Metabolism and disposition of [14C]5-amino-o-cresol in female F344 rats and B6C3F1 mice.

The metabolism and disposition of [(14)C]5-amino-o-cresol (AOC) in female F344 rats following oral, intravenous, and dermal administration and in female B6C3F1 mice following oral administration were studied. Greater than 80% of a single oral dose (4.0-357 mg kg(-1)) or intravenous dose (2.7 mg kg(-1)) was excreted in urine within 24 h. When the dosing site was protected from grooming, less than 10% of the dermal dose (2.5 and 26 mg kg(-1), rinsed off after 6 h) was absorbed within 24 h, and most of the absorbed radioactivity was excreted in urine. For the unprotected dermal dose, grooming played a major role in the absorption of AOC. Very little AOC-derived radioactivity was present in the surveyed tissues after 24 or 72 h regardless of route, dose level, or species. Five urinary metabolites were identified: 5-acetamido-1,4-dihydroxy-2-methylbenzene glucuronide, AOC O-glucuronide, AOC O-sulfate, N-acetyl-AOC O-glucuronide, and N-acetyl-AOC O-sulfate.

One RNA polymerase serving two genomes.

The land plant Arabidopsis thaliana contains three closely related nuclear genes encoding phage-type RNA polymerases (RpoT;1, RpoT;2 and RpoT;3). The gene products of RpoT;1 and RpoT;3 have previously been shown to be imported into mitochondria and chloroplasts, respectively. Here we show that the transit peptide of RpoT;2 possesses dual targeting properties. Transient expression assays in tobacco protoplasts as well as stable transformation of Arabidopsis plants demonstrate efficient targeting of fusion peptides consisting of the N-terminus of RpoT;2 joined to green fluorescent protein to both organelles. Thus, RpoT;2 might be the first RNA polymerase shown to transcribe genes in two different genomes. RNA polymerase activity of recombinant RpoT;2 is uneffected by the inhibitor tagetin, qualifying the gene product of RpoT;2 as a phage-type polymerase.

Inter-organellar crosstalk in higher plants: impaired chloroplast development affects mitochondrial gene and transcript levels.

Co-ordination of gene expression between the three genomes present in plastids, mitochondria and nucleus is of crucial importance for plant cells. Previous studies revealed that in white leaves of the albostrians (Hordeum vulgare cv. Haisa) mutant, photosynthesis-related plastid and nuclear genes are expressed only at an extremely low level. The plastids of this mutant lack ribosomes, photosynthetic activity and have only rudimentary membrane systems. Here we report on the expression of mitochondrial genes in albostrians barley. Steady-state RNA levels of the mitochondrial genes encoding cytochrome oxidase or ATPase subunits, coxII, coxIII, atpA, atp6, atp9 and cob, were observed to be consistently elevated in the white leaves but not in roots. Investigation of mitochondrial DNA revealed an about three-fold enhanced mitochondrial gene copy number in white compared to green leaf cells, but no differential amplification of mitochondrial genes. Analysis of plants in which the white albostrians plastids were combined with a new nuclear background showed that the enhanced transcript levels were a consequence of the impaired plastids and not of the nuclear albostrians allele. Furthermore, plants bleached by the carotenoid biosynthesis inhibitor norflurazon also showed an enhanced mitochondrial transcript level. These findings allow the conclusion that lack of chloroplast activity in an otherwise fully differentiated leaf leads to an increase in mitochondrial gene copy number and an elevated level of mitochondrial transcripts. Our results indicate an influence of plastids on the genetic apparatus of mitochondria in leaves but not in roots.

Green fluorescent protein as a marker to investigate targeting of organellar RNA polymerases of higher plants in vivo.

The recent identification of phage-type RNA polymerases encoded in the nuclear genome of higher plants has provided circumstantial evidence for functioning of these polymerases in the transcription of the mitochondrial and plastid genomes, as demonstrated by sequence analysis and in vitro import experiments. To determine the subcellular localization of the phage-type organellar RNA polymerases in plants, the putative transit peptides of the RNA polymerases RpoT;1 and RpoT;3 from Arabidopsis thaliana and RpoT from Chenopodium album were fused to the coding sequence of a green fluorescent protein (GFP). The constructs were used to stably transform A. thaliana. Transgenic plants were examined for green fluorescence with epifluorescence and confocal laser scanning microscopy. Plants expressing the GFP fusions under control of the CaMV35S promoter exhibited a distinct subcellular localization of the GFP fluorescence for each of the fusion constructs. In plants expressing GFP fusions with the putative transit peptides of ARAth;RpoT;1 and CHEal;RpoT, fluorescence was found exclusively in mitochondria, both in root and leaf cells. In contrast, GFP fluorescence in plants expressing the ARAth;RpoT;3-GFP construct accumulated in chloroplasts of leaf cells and nongreen plastids (leucoplasts) of root cells. By demonstrating targeting in plants, the data add substantial evidence for the phage-type RNA polymerases from C. album and A. thaliana to function in the transcriptional machinery of mitochondria and plastids.

Mitochondrial and chloroplast phage-type RNA polymerases in Arabidopsis.

In addition to the RNA polymerases (RNAPs) transcribing the nuclear genes, eukaryotic cells also require RNAPs to transcribe the genes of the mitochondrial genome and, in plants, of the chloroplast genome. The plant Arabidopsis thaliana was found to contain two nuclear genes similar to genes encoding the mitochondrial RNAP from yeast and RNAPs of bacteriophages T7, T3, and SP6. The putative transit peptides of the two polymerases were capable of targeting fusion proteins to mitochondria and chloroplasts, respectively, in vitro. The results indicate that the mitochondrial RNAP in plants is a bacteriophage-type enzyme. A gene duplication event may have generated the second RNAP, which along with the plastid-encoded eubacteria-like RNAP could transcribe the chloroplast genome.

Cloning and characterization of a cDNA encoding a bacteriophage-type RNA polymerase from the higher plant Chenopodium album.

We have cloned a full-length cDNA from the higher plant Chenopodium album coding for a single subunit bacteriophage-type RNA polymerase. The cDNA isolated from an actively growing cell suspension culture recognized a 3.8 kb transcript on Northern blots. The open reading frame comprises 987 amino acids with a predicted molecular mass of 112 kDa. A comparison of the protein sequence with those of the two known fungal mitochondrial RNA polymerases, from Saccharomyces cerevisiae and Neurospora crassa , reveals extensive homology between the three enzymes. with complete conservation of all catalytically essential amino acids. The putative mitochondrial RNA polymerase from C.album , as well as homologous sequences from rice and barley, which have been partially cloned, lack two catalytically non-essential regions of up to 176 amino acids near the C-terminus present in the two fungal mitochondrial RNA polymerases. The extreme N-terminus of the cloned C.album RNA polymerase displays features of a potential mitochondrial transit sequence. In phylogenetic trees constructed to compare the evolutionary relationships between the different single subunit RNA polymerases the C.album sequence forms a subgroup together with the S.cerevisiae and the N.crassa mitochondrial RNA polymerases, well separating from both bacteriophage enzymes and plasmid-encoded RNA polymerases found in mitochondria of many fungi and some higher plants.