David A. Goldthwait

Gene 5 protein of bacteriophage fd: A dimer which interacts Co-operatively with DNA☆

Abstract Isolated gene 5 protein from bacteriophage fd-infected Escherichia coli has been shown by sedimentation equilibrium to exist primarily as a dimer under non-denaturing conditions. The dimer was stable under conditions of high ionic strength, extremes in pH, dilution to 0.075 mg/ml, and increased temperature. Gene 5 protein did not undergo the indefinite self-association observed with gene 32 protein. Three lines of evidence for co-operative binding of gene 5 protein to DNA were developed. First, the interaction between gene 5 protein and phage T4 DNA was examined using a nitrocellulose filter assay. Scatchard plots of the binding data indicated that the interaction was co-operative. Similar results were obtained with gene 32 protein. Second, the co-operative binding of both proteins to DNA was shown by the sensitivity of the protein-DNA interaction to increasing ionic strength at various ratios of protein to DNA. Finally, by using the cross-linking agent, dimethyl suberixmidate, oligomeric structures containing at least seven monomers were found when the DNA was less than saturated. The possibility that gene 5 protein dimers undergo indefinite self-association in the presence of oligonucleotides was examined by sedimentation equilibrium. With oligo[d(pT)4], the protein dimer was complexed with this oligonucleotide but no self-association was observed. With oligo[d(pT)8], gene 5 protein formed tetramers, but no significant indefinite association was noted. These results do not suggest a DNA-induced conformational change, which results in indefinite association. A model for the co-operative binding of gene 5 protein to DNA is presented.

Impairment of Hemodynamics with Increasing Mean Airway Pressure during High-Frequency Oscillatory Ventilation

ABSTRACT: We investigated the effects of changes in mean airway pressure (Paw), oscillatory frequency and lung compliance on cardiac output (CO) and pulmonary vascular resistance in seven adult cats (3.0 ± 0.6 kg) during high-frequency oscillatory ventilation (HFOV). The cats were anesthetized with chloralose and urethane and ventilated with a high-frequency oscillator at Paw of 4, 8, 12, and 16 cm H2O and frequencies of 3, 6,12,16, and 20 Hz. Saline lavage was used to reduce lung compliance. CO was continuously recorded with an electromagnetic flow probe placed around the aorta and pulmonary vascular resistance was calculated from left atrial and pulmonary artery pressures. Lung lavage reduced static compliance of the respiratory system but did not change CO during pressure-limited ventilation. During HFOV, CO was higher in animals after lung lavage at each Paw. As Paw was raised from 4 to 16 cm H2O during HFOV, CO decreased from 133 ± 36 to 87 ± 31 ml/min kg in animals with normal lungs and decreased from 153 ± 33 to 107 ± 19 ml/min kg after lung lavage (both p < 0.001). Increasing Paw was also associated with an increase in pulmonary vascular resistance both before and after lung lavage (both p < 0.005). Changes in frequency did not significantly alter CO or pulmonary vascular resistance. We conclude that the interaction between the heart and lungs during HFOV is largely mediated by Paw and compliance of the respiratory system. Furthermore, regardless of the degree of lung compliance, cardiac function may be impaired during HFOV as Paw is elevated.

Expression of platelet-derived growth factors, transforming growth factors, and the ros gene in a variety of primary human brain tumors.

Ribonucleic acid was isolated from a wide spectrum of central nervous system tumors to examine the expression of platelet-derived growth factors (PDGF) A and B, tumor growth factors (TGF-beta) 1 and 2, and ros messenger ribonucleic acid. Eight glioblastoma cell lines were examined as well as cell cultures from 22 tumor explants. The explants included 6 glioblastomas, 4 anaplastic astrocytomas, 5 astrocytomas, 3 ependymal tumors, 2 meningiomas, 1 medulloblastoma. and 1 ganglioglioma. For comparison, 2 nontumor glial cell cultures were included. The PDGF B-chain was expressed in 5 of 8 glioblastoma cell lines, 2 of 6 glioblastomas, and in 3 of 4 anaplastic astrocytoma explants. There was no PDGF B expression in 4 astrocytomas, 3 ependymomas of varying malignancy, in the remainder of the tumors, or in the nontumor glial cells. The PDGF A-chain was expressed in all of the tumors, with the exception of the malignant ependymoma and in both nontumor glial cell cultures. TGF-beta 1 was expressed in all of the tumors and in nontumor glial cells. The expression of TGF-beta 2 was expressed in many of the benign and malignant tumors and also in both nontumor glial cell cultures. The ros messenger ribonucleic acid was expressed in 1 of 5 glioblastoma cell lines and in 2 of 6 glioblastoma cell explants, but in none of the other tumors or in the nontumor glial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of the self-association of bacteriophage T4 gene 32 protein by equilibrium sedimentation.

