Biomedical subjects

B M Alberts

Publications and source records attributed to B M Alberts.

At least 55 records · Page 3Linked to original sources

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↗

PubMed1989

F-actin affinity chromatography: technique for isolating previously unidentified actin-binding proteins.

We have developed stable and easy to use filamentous actin (F-actin) affinity-chromatography columns that selectively purify proteins that bind to actin filaments from cell extracts. Most traditional assays for actin-associated proteins screen for their effects on actin polymerization or actin filament crosslinking. Because our technique requires only actin-filament binding, it can identify additional types of proteins involved in the function of the actin cytoskeleton. By chromatographing extracts of several types of cells on these columns, we show that known actin-binding proteins are selectively retained as a subset of a larger group of actin-binding proteins that have not been identified previously.

Actins↗

PubMed1989

Direct cell lineage analysis in Drosophila melanogaster by time-lapse, three-dimensional optical microscopy of living embryos.

One of the first signs of cell differentiation in the Drosophila melanogaster embryo occurs 3 h after fertilization, when discrete groups of cells enter their fourteenth mitosis in a spatially and temporally patterned manner creating mitotic domains (Foe, V. E. and G. M. Odell, 1989, Am. Zool. 29:617-652). To determine whether cell residency in a mitotic domain is determined solely by cell position in this early embryo, or whether cell lineage also has a role, we have developed a technique for directly analyzing the behavior of nuclei in living embryos. By microinjecting fluorescently labeled histones into the syncytial embryo, the movements and divisions of each nucleus were recorded without perturbing development by using a microscope equipped with a high resolution, charge-coupled device. Two types of developmental maps were generated from three-dimensional time-lapse recordings: one traced the lineage history of each nucleus from nuclear cycle 11 through nuclear cycle 14 in a small region of the embryo; the other recorded nuclear fate according to the timing and pattern of the 14th nuclear division. By comparing these lineage and fate maps for two embryos, we conclude that, at least for the examined area, the pattern of mitotic domain formation in Drosophila is determined by the position of each cell, with no effect of cell lineage.

Animals↗

PubMed1989

Actin-binding proteins from Drosophila embryos: a complex network of interacting proteins detected by F-actin affinity chromatography.

By using F-actin affinity chromatography columns to select proteins solely by their ability to bind to actin filaments, we have identified and partially purified greater than 40 proteins from early Drosophila embryos. These proteins represent approximately 0.5% of the total protein present in soluble cell extracts, and 2 mg are obtained by chromatography of an extract from 10 g of embryos. As judged by immunofluorescence of fixed embryos, 90% of the proteins that we have detected in F-actin column eluates are actin-associated in vivo (12 of 13 proteins tested). The distributions of antigens observed suggest that groups of these proteins cooperate in generating unique actin structures at different places in the cell. These structures change as cells progress through the cell cycle and as they undergo the specializations that accompany development. The variety of different spatial localizations that we have observed in a small subset of the total actin-binding proteins suggests that the actin cytoskeleton is a very complex network of interacting proteins.

Actins↗

PubMed1989

Identification of microtubule-associated proteins in the centrosome, spindle, and kinetochore of the early Drosophila embryo.

We have developed affinity chromatography methods for the isolation of microtubule-associated proteins (MAPs) from soluble cytoplasmic extracts and have used them to analyze the cytoskeleton of the early Drosophila embryo. More than 50 Drosophila embryo proteins bind to microtubule affinity columns. To begin to characterize these proteins, we have generated individual mouse polyclonal antibodies that specifically recognize 24 of them. As judged by immunofluorescence, some of the antigens localize to the mitotic spindle in the early Drosophila embryo, while others are present in centrosomes, kinetochores, subsets of microtubules, or a combination of these structures. Since 20 of the 24 antibodies stain microtubule structures, it is likely that most of the proteins that bind to our columns are associated with microtubules in vivo. Very few MAPS seem to be identically localized in the cell, indicating that the microtubule cytoskeleton is remarkably complex.

Alkaloids↗

PubMed1988

The bacteriophage T4 insertion/substitution vector system. A method for introducing site-specific mutations into the virus chromosome.

A bacteriophage T4 insertion/substitution vector system has been developed as a means of introducing in vitro generated mutations into the T4 chromosome. The insertion/substitution vector is a 2638-base pair plasmid containing the pBR322 origin of replication and ampicillin resistance determinant, a T4 gene 23 promoter/synthetic supF tRNA gene fusion, and a polylinker with eight unique restriction enzyme recognition sites. A T4 chromosomal "target" DNA sequence is cloned into this vector and mutated by standard recombinant DNA techniques. Escherichia coli cells containing this plasmid are then infected with T4 bacteriophage that carry amber mutations in two essential genes. The plasmid integrates into the T4 chromosome by recombination between the plasmid-borne T4 target sequence and its homologous chromosomal counterpart. The resulting phage, termed "integrants," are selectable by the supF-mediated suppression of their two amber mutations. Thus, although the integrants comprise 1-3% or less of the total phage progeny, growth on a nonsuppressing host permits their direct selection. The pure integrant phage can be either analyzed directly for a possible mutant phenotype or transferred to nonselective growth conditions. In the latter case, plasmid-free phage segregants rapidly accumulate due to homologous recombination between the duplicated target sequences surrounding the supF sequence in each integrant chromosome. A major fraction of these segregants will retain the in vitro generated mutation within their otherwise unchanged chromosomes and are isolated as stable mutant bacteriophage. The insertion/substitution vector system thereby allows any in vitro mutated gene to be readily substituted for its wild-type counterpart in the bacteriophage T4 genome.

