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At least 865 records · Page 48Linked to original sources
PubMed2001

Umbravirus-encoded movement protein induces tubule formation on the surface of protoplasts and binds RNA incompletely and non-cooperatively.

Various functions of the cell-to-cell movement protein (MP) of Groundnut rosette virus (GRV) were analysed. The GRV ORF4-encoded protein was shown by immunofluorescence microscopy to generate tubular structures that protrude from the surface of the protoplast. The protein encoded by ORF4 was assessed also for RNA-binding properties. This protein was tagged at its C terminus with six histidine residues, produced in Escherichia coli using an expression vector and purified by affinity chromatography. Gel retardation analysis demonstrated that, in contrast to many other viral MPs, including the 3a MP of Cucumber mosaic virus (CMV), the ORF4-encoded protein bound non-cooperatively to viral ssRNA and formed complexes of low protein:RNA ratios. Competition binding experiments showed that the ORF4-encoded protein bound to both ssRNA and ssDNA without sequence specificity, but did not bind to dsDNA. UV cross-linking and nitrocellulose membrane-retention assays confirmed that both the GRV and the CMV MPs formed complexes with ssRNA and that these complexes showed similar stability in NaCl. Probing the MP-RNA complexes by atomic force microscopy demonstrated that the ORF4-encoded protein bound RNA incompletely, leaving protein-free RNA segments of varying length, while the CMV 3a protein formed highly packed complexes. The significance of the two properties of limited RNA binding and tubule formation of the umbraviral MP is discussed.

Arachis↗
PubMed2001

Comparisons among the larger genome segments of six nodaviruses and their encoded RNA replicases.

The Nodaviridae are a family of isometric RNA viruses that infect insects and fish. Their genomes, which are among the smallest known for animal viruses, consist of two co-encapsidated positive-sense RNA segments: RNA1 encodes the viral contribution to the RNA-dependent RNA polymerase (RdRp) which replicates the viral genome, whereas RNA2 encodes the capsid protein precursor. In this study, the RNA1 sequences of two insect nodaviruses - Nodamura virus (the prototype of the genus) and Boolarra virus - are reported as well as detailed comparisons of their encoded RdRps with those of three other nodaviruses of insects and one of fish. Although the 5' and 3' untranslated regions did not reveal common features of RNA sequence or secondary structure, these divergent viruses showed similar genome organizations and encoded RdRps that had from 26 to 99% amino acid sequence identity. All six RdRp amino acid sequences contained canonical RNA polymerase motifs in their C-terminal halves and conserved elements of predicted secondary structure throughout. A search for structural homologues in the protein structure database identified the poliovirus RdRp, 3D(pol), as the best template for homology modelling of the RNA polymerase domain of Pariacoto virus and allowed the construction of a congruent three-dimensional model. These results extend our understanding of the relationships among the RNA1 segments of nodaviruses and the predicted structures of their encoded RdRps.

3' Untranslated Regions↗
PubMed2002

Muscovy duck reovirus sigmaC protein is atypically encoded by the smallest genome segment.

Although muscovy duck reovirus (DRV) shares properties with the reovirus isolated from chicken, commonly named avian reovirus (ARV), the two virus species are antigenically different. Similar to the DRV sigmaB-encoded gene (1201 bp long) previously identified, the three other double-stranded RNA small genome segments of DRV have been cloned and sequenced. They were 1325, 1191 and 1124 bp long, respectively, and contained conserved terminal sequences common to ARVs. They coded for single expression products, except the smallest (S4), which contained two overlapping open reading frames (ORF1 and ORF2). BLAST analyses revealed that the proteins encoded by the 1325 and 1191 bp genes shared high identity levels with ARV sigmaA and sigmaNS, respectively, and to a lesser extent with other orthoreovirus counterparts. No homology was found for the S4 ORF1-encoded p10 protein. The 29.4 kDa product encoded by S4 ORF2 appeared to be 25% identical to ARV S1 ORF3-encoded sigmaC, a cell-attachment oligomer inducing type-specific neutralizing antibodies. Introduction of large gaps in the N-terminal part of the DRV protein was necessary to improve DRV and ARV sigmaC amino acid sequence alignments. However, a leucine zipper motif was conserved and secondary structure analyses predicted a three-stranded alpha-helical coiled-coil feature at this amino portion. Thus, despite extensive sequence divergence, DRV sigmaC was suggested to be structurally and probably functionally related to ARV sigmaC. This work provides evidence for the diversity of the polycistronic S class genes of reoviruses isolated from birds and raises the question of the relative classification of DRV in the Orthoreovirus genus.

