Search PubMed⌕ Search

Biomedical subjects

W G Dougherty

Publications and source records attributed to W G Dougherty.

At least 37 records · Page 2Linked to original sources
PubMed1989

A second proteinase encoded by a plant potyvirus genome.

The RNA genome of tobacco etch virus (TEV) encodes a large polyprotein precursor that is processed to mature proteins by virus-specific proteinases. Cleavage sites located within the carboxyl-terminal two-thirds of the polyprotein are processed by a TEV-encoded 49 kd proteinase, while the enzyme(s) responsible for cleaving the remaining sites has not been found. In this study, a second TEV-encoded proteinase has been identified based on cell-free expression of defined RNA transcripts. The boundaries of this proteinase have been delineated by deletion analysis and site-directed mutagenesis. The proteolytically active domain has been localized to the carboxyl-terminal half of the 56 kd aphid-transmission helper component. A cleavage site that is recognized by this proteinase has been identified in the polyprotein adjacent to the carboxyl-terminus of the enzyme, and the proteinase appears to cleave by an autocatalytic mechanism. Proteolysis in vitro occurs between a Gly-Gly dipeptide as determined by radiochemical sequencing at the amino-terminus of the proteolytic product.

Amino Acid Sequence↗
PubMed1989

Molecular genetic and biochemical evidence for the involvement of the heptapeptide cleavage sequence in determining the reaction profile at two tobacco etch virus cleavage sites in cell-free assays.

Potyviruses express their genetic information from a genome length RNA as a single polyprotein, which is post-translationally processed by at least two different viral-encoded proteolytic activities. Since regulation of the expression of individual genes is not likely to occur at the transcriptional level, we sought to determine if post-translational regulation of gene expression was possible via differential proteolytic processing. Modulating the rate of cleavage at different gene product junctions could be a potential mechanism to regulate the kinetics of formation of specific gene products. We have examined the proteolytic processing of two tobacco etch virus (TEV) cleavage sites using a cell-free system in which synthetic polyprotein precursors were processed by the TEV 49-kDa proteinase. The amino acid sequences at these two sites contained both conserved and variable positions. The cleavage reaction at the TEV 50/71-kDa junction was characterized as "slow" (T1/2 = approximately 27 min) while the cleavage reaction at the 58/30-kDa junction was judged "fast" (T1/2 = approximately 5 min). Similar cleavage reactions were observed whether the cleavage site was in its natural polyprotein context or placed in a foreign position. The slow and fast cleavage sites could be interconverted by changing the nonconserved amino acid positions. The data suggest that the heptapeptide sequence proximal to a TEV cleavage site determines not only cleavage at a particular junction but influences the cleavage reaction profile in cell-free studies.

Amino Acid Sequence↗
PubMed1989

Characterization of the catalytic residues of the tobacco etch virus 49-kDa proteinase.

The 49-kDa proteinase of tobacco etch virus (TEV) cleaves the polyprotein derived from the TEV genomic RNA at five locations. Molecular genetic and biochemical analyses of the 49-kDa TEV proteinase were performed to test its homology to the cellular trypsin-like serine proteases. A cDNA fragment, containing the TEV 49-kDa proteinase gene and flanking sequences, was expressed in a cell-free transcription/translation system and resulted in the formation of a polyprotein precursor that underwent rapid self-processing. Site-directed mutagenesis was used to test the effect of altering individual 49-kDa amino acid residues on proteolysis. The data suggest that the catalytic triad of the TEV 49-kDa proteinase could be composed of the His234, Asp269, and Cys339. These findings are consistent with the hypothesis that the TEV 49-kDa proteinase is structurally similar to the trypsin-like family of serine proteinases with the substitution of Cys339 as the active site nucleophile. A structural model of the TEV 49-kDa proteinase proposes other virus-specific differences in the vicinity of the active site triad and substrate-binding pocket. The structure may explain the observed negligible effect of most cellular proteinase inhibitors on the activity of this viral proteinase.

Aprotinin↗
PubMed1989

Generation and characterization of monoclonal antibodies reactive with the 49-kDa proteinase of tobacco etch virus.

