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T C Wong

Publications and source records attributed to T C Wong.

At least 55 records · Page 3Linked to original sources
PubMed1991

Generation of mammalian cells expressing stably measles virus proteins via bicistronic RNA.

The proteins of measles virus are believed to be cytotoxic, and have never been expressed stably from the cloned genes in cultured cells. We found that measles viral proteins can be expressed via a bicistronic RNA. The dominantly selectable DHFR* protein-coding region encoding a mutant dihydrofolate reductase was inserted into the 3'-untranslated regions of the measles viral genes encoding nucleoprotein (N), matrix (M) protein, and hemagglutinin (H). The tandemly arranged cistrons were placed under control by the inducible promoter of human metallothionein IIA gene, or the noninducible early promoter of simian virus 40. Upon transfecting into mammalian cells, these gene constructs synthesized bicistronic RNAs. The downstream DHFR* gene conferred resistance to methotrexate (MTX). Cells that survived MTX selection expressed stably the N, M, or H protein of measles virus. Expression of N protein was further inducible by cadmium chloride treatment. This system will be useful for studying the protein functions of measles virus, and could be applied to express other potentially toxic gene products.

Animals↗
PubMed1991

Role of biased hypermutation in evolution of subacute sclerosing panencephalitis virus from progenitor acute measles virus.

We identified an acute measles virus (Nagahata strain) closely related to a defective virus (Biken strain) isolated from a patient with subacute sclerosing panencephalitis (SSPE). The proteins of Nagahata strain measles virus are antigenically and electrophoretically similar to the proteins of Edmonston strain measles virus. However, the nucleotide sequence of the Nagahata matrix (M) gene is significantly different from the M genes of all the acute measles virus strains studied to date. The Nagahata M gene is strikingly similar to the M gene of Biken strain SSPE virus isolated several years later in the same locale. Eighty percent of the nucleotide differences between the Nagahata and Biken M genes are uridine-to-cytosine transitions known as biased hypermutation, which has been postulated to be caused by a cellular RNA-modifying activity. These biased mutations account for all but one of the numerous missense genetic changes predicted to cause amino acid substitutions. As a result, the Biken virus M protein loses conformation-specific epitopes that are conserved in the M proteins of Nagahata and Edmonston strain acute measles viruses. These conformation-specific epitopes are also absent in the cryptic M proteins encoded by the hypermutated M genes of two other defective SSPE viruses (Niigata and Yamagata strains). Nagahata-like sequences are found in the M genes of at least five other SSPE viruses isolated from three continents. These data indicate that Biken strain SSPE virus is derived from a progenitor closely resembling Nagahata strain acute measles virus and that biased hypermutation is largely responsible for the structural defects in the Biken virus M protein.

Animals↗
PubMed1990

Detection of Kestoses and Kestose-Related Oligosaccharides in Extracts of Festuca arundinacea, Dactylis glomerata L., and Asparagus officinalis L. Root Cultures and Invertase by C and H Nuclear Magnetic Resonance Spectroscopy.

A previous study (KL Forsythe, MS Feather [1989] Carbohydr Res 185: 315-319) showed that (13)C nuclear magnetic resonance spectroscopy can be used to detect and identify mixtures of 1-kestose and neokestose after conversion to the acetate derivatives. In this study, unequivocal assignments are made for the anomeric carbon and proton signals for the above two trisaccharide acetates as well as for 6-kestose hendecaacetate and for nystose tetradecaacetate (a 1-kestose-derived tetrasaccharide). A number of oligosaccharide fractions were isolated from several plant species, converted to the acetates, and nuclear magnetic resonance spectra obtained. Using the above reference data, the following information was obtained. The trisaccharide fraction from Dactylis glomerata L. stem tissue and Asparagus officinalis L. roots contain both 1-kestose and neokestose, and the tetrasaccharide fractions contain three components, one of which is nystose. Penta- and hexasaccharide acetates were also isolated from A. officinalis L. roots and were found to contain, respectively, four and at least five components. All components of both of the above species appear to contain a kestose residue and to be produced by the sequential addition of fructofuranosyl units to these. The trisaccharide fraction from Festuca arundinacea is complex, and contains at least five different components, two of which appear to be 1-kestose and neokestose.

