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R L Garcea

Publications and source records attributed to R L Garcea.

49 records · Page 3Linked to original sources
PubMed1986

Phosphatidylinositol metabolism and polyoma-mediated transformation.

The effect of polyoma middle-sized tumor antigen (MTAg) on phosphatidylinositol metabolism has been characterized in vivo and in vitro using polyoma-transformed and polyoma-infected cells. Cells infected with transformation-competent polyoma virus exhibit increased levels of inositol phospholipids and the second messenger inositol trisphosphate. MTAg or pp60c-src immunoprecipitates from MTAg-transformed cells contain an activity that phosphorylates phosphatidylinositol and phosphatidylinositol 4-phosphate. This activity is induced in parallel with MTAg when the MTAg synthesis is regulated by hormonal or heavy metal inducers. Immunoprecipitates from one class of polyoma mutants defective in transformation have a reduced level of associated phosphatidylinositol kinase activity in vitro yet are capable of tyrosine phosphorylation on exogenous protein substrates at rates comparable to wild-type virus. Thus, for these mutants, phosphatidylinositol kinase activity is more tightly correlated with transformation than is protein kinase activity. These results suggest that alterations in phosphatidylinositol metabolism by MTAg play a role in transformation by polyoma virus.

Animals↗
PubMed1985

Polyoma virus major capsid protein, VP1. Purification after high level expression in Escherichia coli.

We have expression-cloned in Escherichia coli the major polyoma virus capsid protein, VP1. Under the inducible control of the hybrid tac promoter, VP1 constituted between 2 and 3% of the total host cell protein. The expressed VP1 was purified to near homogeneity with initial yields to 10%. Optimal expression was temperature-dependent, and significant intracellular degradation could be demonstrated. The final product was obtained as one predominant isoelectric focusing species, without the pattern of post-translational modification seen in virus-infected eukaryotic cells. The purified VP1 from E. coli will be useful as a substrate for the purification of VP1 modification enzymes and in the study of inter-VP1 oligomerization.

Cloning, Molecular↗
PubMed1985

Virion assembly defect of polyomavirus hr-t mutants: underphosphorylation of major capsid protein VP1 before viral DNA encapsidation.

The major capsid protein of polyomavirus, VP1, was separated into at least four subspecies by isoelectric focusing. One of these subspecies was selectively extracted from purified virions by mild treatment with sodium dodecyl sulfate, leaving a 140S particle enriched in the other three forms. The two most acidic subspecies were labeled in vivo with [32P]phosphate, and these subspecies are among those identified as being deficient in nontransforming host range (hr-t) mutant virus nonpermissive infection of NIH3T3 cells. Quantitation of VP1 phosphorylation revealed that hr-t mutant virus VP1 is phosphorylated to about 40 to 50% the level of the wild type in NIH3T3 cells, and two-dimensional phosphoamino acid analysis suggested that threonine phosphorylation was affected more than serine phosphorylation. Two results indicate that the VP1 modifications occur before and independent of virus assembly: modified subspecies were detected during wild-type infection within a 2-min pulse-label with [32S]methionine, and VP1 modifications of temperature-sensitive VP1 mutants were the same at both restrictive and permissive temperatures for virus assembly. We conclude that most VP1 modification occurs before viral DNA encapsidation, and that one defect in hr-t mutant virus assembly is in VP1 phosphorylation, primarily affecting threonine.

DNA, Viral↗
PubMed1983

Isolation and characterization of polyoma nucleoprotein complexes.

A method for the isolation of polyoma nucleoprotein complexes has been developed using neuraminidase treatment of infected cell lysates. At least three distinct forms of polyoma virion intermediates were identified by their [3H]thymidine labeling kinetics and sedimentation coefficients: a rapidly labeled 95 S "replicating complex" which chases to a 75 S minichromosome and then to a 240 S virion structure. The general properties of these distinct intermediates were similar to those found for SV40. In contrast to SV40, however, a continuum of labeled polyoma viral DNA sedimented between 240 S and 95 S. These complexes were characterized by their release from cell debris with neuraminidase, precipitation with antivirion antibody, complete disruption in 1 M NaCl, and association with hemagglutinating (HA) activity. These intermediates may represent incremental capsid protein additions to the 75 S minichromosome, hypothesized in the current models for SV40 assembly. The ability to isolate a complete complement of polyoma subviral complexes provides a basis for studying the growth defect of polyoma host-range mutants, and the properties of neuraminidase release, hemagglutination, and specific immunoprecipitation suggest purification steps for further characterization of these virion assembly intermediates.

Animals↗
PubMed1983

Host range transforming gene of polyoma virus plays a role in virus assembly.