Monoclonal antibodies (McAbs) were generated against two tobacco etch virus (TEV)-encoded nonstructural proteins, the 49-kilodalton (kDa) proteinase and the 58-kDa putative RNA-dependent RNA polymerase. This process was facilitated by the fact that these two TEV nonstructural proteins cocrystallize in the nuclei of virus-infected cells to form nuclear inclusion (NI) bodies which can be purified readily. The anti-NI McAbs were shown by Western blot analysis to be specific for either the TEV 49-kDa or the 58-kDa protein. Those McAbs reactive with the 49-kDa proteinase were characterized further with respect to the 49-kDa domain with which they reacted and with respect to their ability to inhibit the autocatalytic or self-processing activity of the 49-kDa proteinase. The 49-kDa antigens were synthesized from a TEV cDNA sequence using cell-free transcription and translation systems. Each anti-49-kDa McAb was used in immunoprecipitation studies with a series of 49-kDa antigens which represented a nested set of 49-kDa proteins with common amino termini but varying in length. Immunoprecipitation results showed that all of the anti-49-kDa proteinase McAbs reacted with one of five binding regions, designated A through E from the carboxy terminus of the proteinase, which were 77, 38, 81, 18, and 61 amino acids long, respectively. The 38-amino-acid binding region B contained the proposed catalytic cysteine 339 residue and was recognized by only one McAb, 4911. McAb 4911 was the only anti-49-kDa McAb capable of inhibiting the self-processing reaction in which the 49-kDa proteinase is released from its 75-kDa polyprotein precursor.

Antibodies, Monoclonal↗
PubMed1989

Molecular genetic analysis of a plant virus polyprotein cleavage site: a model.

The RNA genome of tobacco etch virus (TEV) is expressed as a polyprotein which is co- and post-translationally processed by viral encoded proteinases. The TEV 49,000 dalton (49-kDa) proteinase cleaves the polyprotein at five positions each defined by the seven amino acid consensus sequence, (formula; see text) One of the cleavage sites, the 58-kDa nuclear inclusion/30-kDa capsid protein junction was altered by site-directed mutagenesis and the effects of these alterations on cleavage were determined. Polyprotein precursors were synthesized by translation of T7 polymerase-derived transcripts and processed in a cell-free system using TEV nuclear inclusion bodies as a source of 49-kDa proteolytic activity. A wild-type cleavage site and 61 substrates containing site-directed amino acid replacements at the nonconserved P7, P5, P4, P2, and P'2 positions were examined. Amino acid replacements flanking the putative TEV cleavage sequence at the P7 and P'2 positions had minimal effects on cleavage. Amino acid substitutions at positions P5, P4, and P2 resulted in substrates which were processed by the 49-kDa TEV proteinase, albeit generally at reduced rates. No substitution at any of these five positions resulted in total elimination of cleavage. A model is presented which proposes different roles for conserved and variable positions in the TEV heptapeptide cleavage sequence.

Amino Acid Sequence↗
PubMed1988

Biochemical and mutational analysis of a plant virus polyprotein cleavage site.

The RNA genome of tobacco etch virus (TEV) is organized as a single translational unit coding for a 346,000 (346 kd) mol. wt (Mr) polyprotein. The 346 kd Mr polyprotein is cleaved by a 49 kd Mr virus-encoded proteinase at five different sites between the dipeptides Gln-Ser or Gln-Gly. These cleavage sites or gene product boundaries are defined by the heptapeptide sequence...Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser or Gly.... We have used the 54 kd Mr nuclear inclusion protein/30 kd Mr capsid protein junction as a model to examine the role of these conserved amino acids in defining a cleavage site. The 54 kd/30 kd Mr protein cleavage site sequence of 10 TEV isolates from geographically distinct locations has been deduced. The conserved amino acids are present in all isolates. To determine if these four amino acids are an absolute requirement for polyprotein substrate activity, a site-directed mutational analysis has been performed. A recombinant cDNA molecule encoding the TEV 54 kd/30 kd Mr gene product cleavage site was mutated and polyprotein substrates were synthesized and processed in a cell-free system. Single amino acid substitutions made at the different positions reveal a strong preference for the naturally conserved amino acids.

