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Biomedical subjects

C C Kao

Publications and source records attributed to C C Kao.

At least 73 records · Page 4Linked to original sources
PubMed1996

Initiation of (-)-strand RNA synthesis catalyzed by the BMV RNA-dependent RNA polymerase: synthesis of oligonucleotides.

RNA replication, a process of fundamental importance for pathogenesis by many viruses, remains poorly understood at the mechanistic level because relatively few of the responsible enzymes have been purified and characterized biochemically. Partially purified RNA-dependent RNA polymerase (RdRp) from brome mosaic virus (BMV)-infected barley leaves is able to synthesize (-)-strand RNAs from input (+)-strand templates. In resolving RdRp products generated during (-)-strand BMV RNA synthesis, we found that an RNA of eight nucleotides was generated at approximately 10-fold molar excess to the full-length (-)-strand RNA. Production of the 8-mer was dependent upon and specific to BMV RNA templates. Furthermore, inhibitors of full-length (-)-strand RNA synthesis by RdRp also affected the production of the 8-mer. Analysis of the sequence of the 8-mer suggests that it is initiated at the authentic initiation site on the BMV RNA template, consistent with the possibility that the 8-mer is an abortive initiation product of RNA synthesis by RdRp in vitro. Addition of heparin or Mn2+ differentially affected production of the 8-mer and full-length (-)-strand RNA, as did some nucleotide changes near the site of RNA initiation. These studies further define the initiation process of (-)-strand RNA synthesis.

Bromovirus↗
PubMed1996

Initiation of minus-strand RNA synthesis by the brome mosaicvirus RNA-dependent RNA polymerase: use of oligoribonucleotide primers.

Various DNA- and RNA-dependent RNA polymerases have been reported to use oligoribonucleotide primers to initiate nucleic acid synthesis. For the brome mosaic virus RNA-dependent RNA polymerase (RdRp), we determined that in reactions performed with limited GTP concentrations, minus-strand RNA synthesis can be stimulated by the inclusion of guanosine monophosphate or specific oligoribonucleotides. Furthermore, guanylyl-3',5'-guanosine (GpG) was incorporated into minus-strand RNA and increased the rate of minus-strand RNA synthesis. In the presence of GpG, RdRp's Km for GTP decreased from 50 microM to approximately 3 microM while the Kms for other nucleotides were unaffected. These results have implications for the mechanism of initiation by RdRp.

Bromovirus↗
PubMed1995

Biochemical and genetic analyses of the interaction between the helicase-like and polymerase-like proteins of the brome mosaic virus.

Replication of the three positive-strand genomic RNAs of brome mosaic virus requires the activities of the helicase-like 1a and the polymerase-like 2a proteins. One hundred fifteen amino acids of the 2a N-terminus and the 1a helicase-like region of over 50 kDa are both necessary and sufficient for 1a-2a interaction. Requirement of the large size of the 1a helicase-like domain suggests that higher order structures might be necessary for the protein's interaction with 2a. To explore the structural properties of 1a, we used limited proteolysis of in vitro-translated 1a protein. Treatment of 1a and its deletion derivatives with papain or trypsin revealed that the C-terminal helicase-like segment of approximately 50-60 kDa is highly resistant under our assay conditions to proteolysis, while the N-terminus is rapidly degraded. All tested mutations in the helicase-like region that renders this region protease-sensitive have previously been found to be defective for RNA replication in vivo. To complement the in vitro studies, we examined the interaction of the 1a helicase-like domain and the 2a N-terminus in yeast using the two-hybrid system. Mutations previously known to disrupt 1a-2a interaction also prevented interaction in yeast. Furthermore, results from two-hybrid analysis suggest that the structural domain mapped in vitro is important for 1a-2a interaction. Finally, we found that the helicase-like proteins of three other tripartite RNA viruses also contain equivalently located protease-resistant domains.

