Production of large quantities of highly purified plasmid DNA is essential for gene therapy. A low-copy-number pBR322-derived plasmid (VCL1005) was converted to a high-copy-number plasmid (VCL1005G/A) by incorporating a G-->A mutation that affects initiation of DNA replication from the ColE1 origin of replication. Because the phenotypic effect of this mutation is enhanced at an elevated temperature, a further increase in yield was achieved by changing the growth temperature from 37 degrees C to 42 degrees C at mid-log phase during batch and fed-batch fermentation. The combined effect of the single base-pair change and the elevated growth temperature produced an overall yield of 2.2 grams of plasmid DNA available for recovery from a 10-liter fed-batch fermentation compared to 0.03 grams from a 10-liter batch fermentation, a 70-fold increase in yield. The plasmid DNA isolated from this process contained lower levels of RNA and chromosomal DNA contaminants, simplifying downstream processing.

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In a previous study, we reported the isolation of a lambda gt11 cDNA clone (C3) for a virus message that mapped to the HindIII R fragment (human cytomegalovirus [AD169]). In this report, we further analyze transcription from this region of the genome. C3 was used to probe Northern (RNA) blots of RNA isolated from infected cells. Two abundant messages, 1.3 and 1.6 kilobases (kb) in size, were detected at 62 h postinfection (p.i.). Examination of different time points determined that the 1.6-kb mRNA accumulated in infected cells between 24 and 48 h p.i. and was classified as a late message. The 1.3-kb message was transcribed early in infection and was initially detected around 12 h p.i. Both transcripts were suppressed when infected cells were treated with inhibitors of DNA synthesis. Sequencing and S1 analysis identified the 5' ends of these two messages within 240 nucleotides of each other. Two CAAT-TATA motifs were found upstream of the 1.3- and 1.6-kb mRNA initiation sites, which suggested that the promoters were also closely associated. Antisera made to the fusion protein, synthesized from the C3 clone, detected a 25-kilodalton (kDa) virus protein found in infected cells and purified virions. Western blot (immunoblot) analysis of infected-cell proteins at various times after infection demonstrated that the accumulation of the 25-kDa protein coincided with the appearance of the 1.6-kb message. Therefore, we conclude that the 25-kDa virion protein is translated from the 1.6-kb message.

, we identified a late, unspliced 1.6-kb mRNA that maps to the HindlIl R fragment of human cytomegalovirus (HCMV) AD169. In the present study, the direction of transcription of this mRNA was determined by Northern (RNA) analysis with strand-specific probes. Primer extension was used to precisely map the 5' end of the mRNA. An open reading frame (ORF) designated ORF 2-1, located 176 nucleotides downstream from the cap site of the 1.6-kb mRNA, was identified. A synthetic peptide was made representing a hydrophilic region in the amino terminus of ORF 2-1. Immunoprecipitation and Western immunoblot analysis of infected HEL cell lysates, using affinity-purified antibody to the peptide (anti-P2l), detected a viral protein with an apparent molecular mass of 58 kDa late in infection. Further support for the presence of this protein in infected-cell lysates was obtained by an enzyme-linked immunosorbent assay. Expression of viral antigens in intact infected HEL cells was assessed by immunofluorescence. General cytoplasmic staining was observed at 62 h postinfection, in contrast to a localized staining observed in the nuclear and perinuclear region at 96 h postinfection.

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