Amino Acid Sequence↗
PubMed1985

Characterization of DNA fragments encoding fimbriae of the uropathogenic Escherichia coli strain KS71.

Recombinant plasmids were constructed that expressed the KS71A, KS71B and KS71C fimbrial antigens of the pyelonephritogenic Escherichia coli strain KS71 (O4:K12) in E. coli HB101. The KS71C-encoding genes were located on a 6.4 kb HindIII-XhoI fragment obtained from the recombinant cosmid pKTH145 that expresses this antigen. Spontaneous KS71C-mutants were isolated that contained a 0.8 kb insert in a specific restriction fragment of KS71C-encoding recombinant plasmids. The KS71B-encoding segment was located on a 11.5 kb deletable DNA fragment of recombinant cosmid pKTH144. A DNA fragment encoding the KS71A fimbria was obtained on a 12 kb EcoRI fragment of the recombinant cosmid expressing this antigen in E. coli HB101 and closely resembled the KS71B-encoding fragment. In the recombinant cosmid, the KS71B-expressing region was flanked by homologous DNA segments. A similar stretch of DNA was found close to the KS71A-expressing DNA region.

Cloning, Molecular↗
PubMed1989

Factors affecting conjugal transfer of plasmids encoding mercury resistance from pure cultures and mixed natural suspensions of epilithic bacteria.

Sixty-five pure cultures of epilithic bacteria were examined for their ability to transfer mercury resistance to Pseudomonas aeruginosa; five isolates transferred plasmids encoding mercury resistance with frequencies ranging from 8.4 x 10(-8) to 2.8 x 10(-3) per recipient. Two of the plasmids, pQM3 and pQM4, encoded narrow-spectrum mercury resistance, pQM3 also encoded streptomycin resistance, and both plasmids were broad host range. Maximum transfer frequencies of epilithic plasmids from pure cultures occurred over the range 10-25 degrees C at 3.5 g C l-1 and with donor to recipient ratios of 0.4-30. Transfer occurred over a range of pH values (pH 5.0-8.0) but the effect of pH was most significant at non-optimal temperature. Anaerobiosis inhibited transfer of one epilithic plasmid, pQM1, but not that of pQM3. Plasmids encoding mercury resistance were also transferred from mixed natural suspensions of epilithic bacteria (MNS) to Pseudomonas spp. on agar in the laboratory. Transfer from MNS occurred over a wide range of environmentally relevant conditions with maximum frequencies (2 x 10(-5) per recipient) after 24 h, at 25 degrees C, pH 5.5-8.0 and on a medium containing 10 g C l-1. The optimal initial cell density of MNS and recipient was 1.7 x 10(5) c.f.u. cm-2 and highest frequencies were obtained with donor to recipient ratios ranging from 1.2 x 10(-1) to 1.7 x 10(-3). Most of the plasmids (54%) from MNS transferred from their original P. aeruginosa transconjugants to a Pseudomonas putida strain, with frequencies ranging from 1.1 x 10(-6) to greater than 1.0 x 10(-1) per recipient. The majority (80%) of the plasmids were larger than 300 kb and all of these large plasmids encoded UV resistance in addition to mercury resistance. Twenty-one plasmids greater than 300 kb were analysed by restriction digests and were shown to be similar, with only minor structural alterations. One of these alterations was associated with the acquisition of streptomycin resistance. Overall, these results suggest that the epilithic bacteria examined possess the potential to transfer mercury resistance within the epilithon under a wide range of environmentally relevant conditions.

Bacteria↗
PubMed1999

Sequence analysis of three Bacillus cereus loci carrying PIcR-regulated genes encoding degradative enzymes and enterotoxin.

