Analysis of the nucleotide sequence of the genetic locus for yeast mitochondrial RNA polymerase (RPO41) reveals a continuous open reading frame with the coding potential for a polypeptide of 1351 amino acids, a size consistent with the electrophoretic mobility of this enzymatic activity. The transcription product from this gene spans the singular reading frame. In vivo transcript abundance reflects codon usage and growth under stringent conditions for mitochondrial biogenesis and function results in a several fold higher level of gene expression than growth under glucose repression. A comparison of the yeast mitochondrial RNA polymerase amino acid sequence to those of E. coli RNA polymerase subunits failed to demonstrate any regions of homology. Interestingly, the mitochondrial enzyme is highly homologous to the DNA-directed RNA polymerases of bacteriophages T3 and T7, especially in regions most highly conserved between the T3 and T7 enzymes themselves.

Langerhans cells (LC) are epidermal dendritic cells capable, in several experimental systems, of Ag-presentation for stimulation of cell-mediated immunity. LC have been considered to play a key role in initiation of cutaneous immune responses. Additionally, administration of donor T cells to bone marrow chimeric mice with persistent host LC, but not mice whose LC have been replaced by donor cells, exhibit marked skin graft-vs-host disease, demonstrating that LC can trigger graft-vs-host disease. However, experiments with transgenic mice in which regulatory elements from human langerin were used to drive expression of diphtheria toxin, resulting in absence of LC, suggest that LC may serve to down-regulate cutaneous immunity. LC are associated with nerves containing the neuropeptide calcitonin gene-related peptide (CGRP), and CGRP inhibits LC Ag-presentation in several models including presentation to a Th1 clone. We now report that CGRP enhances LC function for stimulation of Th2 responses. CGRP exposure enhanced LC Ag presentation to a Th2 clone. Upon presentation of chicken OVA by LC to T cells from DO11.10 chicken OVA TCR transgenic mice, pretreatment with CGRP resulted in increased IL-4 production and decreased IFN-γ production. CGRP also inhibited stimulated production of the Th1 chemokines CXCL9 and CXCL10 but induced production of the Th2 chemokines CCL17 and CCL22 by a dendritic cell line and by freshly obtained LC. Changes in production of these chemokines correlated with the effect of CGRP on mRNA levels for these factors. Exposure of LC to nerve-derived CGRP in situ may polarize them toward favoring Th2-type immunity.

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The overall mode of mitochondrial DNA (mtDNA) replication is best understood for the mouse and human systems, and a general model of vertebrate mtDNA replication has been established (9). Mammalian mtDNA replicates by unidirectional synthesis from two distinct origins, the origin of heavy-strand replication and the origin of light-strand replication, which are located two-thirds of the genomic distance apart on the closed circle. The structural features and mechanisms of RNA priming are very different at these two origins (3,7,9,29,30). A characteristic hallmark of the origin of heavy-strand replication is the presence of three evolutionarily conserved sequence blocks (CSBs I, II, and III) at or near the 5' ends of newly synthesized heavy strands. It has been demonstrated for nearly all 5' ends of nascent heavy-strand DNAs that there are RNA species whose 3' ends map immediately adjacent to the DNA 5' ends. It has also been shown that 5' ends of these RNAs map at the initiation site of transcription from the major lightstrand promoter (3, 7). On the basis of these and other findings, Chang and coworkers hypothesized that transcripts from this promoter play a role in heavy-strand replication by serving as primers for DNA synthesis and that individual primer termini were generated by processing of a primary transcript (3, 7).Mammalian complementary to the origin of leading heavy-strand mtDNA replication (4, 6, 28). The endonuclease activity is present in both nuclear and mitochondrial fractions (2,5,14,17,28) and has been proposed to participate in mitochondrial primer RNA metabolism in vivo. The standard mitochondrial RNA (mtRNA) substrate for mouse and human RNase MRPs contains three short regions of sequence that are highly conserved in vertebrate species (CSBs I, II, and III). Initial characterization of the mouse RNase MRP activity demonstrated that the site-specific cleavage of mtRNA substrate in vitro occurred immediately adjacent to CSB II, and it was postulated that CSB II played a role in cleavage site specificity (4). Subsequent deletional analysis and saturation mutagenesis have determined basic substrate requirements for cleavage by mouse RNase MRP; CSB II and CSB III are essential for both efficient and accurate cleavage, whereas CSB I is not (2).In contrast to the mammalian system, our knowledge of yeast wild-type mtDNA replication initiation, at the molecular level, is less advanced. However, studies with hypersuppressive petite strains indicate that putative yeast mitochondrial replication origins (on [or rep {reference 12 and references therein}] sequences) are characterized by a 300-bp A+T-rich segment containing the following regions: a 16-bp A+T-rich sequence containing an active promoter for transcription initiation (termed r), a 17-bp GC cluster C located immediately downstream of the promoter, a central 200-bp A+T-rich stretch (termed C), and GC clusters A and B, which are separated by an A+T-rich region (1, 10, 13). It has also been proposed that the active on sequences of yeast mtDNA are ...

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