Size distribution analysis of recombinant adenovirus using disc centrifugation

Recombinant adenovirus is one of the primary vectors for human gene therapy. However, the aggregation of unstable virus has been a recurring problem during the production of purified virus for human therapeutics. To facilitate the development of a robust manufacturing process for recombinant adenovirus vectors, a convenient and reliable size distribution analytical assay is necessary and we demonstrate here that disc centrifuge sedimentation is applicable to this purpose. Using the disc centrifuge system and the line start method, the assay can provide particle size distribution of adenovirus samples within 30 min. The assay can detect virus concentrations down to 0.01% (w/v) or 3 × 10¹¹ particles per ml. The apparent hydrodynamic diameter of recombinant adenovirus was determined to be about 0.063 μm. Furthermore, the disc centrifuge analysis was able to detect adenovirus dimers, trimers, and tetramers, consistent with a rigid sphere approximation for adenovirus, as well as a large aggregate of 0.35 μm. The appearance of viral aggregates is confirmed by increased light scattering based on A₃₂₀/A₂₆₀ ratios. The technique could be useful for monitoring the kinetics of aggregation for adenovirus and other DNA and RNA viruses in the submicron region. Therefore, this novel assay provides a critical tool for purification development of viral vectors for meeting therapeutic and research needs. Received 18 September 1997/ Accepted in revised form 15 May 1998     

Proliferation of direct repeats near the Oenothera chloroplast DNA origin of replication

The spacer between the 16S and 23S rRNA genes of the chloroplast DNA has been implicated as an origin of replication in several species of plants. In the evening primrose, Oenothera, this site was found to vary greatly in size, with plastid genomes (plastomes) being readily distinguished. To determine whether plastome "strength" in transmission could be correlated with variation at oriB, the 16S rRNA-trnI spacer was sequenced from five plastomes. The size variation was found to be due to differential amplification (and deletion) of combinations of sequences belonging to seven families of direct repeats. From these comparisons, one short series of direct repeats and one region capable of forming a hairpin structure were identified as candidates for the factor that could be responsible for the differences between strong and weak plastome types. Ample sequence variation allowed phylogenetic inferences to be made about the relationships among the plastomes. Phylogenetic trees also could be constructed for most of the families of direct repeats. The amplifications and deletions of repeats that account for the size variation at oriB are proposed to have occurred through extensive replication slippage at this site.      

Internal eliminated sequences interrupting the Oxytricha 81 locus: allelic divergence, conservation, conversions, and possible transposon origins

Internal eliminated sequences (IESs) often interrupt ciliate genes in the silent germline nucleus but are exactly excised and eliminated from the developing somatic nucleus from which genes are then expressed. Some long IESs are transposons, supporting the hypothesis that short IESs are ancient transposon relics. In light of that hypothesis and to explore the evolutionary history of a collection of IESs, we have compared various alleles of a particular locus (the 81 locus) of the ciliated protozoa Oxytricha trifallax and O. fallax. Three short IESs that interrupt two genes of the locus are found in alleles from both species, and thus must be relatively ancient, consistent with the hypothesis that short IESs are transposon relics. In contrast, TBE1 transposon interruptions of the locus are allele-specific and probably the results of recent transpositions. These IESs (and the TBE1s) are precisely excised from the DNA of the developing somatic macronucleus. Each IES interrupts a highly conserved sequence. A few nucleotides at the ends of each IES are also conserved, suggesting that they interact critically with IES excision machinery. However, most IES nucleotide positions have evolved at high rates, showing little or no selective constraint for function. Nonetheless, the length of each IES has been maintained (+/- 3 bp). While one IES is approximately 33 bp long, three other IESs have very similar sizes, approximately 70 bp long. Two IESs are surrounded by direct repeats of the sequence TTCTT. No other sequence similarities were found between any of the four IESs. However, the ends of one IES do match the inverted terminal repeat consensus sequence of the "TA" IESs of Paramecium. Three O. trifallax alleles appear to have been recipients in recent conversion events that could have been provoked by double-strand breaks associated with IES ends subsequent to IES transposition. Our findings support the hypothesis that short IESs evolved from ancient transposons that have lost most of their sequences, except those necessary for precise excision during macronuclear development.      

Mixed Leucocyte Reaction in Multiple Sclerosis

Astorga and Williams¹ have demonstrated that leucocytes from some patients with rheumatoid arthritis (RA) have an impaired one-way mixed leucocyte reaction² (MLR) when stimulated with leucocytes from other such patients. No definite explanation of this phenomenon is given. We have investigated other diseases in which auto-immune aetiology is suspected to see whether similar phenomena are associated with them. Here we report our investigations of multiple sclerosis (MS).