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B M Alberts

Publications and source records attributed to B M Alberts.

At least 19 recordsLinked to original sources
PubMed1997

Isolation of actin-associated proteins from Caenorhabditis elegans oocytes and their localization in the early embryo.

The actin cytoskeleton plays an important, but poorly understood, role in the development of multicellular organisms. To help illuminate this role, we used actin filament affinity chromatography to isolate actin binding proteins from large quantities of Caenorhabditis elegans oocytes. To examine how these proteins might be involved in early development, we prepared antibodies against some of them and determined their distribution in fixed embryos. Three of these proteins co-localize with different subsets of the embryonic actin cytoskeleton. One co-localizes with actin to all cell cortices. The second oscillates between the nucleus and cortex in a cell-cycle-dependent manner. The third is asymmetrically enriched at the anterior cortex of one-cell embryos, showing a temporal and spatial localization suggestive of a function in generating developmental asymmetry. We conclude that biochemistry is a feasible and useful approach in the study of early C. elegans development, and that the embryonic actin cytoskeleton is regulated in a complex fashion in order to carry out multiple, simultaneous functions.

Actins↗
PubMed1997

Workshop on schizophrenia.

On November 29-30, 1995, the National Academy of Sciences and the Institute of Medicine brought together experts in schizophrenia and specialists in other areas of the biological sciences in a workshop aimed at promoting the application of the latest biological information to this clinical problem. The workshop paid particular attention to evidence of pathology in the brains of people with schizophrenia, and to the possibility that this reflects an abnormality in brain development that eventually leads to the appearance of symptoms. The participants were impressed with the complexity of the problem, and felt that multiple approaches would be required to understand this disease. They recommended that a major focus should be on the search for predisposing genes, but that there should be parallel research in many other areas.

Brain↗
PubMed1997

Two proteins that cycle asynchronously between centrosomes and nuclear structures: Drosophila CP60 and CP190.

Both the nucleus and the centrosome are complex, dynamic structures whose architectures undergo cell cycle-specific rearrangements. CP190 and CP60 are two Drosophila proteins of unknown function that shuttle between centrosomes and nuclei in a cell cycle-dependent manner. These two proteins are associated in vitro, and localize to centrosomes in a microtubule independent manner. We injected fluorescently labeled, bacterially expressed CP190 and CP60 into living Drosophila embryos and followed their behavior during the rapid syncytial blastoderm divisions (nuclear cycles 10-13). Using quantitative 3-D wide-field fluorescence microscopy, we show that CP190 and CP60 cycle between nuclei and centrosomes asynchronously with the accumulation of CP190 leading that of CP60 both at centrosomes and in nuclei. During interphase, CP190 is found in nuclei. Immediately following nuclear envelope breakdown, CP190 localizes to centrosomes where it remains until telophase, thereafter accumulating in reforming nuclei. Unlike CP190, CP60 accumulates at centrosomes primarily during anaphase, where it remains into early interphase. During nuclear cycles 10 and 11, CP60 accumulates in nuclei simultaneous with nuclear envelope breakdown, suggesting that CP60 binds to an unknown nuclear structure that persists into mitosis. During nuclear cycles 12 and 13, CP60 accumulates gradually in nuclei during interphase, reaching peak levels just before nuclear envelope breakdown. Once in the nucleus, both CP190 and CP60 appear to form fibrous intranuclear networks that remain coherent even after nuclear envelope breakdown. The CP190 and CP60 networks do not co-localize extensively with each other or with DNA. This work provides direct evidence, in living cells, of a coherent protein network that may represent a nuclear skeleton.

Animals↗
PubMed1996

Two-dimensional gel analysis of rolling circle replication in the presence and absence of bacteriophage T4 primase.

The rolling circle DNA replication structures generated by the in vitro phage T4 replication system were analyzed using two-dimensional agarose gels. Replication structures were generated in the presence or absence of T4 primase (gp61), permitting the analysis of replication forks with either duplex or single-stranded tails. A characteristic arc shape was visualized when forks with single-stranded tails were cleaved by a restriction enzyme with the help of an oligonucleotide that anneals to restriction sites in the single-stranded tail. After calibrating the gel system with this well-studied rolling circle replication reaction, we then analyzed the in vivo replication directed by a T4 replication origin cloned within a plasmid. DNA samples were generated from infections with either wild-type or primase-deletion mutant phage. The only replicative arc that could be detected in the wild-type sample corresponded to duplex Y forms, consistent with very efficient lagging strand synthesis. Surprisingly, we obtained evidence for both duplex and single-stranded DNA tails in the samples from the primase-deficient infection. We conclude that a relatively inefficient mechanism primes lagging strand DNA synthesis in vivo when gp61 is absent.