Journal Article↗
PubMed1990

Leader sequence distinguishes between translatable and encapsidated measles virus RNAs.

The 3'-terminal 55 nucleotides of the negative-strand measles virus RNA genome called the leader sequence is not transcribed into a detectable distinct RNA product. Most of the monocistronic N and bicistronic N-P RNAs lack the leader sequence. However, a subpopulation of the N and N-P RNAs and all of the antigenomes possess this leader. Here, we show that leader-containing subgenomic RNAs are functionally distinct from their leaderless counterparts. In measles virus-infected cells, leaderless monocistronic N and bicistronic N-P RNAs were associated with polysomes. By contrast, leader-containing N and N-P RNAs were found exclusively in nonpolysomal ribonucleoprotein complexes that were resistant to RNase and had a buoyant density of 1.30 g/ml, the same as that of antigenomic ribonucleoprotein complexes. Both antigenomic and subgenomic ribonucleoprotein complexes were specifically immunoprecipitated by antiserum against the N protein, and leaderless RNAs were not found in these complexes. These findings suggest that measles virus distinguishes RNAs destined for encapsidation or translation by the presence or absence of a leader sequence.

Animals↗
PubMed1989

Efficient coupling of glycopeptides to proteins with a heterobifunctional reagent.

A heterobifunctional linking reagent containing a masked aldehydo group and acyl hydrazide was synthesized for coupling of glycopeptides and other amino-containing compounds to proteins. After conversion to acyl azide, the reagent reacts with the amino group of a glycopeptide, and the modified glycopeptide is deacetalized with a weak acid to unmask the aldehydo group, which is then conjugated to bovine serum albumin (BSA) by reductive alkylation with pyridine-borane. The overall reaction scheme proceeds under relatively mild conditions. When the protein amino group was in a large excess (greater than 6-fold) of the aldehyde reagent, the efficiency of conjugation was as high as 88% even at submicromole levels. As a test case for application of this reagent, 6-aminohexyl beta-D-galactopyranoside (Gal-AH) was attached to the linking reagent and conjugated to BSA at various aldehyde-to-protein molar ratios ranging from 25 to 200. The level of O-galactosyl residue incorporated into BSA by this reagent far exceeded that observed in a similar reductive alkylation involving S-galactoside reagents [Lee, R. T., & Lee, Y. C. (1980) Biochemistry 19, 156-163]. By use of the present conjugating procedure, as many as 112 mol of Gal-AH residues were incorporated per mole of BSA, which represents near total modification of the amino groups. Some binding characteristics of the new BSA derivatives were studied in the mammalian hepatic galactose/N-acetylgalactosamine specific lectin system along with other types of BSA derivatives (containing S-galactosyl residues). In general, the behavior of the new derivatives was similar to that of other types. For instance, the affinity increased exponentially at low sugar substitution levels (up to 30 mol of galactosyl residues/mol of BSA), and the slope of exponential increase and affinity at a given sugar substitution level was similar to those of other types.

Amides↗
PubMed1989

Mutations in 3'-untranslated region of avian sarcoma virus mutant LA46 genome confer the cis-acting temperature-sensitive replication defect.

Avian sarcoma virus (ASV) PRCII mutant LA46 carried a unique cis-acting temperature sensitive (ts) defect affecting virus replication. The 3'-untranslated region of the LA46 genome contained multiple base substitutions in the direct repeat region and a long insertion. Although these sequences were not present in PRCII, most of these changes were found in various other ASVs and the long insertion shared extensive homology with the so called E region in certain ASV strains. By constructing chimeric retroviruses, the ts defect was mapped to the altered 3'-noncoding region. However, site-specific mutagenization targeted towards a four-nucleotide substitution unique to LA46 failed to implicate these mutations in the ts phenotype.

Animals↗
PubMed1989

Generalized and localized biased hypermutation affecting the matrix gene of a measles virus strain that causes subacute sclerosing panencephalitis.

The matrix (M) genes of Yamagata-1 strain subacute sclerosing panencephalitis virus passaged in African green monkey kidney cells and human neuroblastoma cells displayed strikingly nonrandom sequence divergence. The genes of both substrains shared a large number of uridine (U) to cytidine (C) transitions, but the latter contained numerous additional U to C changes in a localized region. Over 90% of the additional mutations were identical to the hypermutated nucleotides in the M gene found in a measles inclusion body encephalitis case. The nonrandom nature, the apparent host dependency, and the abrupt boundaries of these mutations suggest that these mutations might be caused by an extrinsic biased mutational activity rather than intrinsic polymerase errors. This mutational activity might account for the extraordinarily high C to U ratios in the non-protein-coding regions of both the M and fusion genes of wild-type measles virus.