PIcR is a pleiotropic regulator of extracellular virulence factors in the opportunistic human pathogen Bacillus cereus and the entomopathogenic Bacillus thuringiensis, and is induced in cells entering stationary phase. Among the genes regulated by PIcR are: pIcA, encoding phosphatidylinositol-specific phospholipase C (PI-PLC); plc, encoding phosphatidylcholine-preferring phospholipase C (PC-PLC); nhe, encoding the non-haemolytic enterotoxin; hbl, encoding haemolytic enterotoxin BL (HBL); and genes specifying a putative S-layer like surface protein and a putative extracellular RNase. By analysing 37.1 kb of DNA sequence surrounding hbl, plcA and plcR, 28 ORFs were predicted. Three novel genes putatively regulated by PlcR and encoding a neutral protease (NprB), a subtilase family serine protease (Sfp) and a putative cell-wall hydrolase (Cwh) were identified. The corresponding sfp and cwh genes were located in the immediate upstream region of plcA and could both be regulated by a putative PlcR-binding site positioned between the inversely transcribed genes. Similarly, nprB was positioned directly upstream and transcribed in the opposite orientation to plcR. Genes surrounding plcA, plcR and hblCDAB that were lacking an upstream PlcR regulatory sequence did not appear to serve functions apparently related to PlcR and did not exhibit a conserved organization in Bacillus subtilis.

Amino Acid Sequence↗
PubMed2001

Acinetobacter baumannii has two genes encoding glutathione-dependent formaldehyde dehydrogenase: evidence for differential regulation in response to iron.

The adhC1 gene from Acinetobacter baumannii 8399, which encodes a glutathione-dependent formaldehyde dehydrogenase (GSH-FDH), was identified and cloned after mapping the insertion site of Tn3-HoHo1 in a recombinant cosmid isolated from a gene library. Sequence analysis showed that this gene encodes a protein exhibiting significant similarity to alcohol dehydrogenases in bacterial, yeast, plant and animal cells. The expression of the adhC1 gene was confirmed by the detection of GSH-FDH enzyme activity in A. baumannii and Escherichia coli cells that expressed the cloned gene. However, the construction and analysis of an A. baumannii 8399 adhC1::Tn3-HoHo1 isogenic derivative revealed the presence of adhC2, a second copy of the gene encoding GSH-FDH activity. Enzyme assays and immunoblot analysis showed that adhC2 encodes a 46.5 kDa protein that is produced in similar amounts under iron-rich and iron-limited conditions. In contrast, the expression of adhC1, which encodes a 45 kDa protein with GSH-FDH activity, is induced under iron limitation and repressed when the cells are cultured in the presence of free inorganic iron. The differential expression of adhC1 is controlled at the transcriptional level and mediated through the Fur iron-repressor protein, which has potential binding sites within the promoter region of this adhC copy. The expression of both adhC copies is significantly enhanced by the presence of sub-inhibitory concentrations of formaldehyde in the culture media. Examination of different A. baumannii isolates indicates that they can be divided into two groups based on the type of GSH-FDH they produce. One group contains only the constitutively expressed 46.5 kDa protein, whilst the other produces this GSH-FDH type in addition to the iron-regulated isoenzyme. Further analysis showed that the presence and expression of the two adhC genes does not confer resistance to exogenous formaldehyde, nor does it enable it to utilize methylated compounds as a sole carbon source when cultured under iron-rich as well as iron-deficient conditions.

Acinetobacter↗
PubMed2002

Characterization of a novel Vibrio pathogenicity island (VPI-2) encoding neuraminidase (nanH) among toxigenic Vibrio cholerae isolates.