Amino Acid Sequence↗
PubMed1995

Disruption of reproductive rhythms and patterns of melatonin and prolactin secretion following bilateral lesions of the suprachiasmatic nuclei in the ewe.

To determine whether the photoperiodic responses of reproductive and prolactin (PRL) rhythms in the ewe requires an intact suprachiasmatic nucleus (SCN) driving the pineal rhythm of melatonin secretion, four groups of ovary-intact ewes over a 6-year period were subjected to bilateral (n = 40) or sham lesions (n = 15) of the SCN. Animals were exposed to an alternating 90-120 day photoregimen of 9L:15D and 16L:8D photoperiods. Blood samples taken twice weekly were assayed for prolactin and for progesterone to monitor oestrous cycles. On several occasions blood samples also were taken at hourly intervals for 24 h and analyzed for melatonin. Melatonin concentrations in sham lesioned ewes were basal during the lights-on period and rose robustly during darkness. Those sheep bearing unilateral lesions of the SCN (n = 13) or where the lesion spared the SCN entirely (n = 8) had patterns of melatonin secretion similar to sham ewes. The remaining ewes, having complete (n = 9) or incomplete bilateral (n = 8) destruction of the SCN, with one exception, had disrupted patterns of melatonin secretion. The nature of this disruption varied from complete suppression to continuously elevated levels. In lesioned ewes where melatonin secretion was not affected the onset and cessation of ovarian cycles were similar to sham ewes; stimulation of oestrous cycles under 9L:15D and cessation of oestrous cycles under 16L:8D. In contrast, 13 of 17 ewes with disrupted melatonin secretion also exhibited disrupted patterns of ovarian activity. In these animals oestrous cycles were no longer entrained by photoperiod but still occurred in distinct clusters, that is, groups of cycles began and ended spontaneously. Sheep with normal melatonin patterns showed low levels of PRL secretion during short days and elevated PRL levels during long days. However, 8 of 13 ewes with disrupted melatonin showed patterns of PRL secretion that were no longer entrained by photoperiod. A minority of ewes with disrupted melatonin patterns still showed reproductive (n = 4) and PRL (n = 5) responses similar to those of sham-lesioned ewes. These results show that bilateral destruction of the SCN in the ewe disrupts the circadian pattern of melatonin secretion and that this disruption usually, but not always, is associated with altered photoperiodic responses. These results strongly suggest that the SCN are important neural elements within the photoperiod time-keeping system in this species. A role for the SCN in the generation of endogenous transitions in reproductive activity (refractoriness) and prolactin secretion is not supported.

Animals↗
PubMed1995

Techniques in the composite reconstruction of extensive thoracoabdominal tumor resections.

BACKGROUND: This report illustrates the unique problems of reconstructing thoracoabdominal wall defects coexisting with diaphragmatic defects. STUDY DESIGN: Two patients with extensive primary chest wall tumors (chondrosarcoma and desmoid tumor) underwent aggressive resection of the hemithorax, hemidiaphragm, and anterior chest wall for cure. The combined chest and abdominal wall defect was greater than 625 cm2 in both cases. RESULTS: Functional restoration of these massive thoracoabdominal defects was accomplished by use of polypropylene-methyl methacrylate prostheses and vascularized tissue coverage. The requirements for rigidity, protection, and esthetic contouring of the chest wall are satisfied by this reconstruction. This technique also offers the flexibility and durability that the abdominal wall requires. Repair of the diaphragm using this technique is secure and simple. CONCLUSIONS: Our report confirms the principle that, with modern thoracic and plastic operative techniques, the extent of tumor resection should not be compromised because of concern over the ability to reconstruct the defect.

Abdominal Neoplasms↗
PubMed1994

Protein-protein interactions and glycerophospholipids in bromovirus and nodavirus RNA replication.