Polyoma virus host range transforming (hr-t) mutants are blocked in virion assembly. In normal 3T3 cells, a nonpermissive host, these mutants synthesize 30-40% as much viral DNA and 80-100% as much capsid proteins as does wild-type virus and yet produce only 1-2% as much infectious virus. Intermediates in virion assembly have been followed by [3H]thymidine incorporation. hr-t mutants synthesize 95S replicating minichromosomes, which accumulate as 75S forms. However, the latter fail to undergo efficient transition to 240S virion structures. This block in encapsidation is overcome in permissive hosts such as primary baby mouse kidney (BMK) epithelial cells. The block in assembly of 240S particles is accompanied by a failure to induce a series of acidic isoelectric forms of the major capsid protein, VP1. Multiple species of post-translationally modified VP1 are seen by two-dimensional gel electrophoresis in wild-type virus-infected cells. These acidic VP1 subspecies are decreased 6- to 10-fold in hr-t mutant-infected 3T3 cells but are produced in normal amounts when the same mutants infect BMK cells. When 3T3 cells are coinfected with hr-t mutant and wild-type viruses, normal amounts of the VP1 subspecies are present, and hr-t mutant viral DNA is efficiently packaged into virions. These studies demonstrate an important role of the hr-t gene of polyoma virus in virus assembly. Specifically, we propose that VP1 is a target for hr-t gene-controlled modification and that modified forms of VP1 are essential for encapsidation of viral minichromosomes.

Animals↗
PubMed1980

Comparative studies of histone acetylation in nucleosomes, nuclei, and intact cells. Evidence for special factors which modify acetylase action.

We have studied the pattern of histone acetylation in intact rat hepatoma tissue culture (HTC) cells, in isolated HTC nuclei, and in chromatin prepared from these cells. The results have been compared with the histone acetylation observed in a reconstituted in vitro system consisting of a variety of purified soluble nucleosomal substrates, [3H]acetyl-CoA, and one of two different purified histone N-acetyltransferases. Acetylase A, a highly purified nuclear enzyme, catalyzed the acetylation of 1) nucleosomally bound histones in the order H4 > H2a = H2b > H3, and 2) free histones in the order H4 > H3 > H2b > H2a. Acetylase B, a cytoplasmic enzyme, modified only free histone H4, and it failed to acetylate histones in nucleosomes. The pattern of histone acetylation obtained by in vitro reaction of purified nucleosomes with the purified nuclear acetylase A differed considerably from the corresponding patterns obtained either by acetate labeling of intact cells, or by the acetyl-CoA labeling of nuclei and crude preparations of nucleosomes, as catalyzed by endogenous chromatin-bound acetylase(s). The most striking difference was in the relative preference for acetylation of histone H4 versus acetylation of histone H3: with the purified acetylase, histone H4 in nucleosomes was acetylated to a much greater extent than was histone H3, whereas the reverse preference was found with the endogenous acetylase(s). This result suggests that either a second nuclear acetylase enzyme, or a separate cofactor for acetylase A, is required for histone H3 acetylation in vivo. In support of this view, we find that the acetylation of histones H4, H2a, and H2b in nuclei is inhibited by urea, salt, or N-ethylmaleimide treatments to a very different extent than is the acetylation of histone H3. By comparing n-butyrate-treated HTC cells with untreated cells, classes of nucleosomes specially accessible and inaccessible to acetylation can be distinguished (Cousens, L. S., Gallwitz, D., and Alberts, B. M. (1979) J. Biol. Chem. 254, 1716-1723). Both types of special nucleosomal reactivities were present in isolated nuclei, but were lost as nucleosomes were purified from these cells. OUr data thus suggest the existence of labile specificity factors or structures, which guide the acetylase(s) to restricted groups of otherwise similar nucleosomes in vivo.

Acetylation↗
PubMed1980

Transcription and in vitro processing of yeast 5 S rRNA.

A method is described for the isolation of a yeast chromatin fraction highly enriched in ribosomal DNA sequences. In the presence of exogenous yeast RNA polymerase III, this purified chromatin actively synthesizes a set of 5 S ribosomal RNAs all of which have 5'-sequences identical with mature 5 S RNA but which end with a variable number (up to 10) of additional residues at the 3'-terminus. These extra nucleotides are precisely removed by a processing nuclease found in the chromatin supernatant fraction.

Base Sequence↗
PubMed1978

Muscle development in Caenorhabditis elegans: mutants exhibiting retarded sarcomere construction.

