Biomedical subjects

P Maliga

Publications and source records attributed to P Maliga.

At least 37 records · Page 2Linked to original sources

PubMed1998

rbcL Transcript levels in tobacco plastids are independent of light: reduced dark transcription rate is compensated by increased mRNA stability.

The plastid rbcL gene, encoding the large subunit of ribulose-1, 5-bisphosphate carboxylase, in higher plants is transcribed from a sigma70 promoter by the eubacterial-type RNA polymerase. To identify regulatory elements outside of the rbcL -10/-35 promoter core, we constructed transplastomic tobacco plants with uidA reporter genes expressed from rbcL promoter derivatives. Promoter activity was characterized by measuring steady state levels of uidA mRNA on RNA gel blots and by measuring promoter strength in run-on transcription assays. We report here that the rbcL core promoter is sufficient to obtain wild-type rates of transcription. Furthermore, the rates of transcription were up to 10-fold higher in light-grown leaves than in dark-adapted plants. Although the rates of transcription were lower in the dark, rbcL mRNA accumulated to similar levels in light-grown and dark-adapted leaves. Accumulation of uidA mRNA from most rbcL promoter deletion derivatives directly reflected the relative rates of transcription: high in the light-grown and low in the dark-adapted leaves. However, uidA mRNA accumulated to high levels in a light-independent fashion as long as a segment encoding a stem-loop structure in the 5' untranslated region was included in the promoter construct. This finding indicates that lower rates of rbcL transcription in the dark are compensated by increased mRNA stability.

Base Sequence↗

PubMed1997

The two RNA polymerases encoded by the nuclear and the plastid compartments transcribe distinct groups of genes in tobacco plastids.

The plastid genome in photosynthetic higher plants encodes subunits of an Escherichia coli-like RNA polymerase (PEP) which initiates transcription from E.coli sigma70-type promoters. We have previously established the existence of a second nuclear-encoded plastid RNA polymerase (NEP) in photosynthetic higher plants. We report here that many plastid genes and operons have at least one promoter each for PEP and NEP (Class II transcription unit). However, a subset of plastid genes, including photosystem I and II genes, are transcribed from PEP promoters only (Class I genes), while in some instances (e.g. accD) genes are transcribed exclusively by NEP (Class III genes). Sequence alignment identified a 10 nucleotide NEP promoter consensus around the transcription initiation site. Distinct NEP and PEP promoters reported here provide a general mechanism for group-specific gene expression through recognition by the two RNA polymerases.

Adenosine Triphosphatases↗

PubMed1997

Targeted deletion of sprA from the tobacco plastid genome indicates that the encoded small RNA is not essential for pre-16S rRNA maturation in plastids.

The small plastid RNA (spRNA) which includes a segment that is complementary to the pre-16S rRNA has been suggested to facilitate maturation of pre-16S rRNA in tobacco. To investigate the function of spRNA, the gene encoding it (sprA) was removed from the plastid genome using targeted gene deletion. We report here that deletion of sprA does not significantly affect pre-16S rRNA maturation, nor does it cause any obvious phenotype. Although the spRNA still may be involved in rRNA maturation, it is non-essential under normal growth conditions.

Gene Deletion↗

PubMed1997

A negative selection scheme based on the expression of cytosine deaminase in plastids.

The enzyme cytosine deaminase (CD) encoded by codA catalyzes deamination of cytosine to uracil. CD is present in prokaryotes and in many eukaryotic micro-organisms, but is absent in higher plants. 5-fluorocytosine (5FC) is metabolized in CD-expressing cells, causing cellular death. A chimeric codA has been introduced into the tobacco plastid genome and 5FC was used to select against tissue culture cells and seedlings expressing CD. This negative selection scheme will be useful in identifying nuclear genes which control plastid gene expression in higher plants.

Catalysis↗

PubMed1996

Sequences directing C to U editing of the plastid psbL mRNA are located within a 22 nucleotide segment spanning the editing site.