Acquisition of virulence genes encoded on mobile genetic elements has played an important role in the emergence of pathogenic isolates of Vibrio cholerae, the causative agent of the diarrhoeal disease cholera. The genes encoding cholera toxin (ctxAB), the main cause of profuse secretory diarrhoea in cholera, are encoded on a filamentous bacteriophage CTXphi. The toxin coregulated pilus (TCP), an essential intestinal colonization factor, was originally designated as part of a pathogenicity island named the Vibrio pathogenicity island (VPI), but this island has more recently been proposed to be the genome of a filamentous phage, VPIphi. In this study, it is shown that nanH, which encodes neuraminidase, maps within a novel pathogenicity island designated VPI-2. The 57.3 kb VPI-2 has all of the characteristic features of a pathogenicity island, including the presence of a bacteriophage-like integrase (int), insertion in a tRNA gene (serine) and the presence of direct repeats at the chromosomal integration sites. Additionally, the G+C content of VPI-2 (42 mol%) is considerably lower than that of the entire genome (47 mol%). VPI-2 encodes several gene clusters, such as a restriction modification system (hsdR and hsdM) and genes required for the utilization of amino sugars (nan-nag region) as well as neuraminidase. To determine the distribution of VPI-2 among V. cholerae, 78 natural isolates were examined using PCR and Southern hybridization analysis for the presence of this region. All toxigenic V. cholerae O1 serogroup isolates examined contained VPI-2, whereas non-toxigenic isolates lacked the island. Of 14 V. cholerae O139 serogroup isolates examined, only one strain, MO2, contained the entire 57.3 kb island, whereas 13 O139 isolates contained only a 20.0 kb region with most of the 5' region of VPI-2 which included nanH deleted in these strains.

Humans↗
PubMed1995

An operon encoding aspartokinase and purine phosphoribosyltransferase in Thermus flavus.

The nucleotide sequence of a 1.1 kb XhoI-HindIII fragment downstream of the malate dehydrogenase (mdh) gene of Thermus flavus revealed the presence of an ORF and an incomplete ORF lacking its NH2-terminal portion, in the opposite orientation to that of the mdh gene. These two genes overlapped with each other, sharing two base pairs, suggesting that these genes are co-transcribed in a single mRNA. One ORF (termed gpt) encoded a protein of 154 amino acids showing significant amino acid sequence similarity to purine phosphoribosyltransferases, such as xanthine-guanine phosphoribosyltransferase of Escherichia coli and human hypoxanthine phosphoribosyltransferase. Cloning and sequencing of the upstream region of the gpt gene, together with sequence comparison of the gene product encoded by the region upstream of gpt, suggested that the upstream ORF encoded two in-frame overlapping aspartokinase genes, askA, encoding the alpha-subunit of 405 amino acids, and askB, encoding the beta-subunit of 161 amino acids, which was part of the 3' portion of askA. Consistent with the sequence data, the askAB and the gpt genes conferred the heat-stable enzyme activities of aspartokinase and phosphoribosyltransferase, respectively, on E. coli. Preliminary characterization of these enzymes produced in E. coli is described.

Amino Acid Sequence↗
PubMed1996

Novel phosphotransferase genes revealed by bacterial genome sequencing: a gene cluster encoding a putative N-acetylgalactosamine metabolic pathway in Escherichia coli.

We have analysed a gene cluster in the 67 center dot 4-76 center dot 0 min region of the Escherichia coli chromosome, revealed by recent systematic genome sequencing. The genes within this cluster include: (1) five genes encoding homologues of the E. coli mannose permease of the phosphotransferase system (IIB, IIB', IIC, IIC' and IID); (2) genes encoding a putative N-acetylgalactosamine 6-phosphate metabolic pathway including (a) a deacetylase, (b) an isomerizing deaminase, (c) a putative carbohydrate kinase, and (d) an aldolase; and (3) a transcriptional regulatory protein homologous to members of the DeoR family. Evidence is presented suggesting that the aldolase-encoding gene within this cluster is the previously designated kba gene that encodes tagatose-1,6-bisphosphate aldolase. These proteins and a novel IIAMan-like protein encoded in the 2 center dot 4-4 center dot 1 min region are characterized with respect to their sequence similarities and phylogenetic relationships with other homologous proteins. A pathway for the metabolism of N-acetylgalactosamine biochemically similar to that for the metabolism of N-acetylglucosamine is proposed.

Acetylgalactosamine↗
PubMed2003

Allelic variation in the contiguous loci encoding Candida albicans ALS5, ALS1 and ALS9.