Base Sequence↗
PubMed1989

Structural defect linked to nonrandom mutations in the matrix gene of biken strain subacute sclerosing panencephalitis virus defined by cDNA cloning and expression of chimeric genes.

Biken strain, a nonproductive measles viruslike agent isolated from a subacute sclerosing panencephalitis (SSPE) patient, contains a posttranscriptional defect affecting matrix (M) protein. A putative M protein was translated in vitro with RNA from Biken strain-infected cells. A similar protein was detected in vivo by an antiserum against a peptide synthesized from the cloned M gene of Edmonston strain measles virus. By using a novel method, full-length cDNAs of the Biken M gene were selectively cloned. The cloned Biken M gene contained an open reading frame which encoded 8 extra carboxy-terminal amino acid residues and 20 amino acid substitutions predicted to affect both the hydrophobicity and secondary structure of the gene product. The cloned gene was expressed in vitro and in vivo into a 37,500 Mr protein electrophoretically and antigenically distinct from the M protein of Edmonston strain but identical to the M protein in Biken strain-infected cells. Chimeric M proteins synthesized in vitro and in vivo showed that the mutations in the carboxy-proximal region altered the local antigenicity and those in the amino region affected the overall protein conformation. The protein expressed from the Biken M gene was unstable in vivo. Instability was attributed to multiple mutations in both the amino and carboxy regions. A surprising number of mutations in both the coding and noncoding regions of the Biken M gene were identical to those in an independently isolated SSPE virus strain with a similar defect. These results offer insights into the basis of the defect in Biken strain and pose intriguing questions about the evolutionary origins of SSPE viruses in general.

Amino Acid Sequence↗
PubMed1989

Measles virus synthesizes both leaderless and leader-containing polyadenylated RNAs in vivo.

The minus-sense RNA genome of measles virus serves as a template for synthesizing plus-sense RNAs of genomic length (antigenomes) and subgenomic length [poly(A)+ RNAs]. To elucidate how these different species are produced in vivo, RNA synthesized from the 3'-proximal N gene was characterized by Northern RNA blot and RNase protection analyses. The results showed that measles virus produced three size classes of plus-sense N-containing RNA species corresponding to monocistronic N RNA, bicistronic NP RNA, and antigenomes. Unlike vesicular stomatitis virus, measles virus does not produce a detectable free plus-sense leader RNA. Instead, although antigenomes invariably contain a leader sequence, monocistronic and bicistronic poly(A)+ N-containing RNAs are synthesized either without or with a leader sequence. We cloned and characterized a full-length cDNA representing a product of the latter type of synthesis. mRNAs and antigenomes appeared sequentially and in parallel with leaderless and leader-containing RNAs. These various RNA species accumulated concurrently throughout infection. However, cycloheximide preferentially inhibited accumulation of antigenomes and leader-containing RNA but not leaderless and subgenomic RNAs late in infection, suggesting that synthesis of the former RNA species requires a late protein function or a continuous supply of structural proteins or both. These results reveal a previously undescribed mechanism for RNA synthesis in measles virus.

Animals↗
PubMed1987

Synthesis of D-galactosamine derivatives and binding studies using isolated rat hepatocytes.

Derivatives of glycosides of D-galactosamine were prepared in order to study further the binding requirement of the Gal/GalNAc receptor in mammalian hepatocytes. These structures included N-propanoyl, N-benzoyl, and N,N-phthaloyl derivatives of 2-hydroxyethyl 2-amino-2-deoxy-beta-D-galactopyranoside, 6-amino-hex-1-yl 2-deoxy-2-(trifluoroacetamido)-beta-D-galactopyranoside, the mono- and di-O-methyl derivatives of allyl 2-acetamido-2-deoxy-beta-D-galactopyranoside, and allyl 2-acetamido-2,4-dideoxy-4-fluoro-alpha-D-galactopyranoside. The inhibition results confirmed some of our previous findings on the involvement of the hydroxyl groups, and provided new information on the involvement of the N-substituent, as well as on the requirement of hydrogen bonding of the 4-hydroxyl group in binding.