In plastids, editing of an ACG codon to an AUG codon creates the translation initiation codon for the psbL and ndhD transcripts in tobacco. To identify the RNA segment required for psbL editing, chimeric kanamycin resistance genes were constructed containing psbL deletion derivatives, and tested in vivo for editing in transgenic plants. We report here that a 22 nucleotide segment is sufficient to direct efficient psbL editing, including 16 nucleotides upstream and five nucleotides downstream of the editing site. Mutation of the A nucleotide to a C upstream of the editing site completely abolished editing, while mutation of the downstream G to a C only reduced the editing efficiency. Out of the 22 nucleotide editing target sequence, the 16 upstream nucleotides were found to compete with the endogenous psbL transcript for a depletable trans-factor. To test whether editing of initiation codons involves a common trans-factor, a chimeric gene containing the ndhD editing site was expressed in tobacco plastids. As for psbL, editing of the ndhD site requires a depletable trans-factor. However, the ndhD trans-factor is distinct from that required for psbL editing. Distinct cissequences and trans-factor requirements for the psbL and ndhD editing sites indicate an individual recognition mechanism for the editing of plastid initiation codons.

Amino Acid Sequence↗

PubMed1996

Deletion of rpoB reveals a second distinct transcription system in plastids of higher plants.

The plastid genome in higher plants encodes subunits of an Escherichia coli-like RNA polymerase which initiates transcription of plastid genes from sequences resembling E.coli sigma70-type promoters. By deleting the gene for the essential beta subunit of the tobacco E.coli-like RNA polymerase, we have established the existence of a second plastid transcription system which does not utilize E.coli-like promoters. In contrast to the E.coli-like RNA polymerase, the novel transcription machinery preferentially transcribes genetic system genes rather than photosynthetic genes. Although the mutant plants are photosynthetically defective, transcription by this polymerase is sufficient for plastid maintenance and plant development.

Base Sequence↗

PubMed1996

Chloroplast mRNA 3'-end processing by a high molecular weight protein complex is regulated by nuclear encoded RNA binding proteins.

In the absence of efficient transcription termination correct 3'-end processing is an essential step in the synthesis of stable chloroplast mRNAs in higher plants. We show here that 3'-end processing in vitro involves endonucleolytic cleavage downstream from the mature terminus, followed by exonucleolytic processing to a stem-loop within the 3'-untranslated region. These processing steps require a high molecular weight complex that contains both endoribonucleases and an exoribonuclease. In the presence of ancillary RNA binding proteins the complex correctly processes the 3'-end of precursor RNA. In the absence of these ancillary proteins 3'-end maturation is prevented and plastid mRNAs are degraded. Based on these results we propose a novel mechanism for the regulation of mRNA 3'-end processing and stability in chloroplasts.

Amino Acid Sequence↗

PubMed1995

Marker rescue from the Nicotiana tabacum plastid genome using a plastid/Escherichia coli shuttle vector.

We recently reported an 868-bp plastid DNA minicircle, NICE1, that formed during transformation in a transplastomic Nicotiana tabacum line. Shuttle plasmids containing NICE1 sequences were maintained extrachromosomally in plastids and shown to undergo recombination with NICE1 sequences on the plastid genome. To prove the general utility of the shuttle plasmids, we tested whether plastid genes outside the NICE1 region could be rescued in Escherichia coli. The NICE1-based rescue plasmid, pNICER1, carries NICE1 sequences for maintenance in plastids, the ColE1 ori for maintenance in E. coli and a spectinomycin resistance gene (aadA) for selection in both systems. In addition, pNICER1 carries a defective kanamycin resistance gene, kan*, to target the rescue of a functional kanamycin resistance gene, kan, from the recipient plastid genome. pNICER1 was introduced into plastids where recombination could occur between the homologous kan/kan* sequences, and subsequently rescued in E. coli to recover the products of recombination. Based on the expression of kanamycin resistance in E. coli and the analysis of three restriction fragment polymorphisms, recombinant kan genes were recovered at a high frequency. Efficient rescue of kan from the plastid genome in E. coli indicates that NICE1-based plasmids are suitable for rescuing mutations from any part of the plastid genome, expanding the repertoire of genetic tools available for plastid biology.

