on the leading strand [Cha, T.-A., & Alberts, B. M. (1989) J. Biol. Chem. 264, 12220-12225]. Coupling an RNA primase to this helicase in the primosome therefore coordinates the leading- and lagging-strand DNA syntheses at a DNA replication fork. Our experiments reveal that the addition of the T4 helix-destabilizing protein (the gene 32 protein) is required to confine the synthesis of RNA primers to those sites where they are used to start an Okazaki fragment, causing many potential priming sites to be passed by the primosome without triggering primer synthesis.

Base Sequence↗
PubMed1990

daughterless-abo-like, a Drosophila maternal-effect mutation that exhibits abnormal centrosome separation during the late blastoderm divisions.

daughterless-abo-like (dal) is a maternal-effect semilethal mutation in Drosophila. The nuclear divisions of embryos derived from homozygous dal females are normal through nuclear cycle 10. However, during nuclear cycles 11, 12 and 13, a total of about half of the nuclei in each embryo either fail to divide or fuse with a neighboring nucleus during telophase. These abnormal nuclei eventually sink into the interior of the embryo, leaving their centrosomes behind on the surface. The loss of about one-half of the peripheral nuclei into the interior of the embryo results in these embryos cellularizing during nuclear cycle 14 with about one-half the normal number of cells. Surprisingly, many of these embryos develop a nearly normal larval cuticle and 8% develop to adulthood. Observations of live embryos doubly injected with tubulin and histones that have been fluorescently labeled allows nuclear and centrosomal behavior to be directly followed as the embryo develops. We find that the abnormal nuclei arise from nuclei whose centrosomes have failed to separate normally in the previous interphase. These incompletely separated centrosomes can cause a non-functional spindle to form, leading to a nuclear division failure. Alternatively, they can form an abnormal spindle with a centrosome from a neighboring nucleus, causing two nuclei to share a common spindle pole. Such nuclei with a shared centrosome will undergo telophase fusions, unequal divisions, or division failures later in mitosis. These findings have helped us to understand the function of the centrosome in the Drosophila embryo.

Animals↗
PubMed1989

Sequence-specific pausing during in vitro DNA replication on double-stranded DNA templates.

Sequence-specific pausing occurs during DNA synthesis catalyzed by the bacteriophage T4 DNA polymerase holoenzyme in the presence of the T4 helix destabilizing protein (gene 32 protein). Two of the six strongest pause sites on a double-stranded bacteriophage fd DNA template are in regions where hairpin helices are predicted to form when the DNA is single stranded. However, the other pause sites are in regions that are not obviously involved in secondary structure. The positions of the DNA chain ends produced at one pause site of each type were determined to within +/- 2 nucleotides. At this resolution, a clustering of sites is observed, suggesting that the polymerase holoenzyme may become destabilized when moving along selected regions of the DNA and then pause at one or more of several closely spaced positions. The addition of the T4 gene 41 protein (a DNA helicase that forms part of the T4 primosome) to the above replication system greatly increases the rate of fork movement and eliminates detectable pausing. In contrast, the addition of the T4 dda protein (a second DNA helicase that increases the rate of fork movement to a similar extent) has no affect on replication fork pausing. This difference could either be due to specific protein-protein interactions formed between the polymerase holoenzyme and the 41 protein or to the highly processive movement of the 41 protein along the displaced DNA strand.

Base Sequence↗
PubMed1989

Enhancement of bacteriophage T4 late transcription by components of the T4 DNA replication apparatus.

The expression of the late genes in bacteriophage T4 development is closely connected to viral DNA replication. Three T4-encoded DNA polymerase accessory proteins are shown to stimulate transcription at T4 late promoters in an adenosine triphosphate (ATP) hydrolysis-requiring process. The properties of the activation resemble those found for enhancers of eukaryotic transcription. However, the nature of the enhancer of T4 late transcription is novel in that it is a structure--a break in the nontranscribed DNA stand--to which the three replication proteins bind, rather than a sequence. Since the three DNA polymerase accessory proteins are carried on the moving replication fork as part of the replisome, we postulate that viral DNA replication forks act, in vivo, as the mobile enhancers of T4 late gene transcription. Whereas Escherichia coli RNA polymerase bearing the T4 gene 55 protein can selectively recognize T4 late promoters, it is only capable of responding to the transcription-enhancing activity of the three replication proteins on acquiring an additional T4-specific modification.

DNA Replication↗
PubMed1989

The bacteriophage T4 DNA replication fork. Only DNA helicase is required for leading strand DNA synthesis by the DNA polymerase holoenzyme.

Seven bacteriophage T4-encoded proteins reconstitute a DNA replication apparatus that catalyzes coupled leading and lagging strand DNA synthesis at a replication fork in vitro. The proteins involved are the T4 DNA polymerase holoenzyme (the products of T4 genes 43, 44/62, and 45), a helix-destabilizing (SSB) protein (gene 32 protein), and the T4 primosome which is composed of a DNA helicase (gene 41 protein) and a primase (gene 61 protein). We show here that the presence of 41 protein on the lagging strand of the fork enables the polymerase holoenzyme to catalyze leading strand DNA synthesis at a maximum rate and with high processivity. This leading strand synthesis is unaffected by the addition of either the gene 32 or the gene 61 protein; the 41 protein cannot be replaced by the dda protein, a second T4-encoded DNA helicase. When the 61 protein is added to the 41 protein to complete the primosome, Okazaki fragment synthesis on the lagging strand accompanies leading strand DNA synthesis in this system even in the absence of the 32 protein. However, the addition of 32 protein decreases the size of the Okazaki fragments made, as expected for an increase in the lagging strand polymerization rate at a fork that has coupled leading and lagging strand DNA polymerase molecules.

DNA Helicases↗