Abstract The self-association of the bacteriophage T4 gene 32 protein has been examined in the analytical ultracentrifuge under varying conditions to determine the nature of the process. The process is not a simple indefinite association with one association constant (monomer dimer trimer etc.). The complexity of the process is shown by (1) peculiarities in the molecular weight versus concentration curves, in the region of the dimer (observed with increasing ionic strength, at pH 10, in 0.04 m -MgCl2, with aged preparations, at 19 °C and in the presence of the oligonucleotide d(pT)10), (2) the increased sigmoidicity of the association curve in the presence of glycerol or oligo[d(pT)4], and (3) the discontinuity in the association curve at the tetramer at a pH value of approximately 9.4. A model with two association constants which could vary independently (monomer dimer tetramer etc.) explained many of the findings. However, a more complex model was required to explain curves which had a plateau at the dimer with increased association at higher protein concentrations. Thus, under all conditions examined there is evidence for more than one type of protein-protein interaction. These different interactions may be involved in a physiological function such as recombination.

The isolation of a dimer of gene 8 protein of bacteriophage fd

Abstract Gene 8 protein was isolated in a highly aggregated form in aqueous solution from purified fd bacteriophage. Gene 8 protein was dissociated to a stable dimeric form in the presence of 10 m m -sodium deoxycholate. The dimer obtained in the presence of the detergent was stable to further dissociation by sodium dodecyl sulfate, and was estimated to have a 51% helical content on the basis of circular dichroism data. When the deoxycholate was removed, the dimers were observed by electron microscopy to self-associate to complex string-like structures. The dimer obtained is thought to be a result of one of the interactions between adjacent monomers in the model proposed by Marvin & Wachtel (1975).

Identification of Alu transposition in human lung carcinoma cells

We have demonstrated genetic transposition in human cells. An experimental system was established in which the Ecogpt (gpt) gene was employed as a target for inactivation. The human lung carcinoma cell line A549 containing this target was fused to UV-irradiated A549 cells that did not contain the target. From the fusion products, sublines carrying an inactivated gpt gene were analyzed. UV irradiation increased the frequency of inactivated gpt genes in the fusion cells by 100-fold. One subline was found to contain a complete Alu sequence in the coding region of the gpt gene. The inserted element differed from the Blur8 sequence by only 7 out of the 270 nucleotides. The insertion of this Alu element created a 5 bp insertion site duplication.

A Search for Gli Expression in Tumors of the Central Nervous System

The gli gene was originally isolated from DNA amplified in double minutes in a glioblastoma (D259MG). Using a sensitive RNA-RNA hybridization, we tested a series of central nervous system tumors for expression of the gli gene. These included 8 glioblastoma cell lines, plus cell cultures of 5 glioblastomas, 4 anaplastic astrocytomas, 3 different ependymal tumors, a malignant meningioma, and a medulloblastoma. Two normal glial cell cultures were also examined. There was no gli expression in any of these specimens. In glioblastoma D259MG, approximately 130 molecules of gli mRNA per cell were present and the half-life of the mRNA was approximately 5 h. By reverse transcription and PCR, gli mRNA was observed in 4 cell lines and in normal human glial cells, but the level was estimated to be less than one five hundredth of that in the D259MG cell line. The results suggest that gli expression in central nervous system tumors is a rare event and mostly likely associated with amplification of the gene.