The plant bromoviruses and animal nodaviruses are distinct groups of positive strand RNA viruses that have proven to be useful models for RNA replication studies. Bromoviruses encode two large proteins required for RNA replication: 1a contains domains implicated in helicase and capping functions, and 2a contains a central polymerase-like domain. Using immunoprecipitation and far-western blotting, we have now shown that 1a and 2a form a specific complex in vitro and have mapped the interacting domains. Molecular genetic data implicate the 1a-2a complex in RNA replication and suggest that it supports coordinate action of the putative helicase, polymerase, and capping domains. The locations of the interacting 1a and 2a domains have implications for replication models and the evolution of virus genomes bearing homologous replication genes in fused vs. divided forms. For the nodavirus Flock house virus (FHV), a true RNA replicase has been isolated that carries out complete, highly active replication of added FHV RNA, producing newly synthesized positive strand RNA in predominantly ssRNA form. Positive strand RNA synthesis in this FHV cell-free system is strongly dependent on the addition of any of several glycerophospholipids. Positive strand RNA synthesis depends on the complete glycerophospholipid structure, including the polar head group and diacyl glycerol lipid portion, and is strongly influenced by acyl chain length.

Bromovirus↗
PubMed1994

Antithrombin III enhances the motility and chemotaxis of boar sperm.

Purified porcine antithrombin III stimulates motility and acts as a chemoattractant of washed boar sperm. Porcine follicular fluid and blood plasma both possess chemotactic activity. Porcine antithrombin III-immunoreactive molecules are distributed on the zona pellucida of ovulated eggs flushed from the swine oviducts. We propose that antithrombin III plays important roles in the mammalian fertilization process in vivo.

Animals↗
PubMed1993

Characterization of a host protein associated with brome mosaic virus RNA-dependent RNA polymerase.

The association of host proteins with viral RNA replication proteins has been reported for a number of (+)-strand RNA viruses. However, little is known about the identity or function of these host proteins in viral replication. In this paper we report the characterization of a host protein associated with the RNA-dependent RNA polymerase (RdRp) from brome mosaic virus (BMV)-infected barley. A host protein was specifically and proportionally enriched with BMV RdRp activity through several purification steps. This RdRp-associated host protein reacted with an antiserum prepared against wheat germ eukaryotic translation initiation factor 3 (eIF-3). The RdRp-associated host protein, the p41 subunit of wheat germ eIF-3, and an antigenically related protein from rabbit reticulocyte lysates were all found to bind with high affinity and specificity to BMV-encoded protein 2a, which is involved in viral RNA replication. Moreover, addition of wheat germ eIF-3 or the p41 subunit from wheat germ to BMV RdRp gave a specific and reproducible 3-fold stimulation of (-)-strand RNA synthesis in vivo. These results suggest that the barley analog of eIF-3 subunit p41, or a closely related protein, associates with BMV RdRp in vivo and is involved in BMV RNA replication. This observation and the established role of translation factors in bacteriophage Q beta RdRp suggest that association with translation factors may be a general feature of RNA replication by (+)-strand RNA viruses.

Animals↗
PubMed1992

Purification of a sperm motility stimulator from porcine follicular fluid.

1. We purified a glycoprotein possessing a potent ability to stimulate porcine sperm motility, from porcine ovarian follicular fluid. 2. The protein, of molecular weight 52,000, has an N-terminal sequence similar to that of antithrombin III, indicating that it is identical to or highly homologous to antithrombin III. 3. Thus it is proposed that antithrombin III or its homologue is involved in the mammalian fertilization process.

Amino Acid Sequence↗
PubMed1992

Extracellular polysaccharide is required for wild-type virulence of Pseudomonas solanacearum.

Several Pseudomonas solanacearum strains which produced no detectable extracellular polysaccharide (EPS) in planta had been reported to remain highly virulent when tested at high inoculum concentrations (P. Xu, M. Iwata, S. Leong, and L. Sequeira, J. Bacteriol. 172:3946-3951, 1990; P. Xu, S. Leong, and L. Sequeira, J. Bacteriol. 170:617-622, 1988). Two of these mutants, KD700 and KD710, have now been molecularly and genetically mapped to the EPSI gene cluster described by Denny and Baek (Mol. Plant-Microbe Interact. 4:198-206, 1991). When a range of inoculum concentrations was used, these two mutants and all other EPS-defective mutants tested were found to be reduced in virulence to eggplants and tobacco relative to the wild-type strain. Thus, EPS consistently is required for the wild-type level of virulence in P. solanacearum.

