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J Virol, July 1998, p. 5565-5572, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Length and Sequence Composition of Vesicular
Stomatitis Virus Intergenic Regions Affect mRNA Levels and the Site
of Transcript Initiation
Elizabeth A.
Stillman and
Michael A.
Whitt*
Department of Microbiology and Immunology,
University of Tennessee, Memphis, Memphis, Tennessee 38163
Received 17 November 1997/Accepted 9 April 1998
In this study, we used a dicistronic vesicular stomatitis virus
(VSV) minigenome to investigate the effects of either single or
multiple nucleotide insertions placed immediately after the nontranscribed intergenic dinucleotide of the M gene on VSV
transcription. Both Northern blot and primer extension analysis showed
that the polymerase responded to the inserted nucleotides in a
sequence-specific manner such that some insertions had no effect on
mRNA synthesis from the downstream G gene, nor on the site of
transcript initiation, whereas other insertions resulted in dramatic
reductions in transcript accumulation. Some of these transcripts were
initiated at the wild-type site, while others initiated within the
inserted sequence. We also examined the transcriptional events that
occurred when a natural, 21-nucleotide intergenic region located
between the G and L genes from the New Jersey (NJ) serotype of VSV was
inserted into the minigenome gene junction. In contrast to the normal
25 to 30% attenuation observed for downstream transcription at gene junctions containing the typical dinucleotide (3'-GA-5') intergenic region, the NJ variant showed greater than 75% attenuation at the gene
junction. In addition, the polymerase initiated transcription at two
major start sites, one of which was located within the intergenic
sequence. Collectively, these data suggest that the polymerase
"samples" the intergenic sequences following polyadenylation and
termination of the upstream transcript by scanning until an appropriate
start site is found. One implication of a scanning polymerase is that
the polymerase presumably switches states from a processive elongation
mode to a stuttering mode for polyadenylation to one in which no
transcription occurs, before it reinitiates at the downstream gene. Our
data support the hypothesis that sequences surrounding the intergenic
region modulate these events such that appropriate amounts of each mRNA
are synthesized.
*
Corresponding author. Mailing address: 858 Madison
Ave., Department of Microbiology and Immunology, University of
Tennessee, Memphis, Memphis, TN 38163. Phone: (901) 448-4634. Fax:
(901) 448-8462. E-mail: MWhitt{at}utmem1.utmem.edu.
J Virol, July 1998, p. 5565-5572, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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