Antigenic and differentiative heterogeneity among human glioblastomas

Increasing evidence suggests that in mammals, astrocytes are a heterogenous family of cells all of which share certain properties, but differ in lineage, biochemical and functional aspects. It seems likely that glioblastomas, arising from glial precursors, may also represent a family of related but distinct cell types. We have examined the antigenic characteristics and differentiative potential of 7 different human glioblastoma cell lines in vitro. All the cell lines were labeled with a monoclonal antibody 7B11 which labels all classes of astrocytes and their precursors in the rat CNS. U138MG and Tm3 cells expressed antigens on their surfaces recognized by the monoclonal antibodies A2B5 and HNK-1. When grown in serum-free medium in the presence of cAMP and theophylline, U138MG cells assumed a process-bearing morphology and some cells expressed the Gal-C antigen specific for oligodendrocytes. Under identical conditions, Tm3 cells converted to process-bearing cells, some of which expressed glial fibrillary acidic protein (GFAP) specific for astrocytes. Other cell lines with similar antigenic characteristics did not respond similarly to cAMP and theophylline. Finally, A2781 cells were GFAP immunoreactive and unlabeled by either A2B5 or HNK-1 antibodies. These observations suggest that individual glioblastoma cell lines may be derived from distinct glial precursor cells in the vertebrate CNS.

Studies with the RNA polymerase I. Factors affecting the binding of nucleic acid polymers to the enzyme

The association of RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) with DNA or RNA as well as some of the factors which affect the enzyme-polymer complex have been examined. 1. 1. The 13-S form of the enzyme was bound to transfer RNA (tRNA). Binding of slightly more than 1 tRNA per enzyme molecule was observed. 2. 2. An order of displacement for nucleic acid polymers was established by experiments which demonstrated the displacement of one polymer from the enzyme by a second polymer. The order was: single-stranded sonicated DNA ≧ double-stranded sonicated DNA > single-stranded DNA > tRNA > native DNA. 3. 3. Ionic strength affected the association of enzyme and polymer. Association was greatest at low ionic strength; in centrifugation experiments, (NH4)2SO4 at 0.066 M caused a 90% dissociation of the enzyme-polymer complex. In kinetic experiments with varying DNA concentrations, (NH4)2SO4 produced noncompetitive inhibition. 4. 4. Divalent cations decreased association. In the absence of divalent cations and in the presence of EDTA, 20–25% more RNA or DNA was bound to the enzyme than in the presence of divalent cations. 5. 5. Nucleoside triphosphates at elevated concentrations produced dissociation of the RNA-enzyme complex. The order for the displacement effect was ATP > GTP > UTP > CTP. Nucleoside triphosphates also produced either dissociation or aggregation of the DNA-enzyme complex. The order for this effect was GTP > ATP > CTP > UTP. A divalent cation was required for this nucleotide activity with both RNA and DNA. 6. 6. Nucleoside triphosphates at elevated concentrations also inactivated the enzyme. The order of activity for this effect was GTP > ATP > CTP > UTP. 7. 7. The effects of the nucleotides lead to a prediction that a site exists on the RNA polymerase which interacts preferentially with the purine nucleotides. It is suggested that this may be the binding site for the 5′-terminal nucleoside triphosphate.

Transcriptional patterns of growth factors and proto-oncogenes in human glioblastomas and normal glial cells.

To document over-expression of proto-oncogenes in tumors, it is necessary to determine the level of expression in the progenitor normal tissue. These studies compare the levels of nuclear transcription of a series of growth-factor related genes and proto-oncogenes in human glioblastoma cell lines with those in three normal glial cell populations. The unusual finding was that levels in the three normal glial cell populations varied considerably for several genes and thus overexpression of a specific gene in a tumor cell when compared to just one normal glial cell population would not necessarily represent overexpression. In this study, we compared the level of 17 genes in 7 tumors to the highest level of each gene found in any of three normal glial cell populations. Over-expression of PDGF-B in 4/7 glioblastoma cell lines, EGFR in 1/7, neu in 1/7 IGF-2 in 1/7 and ros in 2/7 was observed. The variation observed in the normal glial cell populations emphasizes the possibility that the normal glial cell populations represent different glial cell lineages and/or stages of differentiation and that the tumors could have arisen from different normal glial cells. Matching lineages of normal and tumor cells, probably by monoclonal antibody reactions, may be required to accurately define over-expression.