Bacteriophage T4↗
PubMed1995

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.

Bacteriophage T4↗
PubMed1995

Three-dimensional structural characterization of centrosomes from early Drosophila embryos.

An understanding of the mechanism and structure of microtubule (MT)-nucleating sites within the pericentriolar material (PCM) of the centrosome has been elusive. This is partly due to the difficulty in obtaining large quantities of centrosomes for analysis, as well as to the problem of attaining interpretable structural data with conventional EM techniques. We describe a protocol for isolating a large quantity of functional centrosomes from early Drosophila embryos. Using automated electron tomography, we have begun a three-dimensional structural characterization of these intact centrosomes with and without regrown MTs. Reconstructions of the centrosomes to approximately 6-8 nm resolution revealed no large structures at the minus ends of MTs, suggesting that if MT-nucleating material physically contacts the MTs, it must conform closely to the shape of the minus end. While many MTs originate near the centrioles, MT minus ends were found throughout the PCM, and even close to its outer boundary. The MTs criss-crossed the PCM, suggesting that nucleating sites are oriented in many different directions. Reconstructions of centrosomes without MTs suggest that there is a reorganization of the PCM upon MT regrowth; moreover, ring-like structures that have a similar diameter as MTs are apparent in the PCM of centrosomes without MTs, and may be MT-nucleating sites.

Animals↗
PubMed1995

Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex.

We report the cDNA sequence and localization of a protein first identified by actin filament chromatography of Drosophila embryo extracts as ABP8 (Miller, K. G., C. M. Field, and B. M. Alberts. 1989. J. Cell Biol. 109:2963-2975). The cDNA encodes a 1201-amino acid protein which we name anillin. Anillin migrates at 190 kD on SDS-PAGE. Anillin is expressed throughout Drosophila development and in tissue culture cells. By immunofluorescence, anillin localizes to the nucleus of interphase cells, except in the syncytial embryo where it is always cytoplasmic. During metaphase, it is present in the cytoplasm and cortex, and during anaphase-telophase it becomes highly enriched in the cleavage furrow along with myosin II. In the syncytial embryo, anillin, along with myosin-II, is enriched in cortical areas undergoing cell cycle regulated invagination including metaphase furrows and the cellularization front. In contractile rings, metaphase furrows, and nascent ring canals, anillin remains bound to the invaginated cortex suggesting a stabilizing role. Anillin is not expressed in cells that have left the cell cycle. Anillin isolated from embryo extracts binds directly to actin filaments. The domain responsible for this binding has been mapped to a region of 244 amino acids by expression of protein fragments in bacteria. This domain, which is monomeric in solution, also bundles actin filaments. We speculate that anillin plays a role in organizing and/or stabilizing the cleavage furrow and other cell cycle regulated, contractile domains of the actin cytoskeleton.

Actins↗
PubMed1995

CP60: a microtubule-associated protein that is localized to the centrosome in a cell cycle-specific manner.

DMAP190 is a microtubule-associated protein from Drosophila that is localized to the centrosome. In a previous study, we used affinity chromatography to identify proteins that interact with DMAP190, and identified a 60-kDa protein that we named DMAP60 (Kellogg and Alberts, 1992). Like DMAP190, DMAP60 interacts with microtubules and is localized to the centrosome, and the two proteins associate as part of a multiprotein complex. We now report the cloning and sequencing of the cDNA encoding DMAP60. The amino acid sequence of DMAP60 is not homologous to any protein in the database, although it contains six consensus sites for phosphorylation by cyclin-dependent kinases. As judged by in situ hybridization, the gene for DMAP60 maps to chromosomal region 46A. In agreement with others working on Drosophila centrosomal proteins, we have changed the names for DMAP190 and DMAP60 to CP190 and CP60, respectively, to give these proteins a consistent nomenclature. Antibodies that recognize CP60 reveal that it is localized to the centrosome in a cell cycle-dependent manner. The amount of CP60 at the centrosome is maximal during anaphase and telophase, and then drops dramatically during late telophase or early interphase. This dramatic disappearance of CP60 may be due to specific proteolysis, because CP60 contains a sequence of amino acids similar to the "destruction box" that targets cyclins for proteolysis at the end of mitosis. Starting with nuclear cycle 12, CP60 and CP190 are both found in the nucleus during interphase. CP60 isolated from Drosophila embryos is highly phosphorylated, and dephosphorylated CP60 is a good substrate for cyclin B/p34cdc2 kinase complexes. A second kinase activity capable of phosphorylating CP60 is present in the CP60/CP190 multiprotein complex. We find that bacterially expressed CP60 binds to purified microtubules, and this binding is blocked by CP60 phosphorylation.