The ALS gene family of Candida albicans consists of eight genes (ALS1 to ALS7 and ALS9) that encode cell-wall glycoproteins involved in adhesion to host surfaces. Considerable allelic sequence variability has been documented for regions of ALS genes encoding repeated sequences. Although regions of ALS genes encoding non-repeated sequences tend to be more conserved, some sequence divergence has been noted, particularly for alleles of ALS5. Data from the C. albicans genome sequencing project provided the first indication that strain SC5314 encoded two divergent ALS9-like sequences and that three of the ALS genes (ALS5, ALS1 and ALS9) were contiguous on chromosome 6. Data from PCR analysis and construction of both single and double deletion mutants indicated that the divergent sequences were alleles of ALS9, and located downstream of ALS5 and ALS1. Sequences within the 5' domain of ALS9-1 and ALS9-2 varied by 11 %. Within the 3' domain of each allele, extra nucleotides were present in two regions of ALS9-2, designated Variable Block 1 (VB1) and Variable Block 2 (VB2). Analysis of strains from the five major C. albicans genetic clades showed that both ALS9 alleles are widespread among these strains, that the sequences of ALS9-1 and ALS9-2 are conserved among diverse strains and that recombinant ALS9 alleles have been generated during C. albicans evolution. Phylogenetic analysis showed that, although divergent in sequence, ALS9 alleles are more similar to each other than to any other ALS genes. The degree of sequence divergence for ALS9 greatly exceeds that observed previously for other ALS genes and may result in functional differences for the proteins encoded by the two alleles.

Alleles↗
PubMed2005

Sau42I, a BcgI-like restriction-modification system encoded by the Staphylococcus aureus quadruple-converting phage Phi42.

The serotype F phage Phi42 of Staphylococcus aureus is a triple-converting bacteriophage that encodes the staphylokinase gene (sak) and the enterotoxin A gene (entA). Lysogeny results in loss of expression of the chromosomal beta-haemolysin gene (hlb) (negative conversion), the expression of staphylokinase and enterotoxin A (positive conversion), and the acquisition of resistance to lysis by all 23 phages of the International Basic Set (IBS) of S. aureus typing phages. Until this study, the basis of Phi42 resistance to lysis by exogenous phages was unknown. The authors report here that phage Phi42 encodes a restriction-modification (R-M) system, termed Sau42I, adjacent to and in the same orientation to the phage integrase gene int. The genes encoding Sau42I were cloned and sequenced, and found to consist of two overlapping reading frames, ORF S (specificity) and ORF RM (restriction-modification), in the same orientation. The ORFs share a high degree of DNA and amino acid sequence homology with the previously characterized BcgI R-M system of Bacillus coagulans. Expression of the cloned Sau42I ORF S and ORF RM in S. aureus 80CR3 transformants from a plasmid vector conferred resistance to lysis by all 23 IBS phages. Similarly, transformants of S. aureus RN4220 harbouring recombinant plasmids containing both ORFs were resistant to lysis by the IBS typing phages. However, transformants harbouring plasmids encoding either ORF S or ORF RM were susceptible to lysis by the IBS phages, and they had the same phage-susceptibility pattern as the respective parental isolates. In vitro analysis of crude and partially purified extracts of S. aureus transformants harbouring both the Phi42 ORF S and ORF RM genes indicated that Sau42I has endonuclease activity and requires co-factors Mg(2+) and S-adenosylmethionine in order to function, and activity is optimized at pH 8, although the precise recognition sequence has yet to be determined. The findings of this study confirm that Phi42 is a quadruple-converting phage, believed to be the first described for S. aureus, and show that it encodes a novel R-M system termed Sau42I.

Amino Acid Sequence↗
PubMed2005

Chromosome-encoded gene cluster for the metabolic pathway that converts aniline to TCA-cycle intermediates in Delftia tsuruhatensis AD9.