Mutation↗
PubMed1992

Brome mosaic virus RNA replication proteins 1a and 2a from a complex in vitro.

Brome mosaic virus (BMV) is a positive-strand RNA virus that encodes two RNA replication proteins, the helicaselike 1a and the polymeraselike 2a. 1a and 2a share extensive sequence similarities with proteins encoded by many other members of the alphaviruslike superfamily. While further purifying enzymatically active RNA-dependent RNA polymerase from plants infected by BMV, we observed that 1a, 2a, and the polymerase activity all cofractionated through multiple independent purification steps. Moreover, using immunoprecipitation, we found that BMV 1a and 2a proteins synthesized in rabbit reticulocyte lysates or insect cells can form a specific complex in vitro. Complex formation was more efficient when 1a and 2a were cotranslated than when they were mixed after independent synthesis. In an antibody-independent assay, in vitro-translated 1a protein was also found to bind to 2a protein fixed on a nylon membrane. A three-amino-acid insertion in 1a that blocks BMV RNA replication in vivo also blocked in vitro interaction with 2a, while another two-amino-acid insertion that renders the 1a protein temperature sensitive for RNA replication interacted in vitro with 2a at 24 degrees C but not at 32 degrees C. These results and previous genetic data suggest that the 1a-2a interaction observed in vitro is required for BMV RNA replication and may have direct implications for other members of the alphaviruslike superfamily.

Amino Acid Sequence↗
PubMed1992

Identification of the domains required for direct interaction of the helicase-like and polymerase-like RNA replication proteins of brome mosaic virus.

Brome mosaic virus is a positive-strand RNA virus whose RNA replication requires viral protein 1a, which has putative helicase and capping functions, and 2a, which has putative polymerase function. Since domains of related sequence are conserved in a wide range of plus-strand RNA viruses, analysis of 1a and 2a function should have applicability to many other viruses. We have recently demonstrated that 1a and 2a form a complex in vivo and in vitro. Using immune coprecipitation and mutant polypeptides made in reticulocyte lysates, we have now mapped both the 1a and 2a domains necessary for complex formation. The sequences needed to bind 2a map to the carboxy-terminal helicase-like domain of 1a. Truncated polypeptides containing this domain were able to bind to 2a, while several small insertions in the helicase-like domain disrupted binding. The sequence required for binding 1a lies within a 115-residue subset of the 2a N-terminal segment preceding the polymerase-like domain. Truncations or fusion polypeptides containing this segment can bind 1a. We also determined that highly purified 2a protein made in insect cells can form a complex with highly purified 1a helicase-like domain made in Escherichia coli, suggesting that no other factor is required to mediate 1a-2a interaction. Previous genetic analyses of 1a and 2a are consistent with this mapping and show that the newly defined 1a and 2a binding regions are required for RNA synthesis. The locations of these interacting regions are discussed with regard to models of viral replication and the evolution of positive-strand RNA virus genomes.

Base Sequence↗
PubMed1991

Adenovirus E1A activation domain binds the basic repeat in the TATA box transcription factor.

The adenovirus large E1A protein is a potent activator of transcription. We use several different experimental approaches to demonstrate that the large E1A protein binds specifically and stably to the TATA box-binding factor (TFIID), the general polymerase II transcription factor that initiates assembly of transcription complexes. Sedimentation velocity centrifugation revealed that TFIID and E1A form a heterodimer in vitro. We demonstrate that the activation domain of E1A (conserved region 3) binds to TFIID. E1A interacts with a 51 residue region from the conserved C-terminal domain of TFIID that includes a repeat of basic residues between the homologous direct repeats of TFIID. Analysis of TFIID binding by various E1A mutants indicates that TFIID binding is necessary, although not sufficient, for E1A transactivation.