Amino Acid Sequence↗
PubMed1995

Heterochromatin protein 1 distribution during development and during the cell cycle in Drosophila embryos.

Heterochromatin protein 1 (HP1) was initially discovered as a protein that is associated with the heterochromatin at the chromocenter of polytene chromosomes in Drosophila larval salivary glands. In this paper we investigate the localization of heterochromatin protein 1 in the diploid nuclei of Drosophila embryos. We focus on its association with the interphase heterochromatin in fixed embryos before and during cycle 14, the developmental time at which heterochromatin becomes most conspicuous, and also follow its localization during mitosis. The GAGA transcription factor was recently shown to be localized at sequences within alpha-heterochromatin in pre-cycle 14 embryos, and an antibody against this protein serves as a convenient marker for these sequences. We find an enrichment of heterochromatin protein 1 in the intensely DAPI-staining regions near the apical surface of nuclear cycle 10 embryos. At this stage GAGA factor is localized into punctate structures in this same region. This enrichment for HP1 is markedly increased during nuclear cycle 14. Surprisingly, whereas GAGA factor retains its association with the heterochromatin throughout the cell cycle, a significant fraction of HP1 is dispersed throughout the spindle around the segregating chromosomes during mitosis. This dispersed pool of heterochromatin protein 1 was observed during mitosis in both early and late Drosophila embryos and in an analysis of a bacterially produced 6x histidine-heterochromatin protein 1 fusion protein injected into living Drosophila embryos. When Drosophila tissue culture cells were prepared by a method which removes soluble protein and avoids fixation of the mitotic chromosomes, an enrichment for heterochromatin protein 1 in the heterochromatin of the chromosomes was discovered also.

Animals↗
PubMed1995

Heterochromatin protein 1 is required for correct chromosome segregation in Drosophila embryos.

Heterochromatin protein 1 is associated with centromeric heterochromatin in Drosophila, mice, and humans. Loss of function mutations in the gene encoding heterochromatin protein 1 in Drosophila, Suppressor of variegation2-5, decrease the mosaic repression observed for euchromatic genes that have been juxtaposed to centromeric heterochromatin. These heterochromatin protein 1 mutations not only suppress this position-effect variegation, but also cause recessive embryonic lethality. In this study, we analyze the latter phenotype in the hope of gaining insight into heterochromatin function. In our analyses of four alleles of Suppressor of variegation2-5, the lethality was found to be associated with defects in chromosome morphology and segregation. While some of these defects are seen throughout embryonic development, both the frequency and severity of the defects are greatest between cycles 10 and 14 when zygotic transcription of the Suppressor of variegation2-5 gene apparently begins. By this time in development, heterochromatin protein 1 levels are diminished by four-fold in a quarter of the embryos produced by parents that are both heterozygous for a null allele (Suppressor of variegation2-5(05)). In a live analysis of the phenotype, we find prophase to be lengthened by more than two-fold in Suppressor of variegation2-5(05) mutant embryos with subsequent defects in chromosome segregation. The elongated prophase suggests that the segregation phenotype is a consequence of defects in events that occur during prophase, either in chromosome condensation or kinetochore assembly or function. Immunostaining with an antibody against a centromerespecific antigen indicates that the kinetochores of most chromosomes are functional. The immunostaining results are more consistent with defects in chromosome condensation being responsible for the segregation phenotype.

Alleles↗
PubMed1994

The slow dissociation of the T4 DNA polymerase holoenzyme when stalled by nucleotide omission. An indication of a highly processive enzyme.