Animals↗
PubMed1987

Scleral flap surgery for modification of corneal astigmatism.

We performed resections and recessions of scleral flaps on human cadaver eyes in order to measure the induced change in keratometric astigmatism. We prepared 3-mm scleral flaps that were 3, 5, 7, or 10 mm long and resected or recessed these flaps up to 1 mm in 0.25-mm increments. Scleral flap resection resulted in up to 10.1 D of net corneal steepening along the meridian of the incision. Scleral flap recessions resulted in up to 7.7 D of net corneal flattening along the meridian of the incision; the effect increased with increasing flap length in the recessed eyes. There was a tendency toward mean corneal flattening with resections and mean corneal steepening with recessions.

Astigmatism↗
PubMed1987

The measles virus matrix gene and gene product defined by in vitro and in vivo expression.

Sequence analysis of full-length cDNA clones of the measles virus matrix gene revealed three possible open reading frames: M, X1, and X2. The M reading frame differed from the reported sequence by a single nucleotide corresponding to a conservative lysine to arginine amino acid substitution near the carboxy-terminus conserved among the M proteins of paramyxoviruses. The putative X reading frames contained no translational termination codon due to a frame-shift mutation. The protein-coding potential of these reading frames was examined by in vitro translation and DNA-mediated gene transfer into primate cells. The M reading frame produced a 38,000 Mr protein indistinguishable from the M protein in measles virus-infected cells. This protein was not phosphorylated nor processed post-translationally in vivo. The putative X1 and X2 reading frames could be translated into proteins when placed near the 5' terminus of the RNA. The resulting proteins were heterogeneous due to the lack of a termination codon. Translation from the putative X reading frames was adversely affected by an upstream AUG codon and these reading frames were unable to synthesize proteins in their normal 3' locations. At least 146 nucleotides of these 3'-untranslated sequences could be deleted without affecting the expression of M protein in vitro or in vivo. Thus despite the multiple open reading frames, the measles virus M gene is functionally monocistronic.

Animals↗
PubMed1987

Structure and function of bicistronic RNA encoding the phosphoprotein and matrix protein of measles virus.

Two independent full-length replicas of a bicistronic RNA species containing the complete P and M genes of measles virus arranged in tandem were isolated from an expressible cDNA library. Sequences at the 5' and 3' termini suggested that the bicistronic RNA was initiated and terminated at precisely the same locations as the monocistronic mRNAs of the P and M genes, respectively. The P and M cistrons were fused together via an intergenic region which was exactly colinear with and complementary to the intergenic region of the genomic RNA. This RNA species was polyadenylated at the normal polyadenylation site at the 3' terminus of the M cistron, but not in the intergenic region. By DNA-mediated gene transfer, these cDNA clones were expressed into bicistronic RNA containing both P and M sequences in primate cells. RNA thus generated did not undergo nucleolytic processing but was translated into high levels of a 70,000-Mr protein immunoprecipitated by monoclonal antiserum against the measles virus P protein. M protein was not produced in the same cells even though the M cistron could direct M protein synthesis in vitro once excised from the upstream P cistron. These results suggested that bicistronic RNA could direct protein synthesis from the first but not the second cistron and might contribute at least in part to expression of viral genes in vivo.

Base Sequence↗
PubMed1986

Binding of N-linked bovine fetuin glycopeptides to isolated rabbit hepatocytes: Gal/GalNAc hepatic lectin discrimination between Gal beta(1,4)GlcNAc and Gal beta(1,3)GlcNAc in a triantennary structure.

