1887

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Volume 62, Issue 2

Transfection with the Isolated Herpes Simplex Virus Thymidine Kinase Genes. I. Minimal Size of the Active Fragments from HSV-1 and HSV-2 Free

Abstract

SUMMARY

We have defined the minimal size and physical map locations in the genomes of both herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) for DNA sequences capable of conferring stable biochemical transformation under thymidine kinase (TK) selection. The experiments involved transfection of Ltk cells with either isolated virus DNA fragments or cloned BR322 plasmids containing the 3.5 kilobase (kb) HI-O fragment from HSV-1 (MP) or the 5.6 kb I-G fragment from HSV-2(333). Mapping of restriction enzyme sites within these cloned DNAs, followed by assays for colony formation in HAT medium after transfection with cleaved DNA, localized the biologically active TK-transforming sequences to lie between coordinates 0.300 and 0.313 in HSV-1 and between 0.303 and 0.315 in HSV-2. Experiments with a series of cloned plasmids containing deletions of the HI-O fragment towards either the 3′- or 5′-ends of the TK gene indicated that the sequences required for stable HSV-1 TK transformation lay within a 1600 base pair (bp) region at 0.303 to 0.313 map units. An internal deletion mutant plasmid, selected by a novel bacterial transfection assay for the absence of the I site at 0.308, also failed to rescue Ltk cells. With the exception of cleavage at the I site at 0.303 in HSV-2, which reduced activity only eightfold, all cleavages that affected TK transformation reduced the efficiency at least 50-fold. A direct comparison of the HSV-1 and HSV-2 minimal transforming regions with the nucleotide sequence of the structural HSV-1 TK gene indicates that the HSV-2 I site lies 30 bp beyond the poly(A) addition site at the 3′-end of TK mRNA. On the other hand, cleavage at the I site in HSV-1 TK, located 80 bp in front of the poly(A) addition point, abolishes colony formation. Comparison of the putative 5′-end of the HSV-2 TK gene defined by transfection assays, with a 250 bp non-transcribed region at the front of the HSV-1 TK gene, suggests that the promoter regions contain a much higher frequency of conserved cleavage sites than do the coding portions of the two genes. Direct nucleotide sequencing of the 5′-flanking sequences for HSV-2 TK confirmed that large portions of the two promoters possess greater than 95% sequence homology. At least 140 bp, but no more than 200 bp, of this 5′-promoter region are essential for efficient transfer and expression of the viral TK gene. Combining the results from HSV-1 and HSV-2, we conclude that a contiguous sequence of 1480 to 1540 bp is necessary to achieve at least 10% of the maximum transformation efficiency.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): HAT selection , HSV-2 promoter , sequence homology and transformation efficiency
© Society for General Microbiology 1982
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1982-10-01
2024-04-20
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Transfection with the Isolated Herpes Simplex Virus Thymidine Kinase Genes. I. Minimal Size of the Active Fragments from HSV-1 and HSV-2
J. Gen. Virol. 62, 191 (1982); https://doi.org/10.1099/0022-1317-62-2-191
/content/journal/jgv/10.1099/0022-1317-62-2-191
/content/journal/jgv/10.1099/0022-1317-62-2-191
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