We have developed an experimental assay to monitor the rate of dissociation of the T4 DNA polymerase holoenzyme (polymerase plus gene 44/62 and 45 proteins) once it has been stalled by nucleotide omission. Using this assay, we determined that the dissociation of the DNA polymerase holoenzyme follows a first order decay with a half-life of 2.5 min. The long half-life resembles that expected for the holoenzyme processively synthesizing DNA on the leading strand of the replication fork. The holoenzyme dissociation rate is independent of polymerase accessory protein concentration and of ATP hydrolysis. The dissociation rate is increased if the gene 32 protein is omitted or if the primer-template region is shortened from 46 to 28 base pairs. But the rate of holoenzyme dissociation is most strikingly increased when the circular DNA template is converted to a linear form. By analogy with other well studied systems, these results support a model in which ATP hydrolysis by the 44/62 proteins serves to load a ring-like 45 protein onto the DNA. Once loaded, the 45 protein, possibly along with the 44/62 complex, acts as a sliding clamp that tethers the DNA polymerase to the template.

Adenosine Triphosphate↗
PubMed1994

The rapid dissociation of the T4 DNA polymerase holoenzyme when stopped by a DNA hairpin helix. A model for polymerase release following the termination of each Okazaki fragment.

We have examined the molecular mechanism that enables the T4 bacteriophage DNA polymerase holoenzyme to synthesize DNA processively on the leading strand of the replication fork for many minutes, while allowing an identical holoenzyme on the lagging strand to recycle from one Okazaki fragment to the next in less than 4 s. We use a perfect hairpin helix of 15 base pairs to mimic the encounter of the polymerase with the end of a previously synthesized Okazaki fragment. Polymerase dissociation is monitored during the stall at the hairpin helix by the addition of excess T4 gene 32 protein (SSB protein), which rapidly melts the helix and allows a stalled polymerase molecule to continue DNA synthesis. In the accompanying paper, we show that polymerase holoenzyme dissociation is slow (half-life of 2.5 min) when this enzyme is stalled by nucleotide omission (Hacker, K. J., and Alberts, B. M. (1994) J. Biol. Chem. 269, 24209-24220). In contrast, the holoenzyme dissociates with a half-life of 1 s after hitting the hairpin helix, a rate sufficient to allow efficient polymerase recycling on the lagging strand in vivo. We conclude that, upon completing each Okazaki fragment, the holoenzyme senses an encounter with duplex DNA and then switches to a state that rapidly dissociates.

Base Sequence↗
PubMed1994

How targeted should research and higher education be?

The education and research system in the biomedical sciences is tremendously vital. But the scientific community must exert more leadership if this vitality is to continue, for both science education and research are prone to a common villain: inertia. For example, in the education arena, why do medical schools require two semesters of organic chemistry but no cell biology, when the center of biomedical research has shifted to cell biology? And why do so many graduate schools continue to send a strong message to their science students that there is only one really successful career path--the one leading to academia--when most of our PhD students cannot expect to become professors? Inertia in research can be seen in the trend for cell biologists to train researchers just like themselves, which means that the many opportunities to use new cell biological techniques to address important problems in tissue biology are likely to be missed. A solution to such problems is to design funding mechanisms that promote more adventuresome research. As a bottom line, our research system must support the independence of our best young scientists and encourage them to take the risks inherent in highly creative endeavors.

Career Mobility↗
PubMed1993

The DNA replication fork can pass RNA polymerase without displacing the nascent transcript.

Replication proteins encoded by bacteriophage T4 generate DNA replication forks that can pass a molecule of Escherichia coli RNA polymerase moving in the same direction as the fork in vitro. The RNA polymerase ternary transcription complex remains bound to the DNA and retains a transcription bubble after the fork passes. The by-passed ternary complex can resume faithful RNA synthesis, suggesting that the multisubunit RNA polymerase of E. coli has evolved to retain its transcript after DNA replication, allowing partially completed transcripts to be elongated into full-length RNA molecules.