Glycopeptides were isolated from bovine fetuin after digestion with Pronase, aminopeptidase M, and carboxypeptidase Y. The glycopeptides were derivatized with tert-butyloxycarbonyltyrosine and separated on the basis of peptide by using reverse-phase high-performance liquid chromatography. Using 400-MHz 1H NMR, the asialotriantennary oligosaccharides at each of the three N-linked glycosylation sites were found to be combinations of the following two structures in which the third branch is either Gal beta(1,4)GlcNAc or Gal beta(1,3)GlcNAc: (formula; see text) The asialotriantennary glycopeptides containing all beta(1,4)-lactosamine as the branches were designated Gal beta(1,4)GlcNAc-TRI while triantennary glycopeptides containing beta(1,3)-lactosamine as branch III were termed Gal beta(1,3)GlcNAc-TRI. The Gal beta(1,3)GlcNAc unit was localized predominantly to the branch III arm on the basis of a downfield shift (-0.027 ppm) in the H-1 and upfield shift (0.01 ppm) in the NAc methyl signals from the branch III GlcNAc resulting from Gal beta(1,3) instead of Gal beta(1,4) substitution. Revised assignments are proposed for the H-1's of Gal residues 6 (delta 4.464) and 8 (delta 4.471) [Vliegenthart, J. F. G., Dorland, L., & van Halbeek, H. (1983) Adv. Carbohydr. Chem. Biochem. 41, 209-373] in a Gal beta(1,4)GlcNAc-TRI. The proportion of Gal beta(1,3)GlcNAc-TRI glycopeptides from the Asn-Asp, Asn-Gly, and Asn-Cys sites was found to be 40%, 60%, and 20%, respectively. Analysis of the binding of these glycopeptides, containing from 20% to 60% Gal beta(1,3)GlcNAc as branch III, to rabbit hepatocytes revealed that the greater the proportion of Gal beta(1,3)GlcNAc, the lower the affinity of the mixture. The Kd for Gal beta(1,4)GlcNAc-TRI was found to be between 3.6 and 5.4 nM (P = 0.10) with a mean of 4.4 nM from binding data analyzed by using the LIGAND program [Munson, P. J., & Rodbard, D. (1980) Anal. Biochem. 107, 220-239] and computer simulations of the binding of two ligands as a mixture to one receptor site. The Kd of Gal beta(1,3)GlcNAc-TRI oligosaccharide, prepared by hydrazinolysis, was found to be 305 nM from inhibition studies.

Acetylgalactosamine↗
PubMed1986

Functional cDNA library for efficient expression of measles virus-specific gene products in primate cells.

A cDNA library designed for high-level expression of measles virus-specific gene products in mammalian cells was generated. From this library, functional clones which contained the entire protein-coding sequences of the nucleocapsid (N) and the phosphoprotein (P) genes were isolated. By DNA-mediated gene transfer into a line of simian virus 40-transformed monkey kidney cells, the N-specific cDNA was expressed into a single polypeptide of about 60,000 Mr, which was immunoprecipitated by monoclonal antibodies against the measles virus N protein. In contrast, the P-specific cDNA could be expressed into either one or two species of polypeptides of 75,000 or 70,000 Mr, both of which were immunoprecipitated by monoclonal antibodies against the measles virus P protein.

Animals↗
PubMed1985

Synthesis of methyl O-(3-deoxy-3-fluoro-beta-D-galactopyranosyl)-(1----6)-O-beta-D- galactopyranosyl-(1----6)-3-deoxy-3-fluoro-beta-D-galactopyranoside and related N.M.R. studies.

Sequential tritylation, benzoylation, and detritylation of methyl 3-deoxy-3-fluoro-beta-D-galactopyranoside gave crystalline methyl 2,4-di-O-benzoyl-3-deoxy-3-fluoro-beta-D-galactopyranoside (9), which was used as the initial nucleophile in the synthesis of the target oligosaccharide (16). Treatment of 9 with 2,3,4-tri-O-benzoyl-6-O-bromoacetyl-alpha-D-galactopyranosyl bromide gave the corresponding disaccharide derivative 13, having a selectively removable blocking group at O-6'. Debromoacetylation of 13 afforded the disaccharide nucleophile 14 which, when treated with 2,4,6-tri-O-benzoyl-3-deoxy-3-fluoro-alpha-D-galactopyranosyl bromide, gave the fully protected trisaccharide 15. Debenzoylation of 15 gave the title glycoside 16. Condensation reactions were performed with silver trifluoromethane-sulfonate as a promoter in the presence of sym-collidine under base-deficient conditions, and gave excellent yields of the desired beta(trans)-products. Analyses of the 1H- and 13C-n.m.r. spectra, as well as determination of the JCF and JHF coupling constants, were made by using various one- and two-dimensional n.m.r. techniques.

Carbohydrate Conformation↗