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Biomedical subjects

B Alberts

Publications and source records attributed to B Alberts.

At least 19 recordsLinked to original sources
PubMed1998

Ozone-induced airway hyperresponsiveness in patients with asthma: role of neutrophil-derived serine proteinases.

Proteinase inhibitors may be of potential therapeutic value in the treatment of respiratory diseases such as chronic obstructive pulmonary disease (COPD) or asthma. Our aim was to study the role of neutrophils, and neutrophil-derived serine proteinases in an acute model in patients with asthma. Exposure to ozone induces an acute neutrophilic inflammatory reaction accompanied by an increase in airway hyperresponsiveness. It is thought that these two effects of ozone are linked, and that neutrophil-derived serine proteinases (i.e. elastase) may play a role in the ozone-induced airway hyperresponsiveness. Therefore, we examined the effect of recombinant antileukoprotease (rALP), one of the major serine proteinase inhibitors in the lung, on ozone-induced changes in airway hyperresponsiveness in this model. We observed that 16 h after exposure to ozone, airway hyperresponsiveness to methacholine was increased both following placebo and rALP treatment. There was no significant difference between placebo and rALP treatment (change in area under the dose-response curve to methacholine: 117.3+/-59.0 vs 193.6+/-59.6 % fall x DD; p=.12). Moreover, the immediate decrease in FEV1 after ozone exposure was not significantly different between the two groups (placebo: -29.6+/-6.7%; rALP: -20.9+/-3.8%; p=.11). In addition, no significant differences were observed in plasma levels of fibrinogen degradation products generated by neutrophil serine proteinases before and after exposure to ozone. We conclude that neutrophil-derived serine proteinases are not important mediators for ozone-induced hyperresponsiveness.

Administration, Inhalation↗
PubMed1998

The Human Genome Project after a decade: policy issues.

The Human Genome Project began a decade ago, its early momentum fueled by two reports. A report from the National Research Council (NRC) in February 1998 endorsed the project and provided the basis for the first joint plan by the National Institutes of Health (NIH) and the Department of Energy (DOE). A report from the Office of Technology Assessment (OTA) in April 1988, provided Congress with a means to assess the roles of NIH and DOE. Both reports highlighted the importance of genomics and emphasized the need for a concerted research program. The committees did not predict the large investment of private funds or the extensive patenting of sequences, and they underestimated the rate of progress. Overall, though, the consensus-building provided by the committees helped to set the blueprint for one of the great success stories in modern biology.

Advisory Committees↗
PubMed1996

Processivity of the gene 41 DNA helicase at the bacteriophage T4 DNA replication fork.

The gene 41 protein is the DNA helicase associated with the bacteriophage T4 DNA replication fork. This protein is a major component of the primosome, being essential for coordinated leading and lagging strand DNA synthesis. Models suggest that such DNA helicases are loaded only onto DNA at origins of replication, and that they remain with the ensuing replication fork until replication is terminated. To test this idea, we have measured the extent of processivity of the 41 protein in the context of an in vitro DNA replication system composed of eight purified proteins (the gene 43, 44/62, 45, 32, 41, 59, and 61 proteins). After starting DNA replication in the presence of these proteins, we diluted the 41 helicase enough to prevent any association of new helicase molecules and analyzed the replication products. We measured an association half-life of 11 min, revealing that the 41 protein is processive enough to finish replicating the entire 169-kilobase T4 genome at the observed replication rate of approximately 400 nucleotides/s. This processivity of the 41 protein does not require the 59 protein, the protein that catalyzes 41 protein assembly onto 32 protein-covered single-stranded DNA. The stability we measure for the 41 protein as part of the replication fork is greater than estimated for it alone on single-stranded DNA. We suggest that the 41 protein interacts with the polymerase holoenzyme at the fork, both stabilizing the other protein components and being stabilized thereby.

Bacteriophage T4↗
PubMed1996

A purified Drosophila septin complex forms filaments and exhibits GTPase activity.

Septin proteins are necessary for cytokinesis in budding yeast and Drosophila and are thought to be the subunits of the yeast neck filaments. To test whether septins actually form filaments, an immunoaffinity approach was used to isolate a septin complex from Drosophila embryos. The purified complex is comprised of the three previously identified septin polypeptides Pnut, Sep2, and Sep1. Hydrodynamic and sequence data suggest that the complex is composed of a heterotrimer of homodimers. The complex copurifies with one molecule of bound guanine nucleotide per septin polypeptide. It binds and hydrolyzes exogenously added GTP. These observations together with conserved sequence motifs identify the septins as members of the GTPase superfamily. We discuss a model of filament structure and speculate as to how the filaments are organized within cells.

Actin Cytoskeleton↗
PubMed1995

Nucleation of microtubule assembly by a gamma-tubulin-containing ring complex.

The highly conserved protein gamma-tubulin is required for microtubule nucleation in vivo. When viewed in the electron microscope, a highly purified gamma-tubulin complex from Xenopus consisting of at least seven different proteins is seen to have an open ring structure. This complex acts as an active microtubule-nucleating unit which can cap the minus ends of microtubules in vitro.