Bacteriophage T4↗
PubMed1993

Drosophila gamma-tubulin is part of a complex containing two previously identified centrosomal MAPs.

gamma-tubulin is a minor tubulin that is localized to the microtubule organizing center of many fungi and higher eucaryotic cells (Oakley, B. R., C. E. Oakley, Y. Yoon, and M. C. Jung. 1990. Cell. 61: 1289-1301; Stearns, T., L. Evans, and M. Kirschner. 1991. Cell. 65:825-836; Zheng, Y., M. K. Jung, and B. R. Oakley. 1991. Cell. 65:817-823). Here we show that gamma-tubulin is a component of a previously isolated complex of Drosophila proteins that contains at least two centrosomal microtubule-associated proteins called DMAP190 and DMAP60. Like DMAP190 and DMAP60, the gamma-tubulin in extracts of early Drosophila embryos binds to microtubules, although this binding may be indirect. Unlike DMAP190 and DMAP60, however, only 10-50% of the gamma-tubulin in the extract is able to bind to microtubules. We show that gamma-tubulin binds to a microtubule column as part of a complex, and a substantial fraction of this gamma-tubulin is tightly associated with DMAP60. As neither alpha- nor beta-tubulin bind to microtubule columns, the gamma-tubulin cannot be binding to such columns in the form of an alpha:gamma or beta:gamma heterodimer. These observations suggest that gamma-tubulin, DMAP60, and DMAP190 are components of a centrosomal complex that can interact with microtubules.

Animals↗
PubMed1993

Analysis of five presumptive protein-coding sequences clustered between the primosome genes, 41 and 61, of bacteriophages T4, T2, and T6.

In bacteriophage T4, there is a strong tendency for genes that encode interacting proteins to be clustered on the chromosome. There is 1.6 kb of DNA between the DNA helicase (gene 41) and the DNA primase (gene 61) genes of this virus. The DNA sequence of this region suggests that it contains five genes, designated as open reading frames (ORFs) 61.1 to 61.5, predicted to encode proteins ranging in size from 5.94 to 22.88 kDa. Are these ORFs actually genes? As one test, we compared the DNA sequence of this region in bacteriophages T2, T4, and T6 and found that ORFs 61.1, 61.3, 61.4, and 61.5 are highly conserved among the three closely related viruses. In contrast, ORF 61.2 is conserved between phages T4 and T6 yet is absent from phage T2, where it is replaced by another ORF, T2 ORF 61.2, which is not found in the T4 and T6 genomes. As a second, independent test for coding sequences, we calculated the codon base position preferences for all ORFs in this region that could encode proteins that contain at least 30 amino acids. Both the T4/T6 and T2 versions of ORF 61.2, as well as the other ORFs, have codon base position preferences that are indistinguishable from those of known T4 genes (coefficients of 0.81 to 0.94); the six other possible ORFs of at least 90 bp in this region are ruled out as genes by this test (coefficients less than zero). Thus, both evolutionary conservation and codon usage patterns lead us to conclude that ORFs 61.1 to 61.5 represent important protein-coding sequences for this family of bacteriophages. Because they are located between the genes that encode the two interacting proteins of the T4 primosome (DNA helicase plus DNA primase), one or more may function in DNA replication by modulating primosome function.

Amino Acid Sequence↗
PubMed1993

A central role for microtubules in the differentiation of Drosophila oocytes.

Drosophila oocytes develop within cysts containing 16 cells that are interconnected by cytoplasmic bridges. Although the cysts are syncytial, the 16 cells differentiate to form a single oocyte and 15 nurse cells, and several mRNAs that are synthesized in the nurse cells accumulate specifically in the oocyte. To gain insight into the mechanisms that generate the cytoplasmic asymmetry within these cysts, we have examined cytoskeletal organization during oocyte differentiation. Shortly after formation of the 16 cell cysts, a prominent microtubule organizing center (MTOC) is established within the syncytial cytoplasm, and at the time the oocyte is determined, a single microtubule cytoskeleton connects the oocyte with the remaining 15 cells of each cyst. Recessive mutations at the Bicaudal-D (Bic-D) and egalitarian (egl) loci, which block oocyte differentiation, disrupt formation and maintenance of this polarized microtubule cytoskeleton. Microtubule assembly-inhibitors phenocopy these mutations, and prevent oocyte-specific accumulation of oskar, cyclin B and 65F mRNAs. We propose that formation of the polarized microtubule cytoskeleton is required for oocyte differentiation, and that this structure mediates the asymmetric accumulation of mRNAs within the syncytial cysts.

Animals↗