Adenoviridae↗
PubMed1991

A gene cluster required for coordinated biosynthesis of lipopolysaccharide and extracellular polysaccharide also affects virulence of Pseudomonas solanacearum.

Bacterial cell surface components can be important determinants of virulence. At least three gene clusters important for extracellular polysaccharide (EPS) biosynthesis have been previously identified in the plant pathogen Pseudomonas solanacearum. We have found that one of these gene clusters, named ops, is also required for lipopolysaccharide (LPS) biosynthesis. Mutations in any complementation unit of this cluster decreased EPS production, prevented the binding of an LPS-specific phage, and altered the mobility of purified LPS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, restoration of LPS biosynthesis alone was not sufficient to restore virulence to the wild-type level, suggesting that EPS is important for pathogenesis.

Bacteriophages↗
PubMed1991

Two distinct domains in the yeast transcription factor IID and evidence for a TATA box-induced conformational change.

Transcription factor IID from Saccharomyces cerevisiae (YIID) binds the TATA box element present in most RNA polymerase II promoters. In this work, partial proteolysis was used as a biochemical probe of YIID structure. YIID consists of a protease-sensitive amino terminus and a highly stable, protease-resistant carboxy-terminal core. The cleavage sites of the predominant chymotrypsin- and trypsin-derived fragments were mapped to amino acid residues 40 to 41 and 48 to 49, respectively, by amino-terminal peptide sequencing. Removal of the amino terminus resulted in a dramatic increase in the ability of YIID to form a stable complex with DNA during gel electrophoresis mobility shift assays and a two- to fourfold increase in DNA-binding affinity, as assayed by DNase I footprinting analysis. The carboxy-terminal 190-amino-acid core was competent for transcription in vitro and was similar in activity to native YIID. DNA containing a TATA element induced hypersensitive sites in the amino-terminal domain and stabilized the core domain to further proteolytic attack. Native YIID did not bind to a TATA box at 0 degrees C, whereas the carboxy-terminal DNA-binding domain did. These results suggest that YIID undergoes a conformational change upon binding to a TATA box. Southern blotting showed that the carboxy-terminal domain is highly conserved, while the amino-terminal domain diverged rapidly in evolution, even between closely related budding yeasts.

Binding Sites↗
PubMed1990

Cloning of a transcriptionally active human TATA binding factor.

Transcription factor IID (TFIID) binds to the TATA box promoter element and regulates the expression of most eukaryotic genes transcribed by RNA polymerase II. Complementary DNA (cDNA) encoding a human TFIID protein has been cloned. The human TFIID polypeptide has 339 amino acids and a molecular size of 37,745 daltons. The carboxyl-terminal 181 amino acids of the human TFIID protein shares 80% identity with the TFIID protein from Saccharomyces cerevisiae. The amino terminus contains an unusual repeat of 38 consecutive glutamine residues and an X-Thr-Pro repeat. Expression of DNA in reticulocyte lysates or in Escherichia coli yielded a protein that was competent for both DNA binding and transcription activation.

Amino Acid Sequence↗
PubMed1990

Localization of an origin of replication in Corynebacterium diphtheriae broad host range plasmid pNG2 that also functions in Escherichia coli.

Subcloning and protoplast transformation studies identified a 2.6 kb fragment of Corynebacterium diphtheriae plasmid pNG2 which contains an origin of replication (oriR). Molecular combination of the 2.6 kb oriR cartridge with Escherichia coli plasmid pUC18CmR enabled the E. coli cloning vector to replicate within several species of Corynebacterium host cells. A 2.6 kb plasmid formed from the oriR cartridge alone is capable of replicating in E. coli. This suggests that a single origin could be used in vectors shuttling between Corynebacterium spp. and E. coli.

Cloning, Molecular↗