William G. Dougherty

The nucleotide sequence of the coding region of tobacco etch virus genomic RNA: Evidence for the synthesis of a single polyprotein☆

The complete nucleotide sequence of the tobacco etch virus (TEV) RNA genome has been determined excepting only the nucleotide(s) present at the extreme 5 terminus. The assembled TEV genomic sequence is 9496 nucleotides in length followed by a polyadenylated tract ranging from 20 to 140 residues. A computer search of the sequence reveals the following. A 5 untranslated region, rich in adenosine and uridine, is present between nucleotides 1 and 144. A putative initiation codon, at nucleotides 145-147, marks the beginning of a large open-reading frame (ORF) which ends with an opal (UGA) termination codon at positions 9307-9309. A 186-nucleotide untranslated region is present between the termination codon of the ORF and the beginning of the 3 polyadenylated region. The predicted translation product of this ORF is a 3054 amino acid polyprotein with a mol wt of 345,943. A function for the large (54,000 Mr) nuclear inclusion protein is suggested by a comparison of the deduced amino acid sequence with a protein data bank. This protein displays biochemical similarities to other viral RNA-dependent, RNA polymerases.

Biochemical analysis of the capsid protein gene and capsid protein of tobacco etch virus: N-terminal amino acids are located on the virion's surface

The sequence of the 1491 nucleotides found at the 3 end of the genome of the highly aphid-transmissible (HAT) isolate of tobacco etch virus (TEV) has been determined. The nucleotide sequence of the capsid protein gene has been identified and compared with the corresponding region of the not-aphid-transmissible (NAT) isolate of TEV and with pepper mottle virus (PeMV). The deduced amino acid sequences of the two TEV capsid proteins displayed 98% homology and a 66% homology with PeMV capsid protein. Three of the six amino acid differences between the capsid proteins of the two TEV isolates occurred near the N terminus of the protein. Biochemical and immunological evidence suggested the N-terminal 29 amino acids of the capsid protein were hydrophilic and were located at or near the virions surface.

Nucleotide sequence at the 3' terminus of pepper mottle virus genomic RNA: evidence for an alternative mode of potyvirus capsid protein gene organization.

The sequence of the 3-terminal 1481 nucleotides of the pepper mottle virus (PeMV) genome has been determined. The sequence was determined by dideoxy nucleotide sequencing of complementary DNA which had been inserted into M13 bacteriophage cloning vectors and was confirmed by sequencing selected regions of PeMV RNA. A discrete open reading frame of 993 nucleotides, ending 333 nucleotides from the 3-terminal polyadenylate tract, was identified that potentially encoded a 37,669-MW protein. The amino acids predicted at positions 64 through 84 of this putative polypeptide were identical to the amino-terminal 21 amino acids of the PeMV capsid protein ascertained by chemical sequencing. These combined nucleotide and amino acid sequence data suggest that the PeMV capsid protein is encoded by the 3-most cistron on the genomic RNA and that it may be expressed as a precursor that is proteolytically processed to produce the mature capsid protein.

Topographic analysis of tobacco etch virus capsid protein epitopes

Monoclonal antibodies have been prepared, which react with capsid protein of an aphid-transmitted isolate of tobacco etch virus (TEV). Ten different monoclonal antibodies were characterized with reference to (1) antibody class, (2) reactivity with different plant virus antigens, (3) the spatial relationship between epitopes, and (4) whether these epitopes were located on the exterior surface of the virion. Three monoclonal antibodies were specific for TEV isolates. These monoclonal antibodies reacted with epitopes exposed on the external surface of the TEV particle. Seven monoclonal antibodies reacted with a variety of different potyviruses including TEV, potato virus Y, tobacco vein mottling virus, pepper mottle virus, watermelon mosaic virus II, and maize dwarf mosaic virus. In general, these seven monoclonal antibodies defined epitopes not readily accessible on the virion surface.

Analysis of viral RNA isolated from tobacco leaf tissue infected with tobacco etch virus

Total RNA has been isolated from tobacco seedlings systemically infected with the potyvirus, tobacco etch virus (TEV). Analysis of this RNA by agarose gel electrophoresis under denaturing conditions, in conjunction with gel hybridization using various virus-specific nucleic acid probes, revealed genomic length TEV RNA and eight zones of less-than-full-length RNA. However, reconstitution experiments demonstrated that the zones were electrophoretic artifacts and not authentic subgenomic RNAs. Furthermore, only a single polyadenylated viral RNA species, which comigrated with TEV genomic RNA in gel electrophoresis studies, was isolated from infected tobacco leaf tissue. Translation of polyadenylated RNA in a rabbit reticulocyte lysate resulted in the synthesis of four high-molecular-weight virus-specific products. The products had apparent molecular weights and serological reactivities as follows: (1) 120,000, reaction with antiserum to TEV cytoplasmic inclusion protein; (2) 87,000, no serological reaction detected; (3) 85,000, reaction with antisera to TEV capsid protein and the large-molecular-weight component of the nuclear inclusion body; (4) 49,000, reaction with antiserum to the small-molecular-weight component of the nuclear inclusion body.

Identification of an aspartate transfer RNA gene in maize mitochondrial DNA

SummaryA 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 mitocondrial aspartate tRNA genes. This gene is located on a 3,123 base pair EcoRl 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.

Construction of a recombinant vaccinia virus which expresses immunoreactive plant virus proteins.

A chimeric transcription unit was assembled consisting of a vaccinia virus promoter linked to a 2400 bp(3) double-stranded complementary DNA (cDNA) made to the 3 end of the genomic RNA of the plant pathogen, tobacco etch virus (TEV). Marker rescue techniques were used to introduce virus recombinant genes into the vaccinia virus genome. The recombinant virus (designated WNAT) was isolated, purified, and subjected to molecular genetic analyses. The ability of WNAT to direct the expression of plant virus genetic information in a mammalian system was assessed by infecting a line of monkey kidney cells. The plant virus cDNA insert was transcribed into RNA molecules that were correctly processed, and subsequently translated into proteins that were recognized by monospecific antisera directed against either the capsid protein or nuclear inclusion protein of TEV. These results are discussed in relation to using vaccinia virus to investigate other aspects of the plant virus replicative cycle.

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.