Alterations in the regulatory region of the human papillomavirus type 6 genome are generated during propagation in Escherichia coli

Abstract

We analyzed the long control regions (LCRs) of seven human papillomavirus type 6b (HPV-6b) clones, which contained prototype HPV-6b sequences recloned into various plasmid vectors and propagated in different strains of Escherichia coli. Southern blot analysis and DNA sequencing demonstrated three different sequences, each distinct from the published prototype HPV-6b sequence. Two of the plasmids contained insertions of 24 and 94 base pairs (bp) and a 1-bp deletion. Four plasmids contained insertions of 24 and 58 bp and a deletion of 49 bp. One plasmid contained a single insertion of 77 bp. The 94-, 77-, and 58-bp insertions occurred at the same site and had 100% positional identity across their shared lengths. All changes were located in the purine-thymidine-rich region of the LCR (nucleotides 7292 to 7400). Two additional LCR sequences were detected by restriction analysis of two other HPV-6b clones. We conclude that the purine-thymidine-rich region of the LCR is a hot spot for recombination in E. coli and that the alterations are the result of recA-independent events. These results emphasize the need to rigorously prove that a cloned isolate is an authentic copy of the genomic DNA present in the original lesion. In addition, the data indicate that the HPV-6b LCR sequences employed in different laboratories may be different, even if their parental DNAs were identical. Finally, we discuss the need for caution in assigning biological significance to alterations in this region, in view of the limited data available on the true identity of the HPV-6b LCR.

Variation of human papillomavirus type 6 (HPV-6) and HPV-11 genomes sampled throughout the world

“…These additional 94 bp were present after position 7350 and were identical to those observed in an independent isolate and published under the code HPV-6 WV6745 (38). We conclude that this sequence was present in the HPV-6b reference type at the time of isolation and was deleted during amplification, a definite possibility according to the studies of the behavior of HPV-6 sequences in Escherichia coli (37). This 94-bp sequence is apparently identical to the additional 120 bp previously identified by restriction analyses, because no additional sequences were found in the LCR or in the 3Ј segment of the L1 gene.…”

“…The bona fide sequence of the 5Ј segment of the LCR of the HPV-6b reference clone has been a matter of confusion, because this region seems to give rise to frequent mutations in vivo. Furthermore, it has also been claimed that a cloning artifact occurred in the original isolate (7,34,37,55). As judged by restriction fragment analysis, a segment of approximately 120 bp may have become artifactually deleted from the HPV-6b clone that was used to establish the published sequence (51).…”

CCAAT displacement protein, a regulator of differentiation-specific gene expression, binds a negative regulatory element within the 5' end of the human papillomavirus type 6 long control region

“…All of the oligonucleotides used in this study were synthesized on an Applied Biosystems model 380B DNA synthesizer (Biochemistry Biotechnology Facility at the Indiana University School of Medicine). Plasmids were amplified in the BE257recA strain (40) of Escherichia coli and purified by using the Qiagen kit reagents and Q-250 chromatography columns. The sequence of all constructs was verified by dideoxy sequencing using Sequenase kit reagents (United States Biochemical).…”

Two new human papillomavirus types (HPV54 and 55) characterized from genital tumours illustrate the plurality of genital HPVs

“…mixture of typical HPV6a genomes and HPV6a DNA molecules with a deletion of about 0.3kb located in the long control region. HPV6 DNA molecules showing rearrangements in the long control region have previously been reported in some Buschke-Liiwenstein tumours (Boshart and zur Hausen, 1986;Rando et al, 1986;Kasher and Roman, 1988). The patient died from an invasive laryngeal squamous-cell carcinoma which was not available for virologic studies.…”

“…HPV54 is only weakly related to the known HPVs, but blot hybridization experiments performed under non-stringent conditions have revealed that this newly recognized type is more related to the genital HPVs than to the cutaneous HPVs. The role of HPV54 in the genesis of the Buschke-Lowenstein tumour from which it was cloned appears unlikely, since it was not found in 9 additional cases, and since it was detected in association with HPV6, a HPV type frequently found in Buschke-Lowenstein tumours (Boshart and zur Hausen, 1986;Rando et al, 1986;Kasher and Roman, 1988). Interestingly, HPV6 DNA sequences were detected as a…”