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A Weihe

Publications and source records attributed to A Weihe.

12 recordsLinked to original sources
PubMed2000

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.

Amino Acid Sequence↗
PubMed1999

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.

Arabidopsis↗
PubMed1997

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.

Amino Acid Sequence↗
PubMed1997

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.

Amino Acid Sequence↗
PubMed1995

Characterization of the single psbA gene of Prochlorococcus marinus CCMP 1375 (Prochlorophyta).

DNA sequence, copy number, expression and phylogenetic relevance of the psbA gene from the abundant marine prokaryote P. marinus CCMP 1375 was analyzed. The 7 amino acids near the C-terminus missing in higher plant and in Prochlorothrix hollandica D1 proteins are present in the derived amino acid sequence. P. marinus contains only a single psbA gene. Thus, this organism lacks the ability to adapt its photosystem II by replacement of one type of D1 by another, as several cyanobacteria do. Phylogenetic trees suggested the D1-1 iso-form from Synechococcus PCC 7942 as the next related D1 protein and place P. marinus separately from Prochlorothrix hollandica among the cyanobacteria.

Amino Acid Sequence↗
PubMed1983

Phosphorylated nonhistone proteins in different fractions of rat liver nuclei obtained by metrizamide gradient centrifugation.

The distribution of nuclear nonhistone proteins (NHPs) labelled in vivo with 32P was investigated in different subnuclear fractions obtained by metrizamide gradient centrifugation of mildly sonicated rat liver nuclei. The bulk chromatin banding at 1.20-1.22 g to cm3 (fraction C) contained only minor amounts both of non-phosphorylated and 32P-labelled NHPs. Nick-translated DNA sequences were found to sediment at a slightly higher density (1.26 g/cm3) in a minor fraction (A). Fraction A also contained a significant portion of the newly synthesized RNA and was enriched additionally with a broad spectrum of NHPs; some of them were highly 32P-labelled. Two hnRNP fractions (R2 and R1) sedimenting at densities greater than 1.28 g/cm3 were analyzed, too. By SDS-PAGE it was shown that the most abundant 32P-NHPs were found in three groups (fraction A, R1, and R2). Two of them, 32-34 KDa, and 37-40 KDa, are components of the major proteins of the hnRNP particles. A group of four moderately labelled 32P-NHPs of approximately 55 K, 64 K, 70 K, and 90 KDa appeared substantially enriched in fraction A. They were found also in RNP fractions R1 and R2, but to a smaller mount. Therefore, we suggest that in fraction A they exist in a complex with hnRNP. Fraction A was also enriched with numerous 32P-NHPs of very high molecular weight found in other types of experiments nearly exclusively in the nuclear residual structures. The 32P-protein pattern of the nucleoli was shown to be different from that of the other subnuclear fractions. Considering our observations on the accumulation of nick-translated DNA sequences, the presence of newly synthesized RNA, the high content of NHPs, and the association of special 32P-NHPs we suggest that fraction A represents transcriptionally active chromatin.

Animals↗
PubMed1982

Discrimination of several classes of nonhistone proteins in chromatin fractions from liver and thymus nuclei of rats.

Nonhistone proteins (NHPs) of salt-soluble chromatin (Chromatin S) and of the residual nuclei (Chromatin P) from rat liver and thymus were studied by SDS-polyacrylamide gel electrophoresis. The two chromatin fractions of the liver showed significant differences in their NHP patterns with most of the hnRNP and matrix proteins occurring in Chromatin P. In accordance with the low protein content of thymus nuclei, the corresponding thymus fractions exhibited electrophoretic patterns with a markedly lower amount of NHPs than in liver. Chromatin P from thymus, in contrast to the liver fraction, revealed only a very low content of hnRNP-specific proteins of molecular weight 30,000-40,000 (30 K to 40 K) (informosomal proteins) consistent with the significantly lower RNA content of thymus nuclei. In the region of the matrix proteins (60-75 K) Chromatin P showed only two bands of about 64 K and 73 K in thymus, whereas in liver five strong bands at 64 K, 66 K, 69 K, 73 K, and 75 K were found. RNase digestion was employed to discriminate hnRNP-specific protein from "real" chromosomal NHPs. At least about 65% and 25% of the NHPs from Chromatin P and S of liver, respectively, were found to be RNP-specific. The two chromatin fractions were further fractionated by sucrose gradient centrifugation and isopycnic banding in metrizamide. After centrifugation the main peaks, both of Chromatin S and P, contained only minor amounts of NHPs with a predominating protein of 38 K. By the centrifugation procedures described in this paper, a small subfraction of chromatin could be separated which was enriched in newly synthesized RNA, informosomal proteins, matrix- and other high molecular weight proteins. This subfraction might be related to transcriptionally active chromatin.

Animals↗
PubMed1978

Complexes of DNA with arginine-rich and slightly lysine-rich histones. Transcription and electron microscopy.

Calf thymus DNA was reconstituted with arginine-rich histones H3 and H4 and slightly lysine-rich histones H2A and H2B respectively. Complexes containing histones H3 and H4 exhibit nucleosome-like structures when examined in the electron microscope and show a restriction of in vitro transcription similar to that obtained for reconstitutes made up from the four histones H2A, H2B, H3 and H4. One the contrary, complexes of DNA and histones H2A and H2B create different morphological structures of short stretches of bound histones and do not cause a template restriction in vitro.

Animals↗
PubMed1975

Effect of RNA synthesis on the binding of 3H-cortisol to nuclear ribonucleoprotein particles from rat liver carrying DNA-like RNA in vivo.

3H-cortisol was found to associate with rat liver nuclear 30S ribonucleoprotein particles carrying D-RNA in vivo. No interaction was detectable when RNA synthesis was inhibited by alpha-amanitine. The association appears to be specifically for RNP carrying RNA synthesized after the administration of cortisol to adrenalectomized rats. The DNA/protein/RNA ratio of rat liver nuclei was not effected by alpha-amanitine under our conditions. However, the drug caused a 5-10 fold decrease in nuclear uptake of cortisol. The results are discussed in relation to a supposed transfer of cortisol-receptor complexes from the chromatin template to the nascent RNA chains.

Adrenalectomy↗