Delftia tsuruhatensis AD9 was isolated as an aniline-degrading bacterium from the soil surrounding a textile dyeing plant. The gene cluster involved in aniline degradation was cloned from the total DNA of strain AD9 into Escherichia coli JM109. After shotgun cloning, two recombinant E. coli strains showing aniline oxidation activity or catechol meta-cleavage activity were obtained by simple plate assays. These strains contained 9.3 kb and 15.4 kb DNA fragments, respectively. Sequence analysis of the total 24.7 kb region revealed that this region contains a gene cluster (consisting of at least 17 genes, named tadQTA1A2BRD1C1D2C2EFGIJKL) responsible for the complete metabolism of aniline to TCA-cycle intermediates. In the gene cluster, the first five genes (tadQTA1A2B) and the subsequent gene (tadR) were predicted to encode a multi-component aniline dioxygenase and a LysR-type regulator, respectively, while the others (tadD1C1D2C2EFGIJKL) were expected to encode meta-cleavage pathway enzymes for catechol degradation. In addition, it was found that the gene cluster is surrounded by two IS1071 sequences, indicating that it has a class I transposon-like structure. PFGE and Southern hybridization analyses confirmed that the tad gene cluster is encoded on the chromosome of strain AD9 in a single copy. These results suggest that, in strain AD9, aniline is degraded via catechol through a meta-cleavage pathway by the chromosome-encoded tad gene cluster. The tad gene cluster showed significant similarity in nucleotide sequence and genetic organization to the plasmid-encoded aniline degradation gene cluster of Pseudomonas putida UCC22.

Aniline Compounds↗
PubMed1997

A bacterial group II intron encoding reverse transcriptase, maturase, and DNA endonuclease activities: biochemical demonstration of maturase activity and insertion of new genetic information within the intron.

The Lactococcus lactis group II intron Ll.ltrB is similar to mobile yeast mtDNA group II introns, which encode reverse transcriptase, RNA maturase, and DNA endonuclease activities for site-specific DNA insertion. Here, we show that the Lactococcal intron can be expressed and spliced efficiently in Escherichia coli. The intron-encoded protein LtrA has reverse transcriptase and RNA maturase activities, with the latter activity shown both in vivo and in vitro, a first for any group II intron-encoded protein. As for the yeast mtDNA introns, the DNA endonuclease activity of the Lactococcal intron is associated with RNP particles containing both the intron-encoded protein and the excised intron RNA. Also, the intron RNA cleaves the sense-strand of the recipient DNA by a reverse splicing reaction, whereas the intron-encoded protein cleaves the antisense strand. The Lactococcal intron endonuclease can be obtained in large quantities by coexpression of the LtrA protein with the intron RNA in E. coli or reconstituted in vitro by incubating the expressed LtrA protein with in vitro-synthesized intron RNA. Furthermore, the specificity of the endonuclease and reverse splicing reactions can be changed predictably by modifying the RNA component. Expression in E. coli facilitates the use of group II introns for the targeting of specific foreign sequences to a desired site in DNA.

Bacterial Proteins↗
PubMedDate not supplied

Cholinergic modulation of the hippocampus during encoding and retrieval of tone/shock-induced fear conditioning.

We investigated the role of acetylcholine (ACh) during encoding and retrieval of tone/shock-induced fear conditioning with the aim of testing Hasselmo's cholinergic modulation model of encoding and retrieval using a task sensitive to hippocampal disruption. Lesions of the hippocampus impair acquisition and retention of contextual conditioning with no effect on tone conditioning. Cholinergic antagonists also impair acquisition of contextual conditioning. Saline, scopolamine, or physostigmine was administered directly into the CA3 subregion of the hippocampus 10 min before rats were trained on a tone/shock-induced fear conditioning paradigm. Freezing behavior was used as the measure of learning. The scopolamine group froze significantly less during acquisition to the context relative to controls. The scopolamine group also froze less to the context test administered 24 h posttraining. A finer analysis of the data revealed that scopolamine disrupted encoding but not retrieval. The physostigmine group initially froze less during acquisition to the context, although this was not significantly different from controls. During the context test, the physostigmine group froze less initially but quickly matched the freezing levels of controls. A finer analysis of the data indicated that physostigmine disrupted retrieval but not encoding. These results suggest that increased ACh levels are necessary for encoding new spatial contexts, whereas decreased ACh levels are necessary for retrieving previously learned spatial contexts.

Acetylcholine↗
PubMed1993

Plastid Genes Encoding the Transcription/Translation Apparatus Are Differentially Transcribed Early in Barley (Hordeum vulgare) Chloroplast Development (Evidence for Selective Stabilization of psbA mRNA).