We have studied the structural changes within the body-wall muscle cells of Caenorhabditis elegans during postmitotic development. In wildtype, the number of sarcomeres progressively increases, and each sarcomere appears to grow in length and depth continuously during this period. In mature wild-type cells, the anterior-most body-wall muscle cells have 6--7 sarcomeres; the rest have 9--10 sarcomeres per cell. Twelve mutants in the unc-52 II gene exhibit markedly retarded sarcomere construction and progressive paralysis. Several unc-52 mutants, such as the severely paralyzed SU200, produced only 2--3 sarcomeres per body-wall muscle cell, while the other mildly paralyzed unc-52 mutants, such as SU250, build 3--4 sarcomeres per muscle cell. Other structures such as the pharynx and even the noncontractile organelles of the body-wall muscle cells do not appear to be structurally or functionally altered. The unc-52 body-wall sarcomeres become moderately disorganized as they are outstripped by cell growth; sufficient order is preserved, however, so that the majority of thick and thin filaments still interdigitate. The myosin heavy chains of SU200 body-wall muscle fail to accumulate normally, while the pharyngeal myosin heavy chains do not appear to be specifically affected. This biochemical result correlates well with the specificity of morphological changes in the mutant. A model is discussed in which the biochemical and morphological deficits are explained by a simple regulatory mechanism.

Animals↗
PubMed1976

Interaction of glucocorticoid receptor-steroid complexes with acceptor sites.

The binding of the "activated" receptor-glucocorticoid complexes of cultured rat hepatoma cells to nuclei, chromatin, and DNA has been studied under cell-free conditions. A critical factor in determining the shape of the binding curve is shown to be an inhibitory material which is present in crude cytosol and which can be removed without destroying the receptor-steroid complex. These and other results argue that the apparent saturation observed in earlier experiments may have been due to the inhibitors. Thus, the actual number of acceptor sites in hepatoma tissue culture cell nuclei is much larger than previously estimated and their affinity for the complex is lower. Nuclear binding experiments indicate that the inhibitory material interacts with the receptor-steroid complex. The inhibitors appear to be macromolecular; but their effects cannot be mimicked by albumin or hemoglobin. The acceptor capacity at low ionic strength for binding receptor-glucocorticoid complexes increases when proceeding from nuclei to DNA. An analysis of the kinetics of association and dissociation and of the relative binding behavior of nuclei and DNA argues that the affinity of complex for nuclei is much greater than for DNA. DNA-associated histones reduce the amount of complex that binds to DNA. These and perhaps other chromosomal proteins may be responsible for the ordering of acceptor capacity. Evidence is presented that the difference in affinities of nuclear and DNA acceptors could also be due to chromosomal proteins. In nuclei, these proteins may thus both reduce the amount of complex binding by rendering regions of DNA less accessible and increase the binding affinity of some, or all, of those DNA binding sites which remain exposed.

Binding Sites↗
PubMed1972

Role of DNA and specific cytoplasmic receptors in glucocorticoid action.

Glucocorticoids induce tyrosine aminotransferase (EC 2.6.1.5) synthesis in cultured rat hepatoma cells. These steroids penetrate the cell membrane and bind to specific cytoplasmic receptor proteins. The resulting complex binds to the nucleus. This nuclear binding has now been studied in a cell-free preparation. The reaction appears to require a temperature-dependent modification of the steroid-receptor complex. There is a fixed number of nuclear sites that are half saturated at a complex concentration of 6 to 24 x 10(-11) M. Treatment with deoxyribonuclease destroys nuclear-binding capacity. The complex also binds to purified HTC cell DNA with characteristics similar to the binding to isolated nuclei, and, as in intact cells, receptors complexed with an anti-inducer steroid bind very poorly to DNA. These data suggest that the nuclear sites for binding steroid-receptor complexes are on the DNA. Since the extent of complex binding to purified DNA exceeds that observed with isolated nuclei, chromosomal proteins may act to restrict binding to certain regions of the DNA. These studies suggest that steroid hormones stimulate the synthesis of specific proteins by affecting the transcription of structural or regulatory genes.

Binding Sites↗
PubMedDate not supplied

Site-directed mutation affecting polyomavirus capsid self-assembly in vitro.

Nonequivalent bonding of identical protein subunits occurs in the polyomavirus capsid were identical pentameric capsomeres occupy both hexavalent and pentavalent positions in the icosahedral surface lattice. The polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in Escherichia coli, has been isolated as capsomeres that self-assemble into capsid-like structures in vitro. The ability to switch bonding specificity in different symmetry environments therefore must be intrinsic to the VP1 molecule. In vitro self-assembly provides an assay for VP1 mutations affecting capsomere and capsid formation. We report here that a directed mutation in the VP1 expression vector, leading to a protein truncated at the carboxy terminus, results in a mutant VP1 that forms capsomeres, but not capsids, in the in vitro assembly assay. The carboxy terminus of VP1 therefore appears to be involved in the specific bonding responsible for the non-equivalent association of capsomeres.

Amino Acid Sequence↗