Amino Acid Sequence↗
PubMed1995

The cell cycle-dependent localization of the CP190 centrosomal protein is determined by the coordinate action of two separable domains.

CP190, a protein of 1,096 amino acids from Drosophila melanogaster, oscillates in a cell cycle-specific manner between the nucleus during interphase, and the centrosome during mitosis. To characterize the regions of CP190 responsible for its dynamic behavior, we injected rhodamine-labeled fusion proteins spanning most of CP190 into early Drosophila embryos, where their localizations were characterized using time-lapse fluorescence confocal microscopy. A single bipartite 19-amino acid nuclear localization signal was detected that causes nuclear localization. Robust centrosomal localization is conferred by a separate region of 124 amino acids; two adjacent, nonoverlapping fusion proteins containing distinct portions of this region show weaker centrosomal localization. Fusion proteins that contain both nuclear and centrosomal localization sequences oscillate between the nucleus and the centrosome in a manner identical to native CP190. Fusion proteins containing only the centrosome localization sequence are found at centrosomes throughout the cell cycle, suggesting that CP190 is actively recruited away from the centrosome by its movement into the nucleus during interphase. Both native and bacterially expressed CP190 cosediment with microtubules in vitro. Tests with fusion proteins show that the domain responsible for microtubule binding overlaps the domain required for centrosomal localization. CP60, a protein identified by its association with CP190, also localizes to centrosomes and to nuclei in a cell cycle-dependent manner. Experiments in which colchicine is used to depolymerize microtubules in the early Drosophila embryo demonstrate that both CP190 and CP60 are able to attain and maintain their centrosomal localization in the absence of microtubules.

Amino Acid Sequence↗
PubMed1994

Purification and characterization of bacteriophage T4 gene 59 protein. A DNA helicase assembly protein involved in DNA replication.

The T4 bacteriophage gene 59 protein is required for normal T4 DNA replication. We have purified this protein to homogeneity in two steps and show that it binds both to single-stranded DNA and to the T4 gene 32 protein, a DNA single strand binding protein. In in vitro assays, covering DNA with 32 protein makes this DNA inaccessible to the 41 protein, the highly processive DNA helicase, that associates with the T4 DNA primase (gene 61 protein) to form an active primosome. However, the 59 protein brings about the rapid assembly of 41 protein onto single-stranded DNA, even if this DNA is covered with 32 protein. The 59 protein is therefore a DNA helicase assembly protein. The observed requirements for the 59 protein in the vivo T4 DNA replication are explained by there being two alternative pathways for loading the 41 protein onto a replication fork at early times of T4 DNA synthesis, with only a 59 protein-mediated pathway remaining operative for the recombination-mediated replication that dominates later in infection (Barry, J., and Alberts, B. M. (1994) J. Biol. Chem. 269, 33063-33068).

Adenosine Triphosphate↗
PubMed1994

A role for two DNA helicases in the replication of T4 bacteriophage DNA.

The T4 bacteriophage gene 41 protein is the highly processive DNA helicase of the T4 primosome, a central part of the protein machinery that moves the T4 DNA replication fork. The T4 gene 59 protein accelerates the loading of 41 protein onto DNA covered with 32 protein (the T4 single strand binding protein), and it makes the 41 protein DNA helicase activity rapidly available to catalyze replication fork movement through a DNA double helix (Barry, J., and Alberts, B.M. (1994) J. Biol. Chem. 269, 33049-33062). With the aid of the 59 protein, we show that the T4 primosome (the T4 gene 41 and 61 proteins) can move rapidly through a promoter-bound RNA polymerase molecule that would otherwise stop replication fork movement. A second, very different DNA helicase, the T4 dda protein, provides an alternative pathway for replication past this DNA-bound RNA polymerase (Bedinger, P., Hochstrasser, M., Jongeneel, C. V., and Alberts, B.M. (1983) Cell 34, 115-123). Combined with other data, these in vitro experiments allow us to propose a model that explains why either the 59 protein or the dda protein, but not both, are required to begin efficient DNA replication inside the T4 bacteriophage-infected cell.

Bacteriophage T4↗
PubMed1994

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.

Animals↗
PubMed1994

A transcribing RNA polymerase molecule survives DNA replication without aborting its growing RNA chain.

We have demonstrated elsewhere that a precisely placed, stalled Escherichia coli RNA polymerase ternary transcription complex (polymerase-RNA-DNA) stays on the DNA template after passage of a DNA replication fork. Moreover, the bypassed complex remains competent to resume elongation of its bound RNA chain. But the simplicity of our experimental system left several important questions unresolved: in particular, might the observation be relevant only to the particular ternary complex that we studied, and can the finding be generalized to a transcribing instead of a stalled RNA polymerase? To address these issues, we have created three additional ternary transcription complexes and examined their fates after passage of a replication fork. In addition, we have examined the fate of moving RNA polymerase molecules during DNA replication. The results suggest that our previous finding applies to all transcription intermediates of the E. coli RNA polymerase.

Adenosine Triphosphate↗
PubMed1994

Scientific conduct.

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Biomedical Research↗