Cloning, Molecular↗

PubMed1995

Light-responsive and transcription-enhancing elements regulate the plastid psbD core promoter.

The psbD operon of higher plant plastids is regulated transcriptionally through the activity of an upstream light-responsive promoter. To identify promoter elements important for the regulation, portions of the tobacco psbD 5' region were fused to the reporter gene, uidA, and were introduced into the tobacco plastid genome by targeted gene insertion. Examination of uidA mRNA accumulation in dark-adapted and light-treated transplastomic plants revealed that a 107 bp segment of psbD 5' sequence was sufficient to promote light-responsive expression of the reporter gene in vivo. The 107 bp promoter region contains three pairs of short, repeated sequences upstream of the core promoter -10/-35 elements. Deletion of the upstream-most A-rich sequences resulted in a 5-fold decrease in reporter gene mRNA accumulation, but did not affect the light response. Additional removal of the second and third repeated elements further reduced the promoter strength approximately 30-fold and almost eliminated the light-dependent accumulation of uidA transcripts. These data indicate that the architecture of chloroplast promoters is more complex than previously assumed, and may comprise general enhancer and regulatory elements in addition to the core promoter motifs. Transcriptional regulation of psbD may be mediated by the chloroplast proteins which were shown to interact with the repeated sequences.

Base Sequence↗

PubMed1995

Correct splicing of a group II intron from a chimeric reporter gene transcript in tobacco plastids.

An in vivo test system was developed to study group II intron splicing in higher plant chloroplasts. The chimeric reporter gene uidA was constructed by translational fusion of an intron-containing segment of the plastid atpF gene with the coding region of a plastid uidA reporter gene. The chimeric uidA gene was inserted into the tobacco plastid genome by the biolistic transformation procedure using a plastid targeting vector. Correct intron excision was confirmed by Northern blot analysis, by sequencing amplified cDNAs and by accumulation of the encoded beta-glucuronidase (GUS), the expression of which was dependent on intron removal. Removal of the intron from the uidA mRNA is less efficient (< 50%) than from the atpF mRNA (> 90%). The efficiency of atpF mRNA splicing is not affected in the plasmid transformants indicating that inefficient splicing of the highly-expressed uidA mRNA is not due to depletion of factor(s) required for the atpF intron removal. A derivative of uidA, with a stop codon introduced into the loop of domain VI, was also tested. The mutations did not affect the splicing efficiency. The chimeric uidA splicing system will facilitate the study of structural and sequence requirements for group II intron splicing in plastids of higher plants.

Base Composition↗

PubMed1995

Site-specific factor involved in the editing of the psbL mRNA in tobacco plastids.

In tobacco plastids, functional psbL mRNA is created by editing an ACG codon to an AUG translation initiation codon. To determine if editing may occur in a chimeric mRNA, the N-terminal part of psbL containing the editing site was translationally fused with the aadA and kan bacterial genes. The chimeric constructs were introduced into the tobacco plastid genome by targeted gene insertion. Editing of the chimeric mRNAs indicated that the 98 nt fragment spanning the psbL editing site contains all cis information required for editing. Expression of the chimeric gene transcripts led to a significant decrease in the editing efficiency of the endogenous psbL mRNA. However, the efficiency of editing in the transplastomic lines was unchanged for four sites in the rpoB and ndhB mRNAs. Reduced efficiency of psbL editing, but not of the other four sites, in the transplastomic lines indicates depletion of psbL-specific editing factor(s). This finding implicates the involvement of site-specific factors in editing of plastid mRNAs in higher plants.

Amino Acid Sequence↗

PubMed1995

In vivo testing of a tobacco plastid DNA segment for guide RNA function in psbL editing.