Chloroplast genomes encode rRNAs, tRNAs, and proteins involved in transcription, translation, and photosynthesis. The expression of 15 plastid genes representing each of these functions was quantitated during chloroplast development in barley (Hordeum vulgare). The transcription of all plastid genes increased during the initial phase of chloroplast development and then declined during chloroplast maturation. RNAs corresponding to rpoB- rpoC1-rpoC2, which encode subunits of a plastid RNA polymerase, and rps16, which encodes a ribosomal protein, reached maximal abundance early in chloroplast development prior to genes encoding subunits of the photosynthetic apparatus (rbcL, atpB, psaA, petB). Transcription of rpoB as well as 16S rRNA, trnfM-trnG, and trnK was high early in chloroplast development and declined 10-fold relative to rbcL transcription during chloroplast maturation. RNA hybridizing to psbA and psbD, genes encoding reaction center proteins of photosystem II, was differentially maintained in mature chloroplasts of illuminated barley. Differential accumulation of psbD mRNA relative to rbcL mRNA was due to light-stimulated transcription of psbD. In contrast, enhanced levels of psbA mRNA in mature chloroplasts were due primarily to selective stabilization of the psbA mRNA. These data document dynamic modulation of plastid gene transcription and mRNA stability during barley chloroplast development.

Journal Article↗
PubMed2003

AtSig5 is an essential nucleus-encoded Arabidopsis sigma-like factor.

Transcription of chloroplast genes is subject to control by nucleus-encoded proteins. The chloroplast-encoded RNA polymerase (PEP) is a eubacterial-type RNA polymerase that is presumed to assemble with nucleus-encoded sigma-factors mediating promoter recognition. Recently, families of sigma-factor genes have been identified in several plants including Arabidopsis. One of these genes, Arabidopsis SIG5, encodes a sigma-factor, AtSig5, which is phylogenetically distinct from the other family members. To investigate the role of this plant sigma-factor, two different insertional alleles of the SIG5 gene were identified and characterized. Heterozygous mutant plants showed no visible leaf phenotype, but exhibited siliques containing aborted embryos and unfertilized ovules. Our inability to recover plants homozygous for a SIG5 gene disruption indicates that SIG5 is an essential gene. SIG5 transcripts accumulate in flower tissues, consistent with a role for AtSig5 protein in reproduction. Therefore, SIG5 encodes an essential member of the Arabidopsis sigma-factor family that plays a role in plant reproduction in addition to its previously proposed role in leaf chloroplast gene expression.

Arabidopsis↗
PubMed2006

The SCABRA3 nuclear gene encodes the plastid RpoTp RNA polymerase, which is required for chloroplast biogenesis and mesophyll cell proliferation in Arabidopsis.

In many plant species, a subset of the genes of the chloroplast genome is transcribed by RpoTp, a nuclear-encoded plastid-targeted RNA polymerase. Here, we describe the positional cloning of the SCABRA3 (SCA3) gene, which was found to encode RpoTp in Arabidopsis (Arabidopsis thaliana). We studied one weak (sca3-1) and two strong (sca3-2 and sca3-3) alleles of the SCA3 gene, the latter two showing severely impaired plant growth and reduced pigmentation of the cotyledons, leaves, stem, and sepals, all of which were pale green. The leaf surface was extremely crumpled in the sca3 mutants, although epidermal cell size and morphology were not perturbed, whereas the mesophyll cells were less densely packed and more irregular in shape than in the wild type. A significant reduction in the size, morphology, and number of chloroplasts was observed in homozygous sca3-2 individuals whose photoautotrophic growth was consequently perturbed. Microarray analysis showed that several hundred nuclear genes were differentially expressed in sca3-2 and the wild type, about one-fourth of which encoded chloroplast-targeted proteins. Quantitative reverse transcription-PCR analyses showed that the sca3-2 mutation alters the expression of the rpoB, rpoC1, clpP, and accD plastid genes and the SCA3 paralogs RpoTm and RpoTmp, which respectively encode nuclear-encoded mitochondrion or dually targeted RNA polymerases. Double-mutant analysis indicated that RpoTmp and SCA3 play redundant functions in plant development. Our findings support a role for plastids in leaf morphogenesis and indicate that RpoTp is required for mesophyll cell proliferation.

Alleles↗