Base Sequence↗

PubMed1988

The mechanism of homologous DNA strand exchange catalyzed by the bacteriophage T4 uvsX and gene 32 proteins.

A strand exchange reaction between a single-stranded DNA circle and a homologous linear double-stranded DNA molecule is catalyzed by a mixture of two T4 bacteriophage proteins, the uvsX protein (a DNA-dependent ATPase that resembles the recA protein) and the gene 32 protein (a helix-destabilizing protein). The products are different from those formed in the corresponding recA protein-catalyzed reaction; rather than producing a linear single strand plus a nicked circular double-stranded (form II) DNA molecule as the final products, interlinked DNA networks are rapidly generated. Electron microscopy reveals that these networks form from multiple pairing reactions that involve the recombination intermediates. Since the uvsX protein is present in substoichiometric quantities, it presumably recycles to catalyze these successive pairing events. Recycling of the uvsX protein has been more directly examined in an assay that monitors the rate of uvsX protein-catalyzed branch migration. The branch migration reaction is rapidly inhibited by dilution of the uvsX protein or by the addition of a heterologous competitor DNA, showing that the uvsX protein-DNA filaments that catalyze strand exchange are dynamic structures. The evidence suggests that individual uvsX protein monomers are continuously entering and leaving the cooperatively formed filament in a cycle that is strongly affected by their ATP hydrolysis.

Adenosine Triphosphatases↗

PubMed1988

Behaviour of microtubules and actin filaments in living Drosophila embryos.

We describe the preparation of novel fluorescent derivatives of rabbit muscle actin and bovine tubulin, and the use of these derivatives to study the behaviour of actin filaments and microtubules in living Drosophila embryos, in which the nuclei divide at intervals of 8 to 21 min. The fluorescently labelled proteins appear to function normally in vitro and in vivo, and they allow continuous observation of the cytoskeleton in living embryos without perturbing development. By coinjecting labelled actin and tubulin into the early syncytial embryo, the spatial relationships between the distinct filament networks that they form can be followed second by second. The dynamic rearrangements of actin filaments and microtubules observed confirms and extends results obtained from previous studies, in which fixation techniques and specific staining were used to visualize the cytoskeleton in the Drosophila embryo. However, no tested fixation method produces an exact representation of the in vivo microtubule distribution.

Actins↗

PubMed1987

Prokaryotic DNA replication mechanisms.

The three different prokaryotic replication systems that have been most extensively studied use the same basic components for moving a DNA replication fork, even though the individual proteins are different and lack extensive amino acid sequence homology. In the T4 bacteriophage system, the components of the DNA replication complex can be grouped into functional classes as follows: DNA polymerase (gene 43 protein), helix-destabilizing protein (gene 32 protein), polymerase accessory proteins (gene 44/62 and 45 proteins), and primosome proteins (gene 41 DNA helicase and gene 61 RNA primase). DNA synthesis in the in vitro system starts by covalent addition onto the 3'OH end at a random nick on a double-stranded DNA template and proceeds to generate a replication fork that moves at about the in vivo rate, and with approximately the in vivo base-pairing fidelity. DNA is synthesized at the fork in a continuous fashion on the leading strand and in a discontinuous fashion on the lagging strand (generating short Okazaki fragments with 5'-linked pppApCpXpYpZ pentaribonucleotide primers). Kinetic studies reveal that the DNA polymerase molecule on the lagging strand stays associated with the fork as it moves. Therefore the DNA template on the lagging strand must be folded so that the stop site for the synthesis of one Okazaki fragment is adjacent to the start site for the next such fragment, allowing the polymerase and other replication proteins on the lagging strand to recycle.

DNA Replication↗

PubMed1986

DNA synthesis dependent on genetic recombination: characterization of a reaction catalyzed by purified bacteriophage T4 proteins.

To simulate a reaction that occurs in T4-infected cells, we have developed an in vitro DNA synthesis system that requires seven highly purified proteins encoded by this bacteriophage: the DNA polymerase "holoenzyme" (four proteins), gene 32 protein, dda DNA helicase, and uvsX protein - an enzyme that catalyzes homologous DNA pairing and is functionally homologous to the recA protein. In the reaction observed, the 3'OH end of one single-stranded DNA molecule primes DNA synthesis using a double-stranded DNA molecule of homologous sequence as the template. The uvsX protein continuously removes the new DNA chain from its template, so that DNA is synthesized by a conservative mechanism. This type of reaction, which requires the cooperation of recombination and replication enzymes, seems likely to be a general feature of DNA metabolism.

