The quality of virus-specific CD8+ CTL immune responses generated by mucosal and systemic poxvirus prime-boost vaccines were evaluated in terms of T cell avidity and single-cell analysis of effector gene expression. Intranasal (I.N.) immunization regimes generated higher avidity CTL responses specific for HIV KdGag197–205 (amino acid sequence AMQMLKETI; H-2Kd binding) compared with i.m. immunization regime. Single-cell RT-PCR of KdGag197–205-specific mucosal and systemic CTL revealed that the cytokine and granzyme B expression profiles were dependent on both the route and time after immunization. The I.N./i.m.-immunized group elicited elevated number of CTL-expressing granzyme B mRNA from the genitomucosal sites compared with the i.m./i.m. regime. Interestingly, CTL generated after both I.N. or i.m. immunization demonstrated expression of Th2 cytokine IL-4 mRNA that was constitutively expressed over time, although lower numbers were observed after I.N./I.N. immunization. Results suggest that after immunization, Ag-specific CTL expression of IL-4 may be an inherent property of the highly evolved poxvirus vectors. Current observations indicate that the quality of CTL immunity generated after immunization can be influenced by the inherent property of vaccine vectors and route of vaccine delivery. A greater understanding of these factors will be crucial for the development of effective vaccines in the future.

Determination of sequences from the nine regions separating the large genes in the 19‐kbp mitochondrial DNA from Torulopsis glabrata has led to the identification of 23 tRNA genes and to the recognition of two types of short repeated sequence implicated in mitochondrial genome expression. The two short repeated sequences are a nonanucleotide motif, 5′‐TATAAGTAA‐3′ and a dodecanucleotide motif, 5′‐TATAATATTCTT‐3′. By RNA sequence determination it has been found that primary transcripts of the small and large rRNAs commence at the 3′ penultimate adenine of the nonanucleotide sequence. This motif has also been found in the DNA sequence upstream from f‐methionine, phenylalanine, leucine, tyrosine and glycine tRNAs, cytochrome oxidase subunit 2 and ATPase subunit 9. The dodecanucleotide sequence is found at least once in each of the nine regions between the large genes. Determination of the 3′ ends of the small and large rRNAs has shown their location to be 8 and 23 nucleotides downstream from the dodecanucleotide sequence. This motif is thought to be involved in signalling processing of polycistronic transcripts. Such transcripts are invoked to account for the production of mRNAs for cytochrome b, cytochrome oxidase subunits 1 and 3, and the joint mRNA for ATPase subunits 8 and 6 genes that lack an adjacent upstream nonanucleotide transcription initiation signal sequence. Processing of polycistronic transcripts at tRNA sequences is also implicated in the formation of mature mRNAs. From the position of tRNA genes relative to the nonanucleotide motif it appears that clusters of these genes are co‐transcribed with downstream sequences for cytochrome oxidase subunits 1 and 3.

Closed‐circular DNA has been isolated from mitochondrial‐enriched fractions from four petite‐negative' yeasts. Electron microscope analysis has shown in each case the presence of a large discrete size class of circular DNA greater than 6 μm in length and smaller heterodisperse circular DNA less than 6 μm. Length and molecular weight measurements of the large circular DNA are: Candida parapsilosis, 11.14 ± 0.45 μ and 23.1 × 106; Hansenula wingei, 8.22 ± 0.43 μm and 17.3 × 106; Kluyveromyces lactis, 11.44 ± 0.20 μm and 24.0 × 106 and Schizosaccharomyces pombe, 6.04 ± 0.16 μm and 12.5 × 106. These circular DNAs are thought to be mitochondrial DNA on the basis of their buoyant densities and their enrichment with mitochondria. The finding of a gross size difference between mitochondrial DNA from petite‐negative yeasts and Saccharomyces cerevisiae focuses attention on the suggestion that certain unique properties of the latter organism's mitochodrial DNA may be of relevance to the mechanism of the petite mutation in yeast.

Results from the theory of random walks applied to the random excision hypothesis for production of petite mutants in yeast suggest that frequency of excision should increase as a linear function of mitochondrial DNA length (see appendix). For a series of petite positive yeasts we have determined the spontaneous petite frequency (ranging from about 0.003% to 9%) and length of mtDNA (ranging from about 19 Kbp to c. 108 Kbp) and found that, while the frequency of petite mutants does generally increase with mtDNA length, the relationship is far from linear. Although these results are inconclusive concerning the random excision hypothesis they do indicate that amongst related yeasts other factors have a greater influence than mtDNA length in determining the frequency of petite mutants.

and maintained on GYP slopes (see below). Media. The basic yeast extract, peptone medium (YP), contains per liter: 3 g of KH2PO4, 5 g of yeast 959