Human herpesvirus 7 (HHV-7) is a recently isolated herpesvirus that has been shown to be related to human cytomegalovirus and human herpesvirus 6 and to be a member of the betaherpesvirus subgroup. Here we report the cloning, restriction endonuclease mapping and partial sequence analysis of HHV-7 strain JI DNA. Virus particles were obtained from the supernatant of infected SupT1 cells, the DNA isolated by proteinase K treatment-phenol extraction, and full-length viral DNA was purified and isolated on a pulsed-field gel. Aliquots of this highly purified material were treated in the following ways: (i) sonicated and end-repaired to create short randomly sheared fragments for cloning into M13mp 18-Smal vector DNA; (ii) cut with EcoRI for cloning into EcoRI-cut lambda ZAPII or lambda DASHII vectors; (iii) cut with BamHI for cloning into BamHI-cut lambda ZAP-Express or lambda DASHII vectors. Partial nucleotide sequencing of the M13 clones followed by detection of open reading frames and their translation allowed the identification of homologues through FASTA searches of the database. Relevant M13 clones were used as probes to isolate corresponding lambda phage clones, which could tentatively be mapped to the genome on the basis of presumed genetic collinearity between HHV-7 and HHV-6. Genomic "walking' between EcoRI and BamHI lambda genomic libraries enabled overlapping neighbouring clones to be identified and mapped. Each of these clones was analysed to map BamHI, EcoRI, Sa/l, Smal and Xhol restriction endonuclease sites to provide complete endonuclease maps for the entire genome.
Cloning, restriction endonuclease mapping and partial sequence analysis of the genome of human herpesvirus 7 strain JI
SECCHIERO, Paola;
1996
Abstract
Human herpesvirus 7 (HHV-7) is a recently isolated herpesvirus that has been shown to be related to human cytomegalovirus and human herpesvirus 6 and to be a member of the betaherpesvirus subgroup. Here we report the cloning, restriction endonuclease mapping and partial sequence analysis of HHV-7 strain JI DNA. Virus particles were obtained from the supernatant of infected SupT1 cells, the DNA isolated by proteinase K treatment-phenol extraction, and full-length viral DNA was purified and isolated on a pulsed-field gel. Aliquots of this highly purified material were treated in the following ways: (i) sonicated and end-repaired to create short randomly sheared fragments for cloning into M13mp 18-Smal vector DNA; (ii) cut with EcoRI for cloning into EcoRI-cut lambda ZAPII or lambda DASHII vectors; (iii) cut with BamHI for cloning into BamHI-cut lambda ZAP-Express or lambda DASHII vectors. Partial nucleotide sequencing of the M13 clones followed by detection of open reading frames and their translation allowed the identification of homologues through FASTA searches of the database. Relevant M13 clones were used as probes to isolate corresponding lambda phage clones, which could tentatively be mapped to the genome on the basis of presumed genetic collinearity between HHV-7 and HHV-6. Genomic "walking' between EcoRI and BamHI lambda genomic libraries enabled overlapping neighbouring clones to be identified and mapped. Each of these clones was analysed to map BamHI, EcoRI, Sa/l, Smal and Xhol restriction endonuclease sites to provide complete endonuclease maps for the entire genome.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