Amino Acid Sequence↗
PubMed1988

A viral cleavage site cassette: identification of amino acid sequences required for tobacco etch virus polyprotein processing.

Mature viral-encoded proteins of tobacco etch virus (TEV) arise by proteolytic processing of a large precursor. The proteinase responsible for most of these cleavages is a viral-encoded 49-kDa protein. All known or predicted cleavage sites in the TEV polyprotein are flanked by the conserved sequence motif Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser or Gly, with the scissile bond located between the Gln-Ser or Gly dipeptide. By using cell-free systems to manipulate and express cloned cDNA sequences, a 25-amino acid segment containing a putative proteolytic cleavage site of the TEV polyprotein has been introduced into the TEV capsid protein sequence. This recombinant protein is cleaved by the 49-kDa proteinase at the introduced cleavage site, thus demonstrating portability of a functional cleavage site. The role of the conserved amino acid sequence in determining substrate activity was tested by construction of engineered proteins that contained part or all of this motif. A protein that harbored an insertion of the conserved 7-amino acid segment was cleaved by the 49-kDa TEV proteinase. Cleavage of the synthetic precursor was shown to occur accurately between the expected Gln-Ser dipeptide by microsequence analysis. Proteins containing insertions that generated only the Gln-Ser, or only the serine moiety of the conserved sequence, were insensitive to the 49-kDa proteinase.

Amino Acid Sequence↗
PubMed1988

Mutational analysis of tobacco etch virus polyprotein processing: cis and trans proteolytic activities of polyproteins containing the 49-kilodalton proteinase.

The genome of tobacco etch virus contains a single open reading frame with the potential to encode a 346-kilodalton (kDa) polyprotein. The large polyprotein is cleaved at several positions by a tobacco etch virus genome-encoded, 49-kDa proteinase. The locations of the 49-kDa proteinase-mediated cleavage sites flanking the 71-kDa cytoplasmic pinwheel inclusion protein, 6-kDa protein, 49-kDa proteinase, and 58-kDa putative polymerase have been determined by using cell-free expression, proteolytic processing, and site-directed mutagenesis systems. Each of these sites is characterized by the conserved sequence motif Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser or Gly (in which cleavage occurs after the Gln residue). The amino acid residue (Gln) predicted to occupy the -1 position relative to the scissile bond has been substituted, by mutagenesis of cloned cDNA, at each of four cleavage sites. The altered sites were not cleaved by the 49-kDa proteinase. A series of synthetic polyproteins that contained the 49-kDa proteinase linked to adjoining proteins via defective cleavage sites were expressed, and their proteolytic activities were analyzed. As part of a polyprotein, the proteinase was found to exhibit cis (intramolecular) and trans (intermolecular) activity.

Amino Acid Sequence↗
PubMed1987

Small nuclear inclusion protein encoded by a plant potyvirus genome is a protease.

Tobacco etch virus, a plant potyvirus, expresses its RNA genome as a large polyprotein precursor which undergoes extensive proteolytic processing to yield seven or more mature products. Two of these products, proteins with apparent molecular weights of 49,000 and 54,000 (49K and 54K proteins), aggregate in the form of crystalline inclusions within the nuclei of infected cells. Cell-free translation of synthetic transcripts was used to map the genes for these two products on the viral genome and to express an enzymatically active protein. The 49K protein was determined to be a viral protease responsible for several cleavages of the polyprotein, including its own autocatalytic excision. Analyses of products expressed from the 49K protein genes which were altered by deletion revealed that only the carboxyl-terminal half was required for proteolytic activity.

Journal Article↗
PubMed1986

Assembly of overlapping DNA sequences by a program written in BASIC for 64K CP/M and MS-DOS IBM-compatible microcomputers.