Adenosine Triphosphatases↗

PubMed1986

Studies of the DNA helicase-RNA primase unit from bacteriophage T4. A trinucleotide sequence on the DNA template starts RNA primer synthesis.

The purified DNA replication proteins encoded by genes 41 and 61 of bacteriophage T4 catalyze efficient RNA primer synthesis on a single-stranded DNA template. In the presence of additional T4 replication proteins, we demonstrate that the template sequences 5'-GTT-3' and 5'-GCT-3' serve as necessary and sufficient signals for RNA primer-dependent initiation of new DNA chains. These chains start with primers that have the sequences pppApCpNpNpN and pppGpCpNpNpN, where N can be any one of the four ribonucleotides. Each primer is initiated from the T (A-start primers) or C (G-start primers) in the center of the recognized template sequence. A subset of the DNA chain starts is observed when one of the four ribonucleoside triphosphates used as the substrates for primer synthesis is omitted; the starts observed reveal that both pentaribonucleotide and tetraribonucleotide primers can be used for efficient initiation of new DNA chains, whereas primers that are only 3 nucleotides long are inactive. It was known previously that, when 61 protein is present in catalytic amounts, the 41 and 61 proteins are both required for observing RNA primer synthesis. However, by raising the concentration of the 61 protein to a much higher level, a substantial amount of RNA-primed DNA synthesis is obtained in the absence of 41 protein. The DNA chains made are initiated by primers that seem to be identical to those made when both 41 and 61 proteins are present; however, only those template sites containing the 5'-GCT-3' sequence are utilized. The 61 protein is, therefore, the RNA primase, whereas the 41 protein should be viewed as a DNA helicase that is required (presumably via a 41/61 complex) for efficient primase recognition of both the 5'-GCT-3' and 5'-GTT-3' DNA template sequences.

Base Sequence↗

PubMed1986

Purification and characterization of the T4 bacteriophage uvsX protein.

Gene uvsX of bacteriophage T4 encodes a 40,000-dalton protein that plays a key role in the major pathway for genetic recombination in T4-infected cells. Mutations at the uvsX locus lead to increased sensitivity to various DNA-damaging agents, reduced phage bursts, decreased genetic recombination, and early arrest of DNA synthesis. Like the Escherichia coli recA protein, the purified uvsX protein is a DNA-dependent ATPase that catalyzes pairing between homologous single- and double-stranded DNA molecules in vitro (Yonesaki, T., Ryo, Y., Minagawa, T., and Takahashi, H., (1985) Eur. J. Biochem. 148, 127-134). At physiological salt concentrations, the uvsX protein binds tightly and cooperatively to single-stranded DNA, covering about five nucleotides per protein monomer; at lower salt concentrations, a similar type of binding to double-stranded DNA is detected (Griffith, J., and Formosa, T., (1985) J. Biol. Chem. 260, 4484-4491). We show here that the ATPase activity of this protein is unusual in producing both ADP plus Pi and AMP plus PPi as products. Generating the fully active form of the ATPase is a cooperative process, apparently requiring that a protein monomer be bound to single-stranded DNA while surrounded by other ATP-bound monomers. The catalysis of homologous pairing by the uvsX protein is shown to be greatly stimulated by the presence of the T4 gene 32 protein, a helix-destablizing protein previously studied in this laboratory, and it requires continued ATP hydrolysis. We present a method that allows the purification of the uvsX protein to essential homogeneity. We also describe the complete purification of two proteins that bind to the uvsX protein: the T4 uvsY protein (16,000 daltons) and an E. coli host protein of 32,000 daltons whose gene is unknown. The host protein is likely to play a role in DNA metabolism, because it also binds to the T4 gene 32 protein and to DNA; the sequence of its amino-terminal 29 amino acids has been determined.

Adenosine Triphosphatases↗

PubMed1986

Characterization of a defective phage system for the analysis of bacteriophage T4 DNA replication origins.

We have developed a defective phage system for the isolation and analysis of phage T4 replication origins based on the T4-mediated transduction of plasmid pBR322. During the initial infection of a plasmid-containing cell, recombinant plasmids with T4 DNA inserts are converted into fully modified linear DNA concatamers that are packaged into T4 phage particles, to create defective phage (transducing particles). In order to select T4 replication origins from genomic libraries of T4 sequences cloned into the plasmid pBR322, we searched for recombinant plasmids that transduce with an unusually high efficiency, reasoning that this should select for T4 sequences that function as origins on plasmid DNA after phage infection. We also selected for defective phage that can propagate efficiently with the aid of a coinfecting helper phage during subsequent rounds of phage infection, which should select for T4 sequences that can function as origins on the linear DNA present in the defective phage. Several T4 inserts were isolated repeatedly in one or both of these selective procedures, and these were mapped to particular locations on the T4 genome. When plasmids were selected in this way from genomic libraries constructed using different restriction nucleases, they contained overlapping segments of the T4 genome, indicating that the same T4 sequences were selected. The inserts in two of the selected plasmids permit a very high frequency of transduction from circular plasmids; these have been shown to contain a special type of T4 replication origin.

DNA Replication↗