The SEQALIGN programs1 described in this report aid in the assembly of up to 100 individual overlapping DNA sequences generated by M-13 subcloning and sequencing methods. The program produces a printout of the aligned sequences presented in register. Use of the program will be facilitated because 1) it is written with the Microsoft BASIC interpreter, 2) sequence data may be entered and edited using WORDSTAR or similar word processing programs, and 3) hardware requirements for execution of the program on CP/M or MS-DOS (IBM-PC compatible) systems are minimal.

Base Sequence↗
PubMed1986

A single nucleotide change in the E2 glycoprotein gene of Sindbis virus affects penetration rate in cell culture and virulence in neonatal mice.

The nucleotide sequence of the glycoprotein genes of fully virulent Sindbis virus and derived mutants that have reduced neurovirulence for neonatal mice (attenuated mutants) has been determined. A single amino acid difference, arginine instead of serine at position 114 of the mature E2 glycoprotein, distinguished the prototype attenuated mutant from its virulent wild-type parent. Virulent revertants of the attenuated mutant showed same-site reversion to the wild-type sequence. An identical single amino acid substitution, an arginine for the serine at E2 position 114, was found in a second independently selected attenuated mutant. The strains are characterized by genetic linkage between attenuation, accelerated penetration of baby hamster kidney cells, and efficient neutralization by the E2-specific monoclonal antibodies R6 and R13; selection for change in one property simultaneously selected for change in the other two (Olmsted, R. A., Baric, R. S., Sawyer, B. A. & Johnston, R. E. (1984) Science 225, 424-427 and Olmsted, R. A., Meyer, W. J. & Johnston, R. E. (1986) Virology 148, 1-10). The nucleotide sequence data suggest that a single mutation in the E2 gene is sufficient to cause these coordinate phenotypic changes. These findings identify a single locus in a Sindbis virus surface glycoprotein gene that determines both efficiency of interaction with cultured baby hamster kidney cells and degree of virulence in neonatal mice.

Amino Acid Sequence↗
PubMed1985

Identification of an aspartate transfer RNA gene in maize mitochondrial DNA.

A gene for a transfer RNA (tRNA) specific for aspartic acid was identified in maize mitochondrial DNA. The nucleotide sequence and predicted secondary structure of this tRNA more closely resemble eubacterial and chloroplast aspartate tRNA genes than other mitochondrial aspartate tRNA genes. This gene is located on a 3,123 base pair EcoRI DNA fragment that also contains an elongator methionine tRNA gene. These two tRNA genes are separated by 726 nucleotides and are located on opposite strands of DNA.

Base Sequence↗
PubMed1985

Sequence determination of the capsid protein gene and flanking regions of tobacco etch virus: Evidence for synthesis and processing of a polyprotein in potyvirus genome expression.

The nucleotide sequence of the 3'-terminal portion of the tobacco etch virus (TEV) genome was determined. The 2324-nucleotide sequence represented approximately one-fourth of the TEV genome and included the capsid protein gene and flanking regions. An open reading frame of 2135 nucleotides and an untranslated region of 189 nucleotides adjacent to a polyadenylate tract were identified. The sequence began within an open reading frame, indicating that the initiation codon was upstream of the available sequence data. The sequence of the 20 NH(2)-terminal amino acids of the TEV capsid protein was established chemically. An identical amino acid sequence, predicted from the nucleotide sequence, was located, commencing at amino acid - 263. These data indicated that maturation of the capsid protein required a post-translational cleavage of a larger protein precursor, with a probable cleavage site between the amino acids glutamine and glycine.

Journal Article↗
PubMed1984

Identification of two methionine transfer RNA genes in the maize mitochondrial genome.

Two methionine transfer RNA (tRNA) genes were identified in the maize mitochondrial genome by nucleotide sequence analysis. One tRNA gene was similar in nucleotide sequence and secondary structure to the initiator methionine tRNA genes of eubacteria and higher plant chloroplast genomes. This tRNA gene also had extensive nucleotide homology (99%) with an initiator methionine tRNA gene described for the wheat mitochondrial genome. The other methionine tRNA gene sequence was distinct and more closely resembled an elongator methionine tRNA.

Journal Article↗