A C-to-U RNA editing event creates a functional initiation codon for translation of the psbL mRNA in tobacco plastids. Small trans-acting guide RNAs (gRNAs) have been shown to be involved in editing site selection in kinetoplastid mitochondria. A computer search of the tobacco plastid genome (ptDNA) identified such a putative gRNA, a 14-nucleotide sequence motif that is complementary to the psbL mRNA, including the A nucleotide required to direct the C-to-U change. The critical A nucleotide of the putative gRNA gene was changed to G by plastid transformation. We report here that the introduced mutation did not abolish psbL editing. Since no other region of the plastid genome contains significant complementarity to the psbL editing site we suggest that, if gRNAs serve as trans-acting factors for plastid psbL mRNA editing, they either have only a limited complementarity to the editing site, or are encoded in the nuclear genome.

Base Sequence↗

PubMed1995

Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco.

The Bacillus thuringiensis (Bt) crystal toxins are safe biological insecticides, but have short persistance and are poorly effective against pests that feed inside plant tissues. Production of effective levels of these proteins in plants has required resynthesis of the genes encoding them. We report that amplification of an unmodified crylA(c) coding sequence in chloroplasts up to approximately 10,000 copies per cell resulted in the accumulation of an unprecedented 3-5% of the soluble protein in tobacco leaves as protoxin. The plants were extremely toxic to larvae of Heliothis virescens, Helicoverpa zea, and Spodoptera exigua. Since the plastid transgenes are not transmitted by pollen, this report has implications for containment of Bt genes in crop plants. Furthermore, accumulation of insecticidal protein at a high level will facilitate improvement in the management of Bt resistant insect populations.

Animals↗

PubMed1995

Expression of a chimeric uidA gene indicates that polycistronic mRNAs are efficiently translated in tobacco plastids.

Polycistronic mRNAs are the predominant form of plastid primary transcripts. To determine if there is internal initiation of translation of promoter-distal open reading frames, a promoterless uidA reporter gene was integrated into the tobacco plastid genome downstream of the rbcL gene. Monocistronic uidA mRNA does not accumulate from the promoterless uidA construct. However, due to inefficient rbcL transcription termination, a polycistronic transcription unit is created that contains the uidA gene as the second cistron. Numerous stop codons in all three reading frames between the rbcL and uidA coding regions ensure that translation of uidA initiates only from the correct start codon. The encoded reporter gene product, beta-glucuronidase (GUS) accumulates to high levels in the transplastomic plants indicating that promoter-distal cistrons can be efficiently translated in plastids.

Base Sequence↗

PubMed1995

A plant mitochondrial sequence transcribed in transgenic tobacco chloroplasts is not edited.

RNA editing occurs in two higher-plant organelles, chloroplasts and mitochondria. Because chloroplasts and mitochondria exhibit some similarity in editing site selection, we investigated whether mitochondrial RNA sequences could be edited in chloroplasts. We produced transgenic tobacco plants that contained chimeric genes in which the second exon of a Petunia hybrida mitochondrial coxII gene was under the control of chloroplast gene regulatory sequences. coxII transcripts accumulated to low or high levels in transgenic chloroplasts containing chimeric genes with the plastid ribosomal protein gene rps16 or the rRNA operon promoter, respectively. Exon 2 of coxII was chosen because it carries seven editing sites and is edited in petunia mitochondria even when located in an abnormal context in an aberrant recombined gene. When editing of the coxII transcripts in transgenic chloroplasts was examined, no RNA editing at any of the usual sites was detected, nor was there any novel editing at any other sites. These results indicate that the RNA editing mechanisms of chloroplasts and mitochondria are not identical but must have at least some organelle-specific components.

Amino Acid Sequence↗

PubMed1994

Introduction of a heterologous editing site into the tobacco plastid genome: the lack of RNA editing leads to a mutant phenotype.

The psbF mRNA is edited in spinach plastids by a C to U conversion, changing a serine to a conserved phenylalanine codon. In tobacco at this position a phenylalanine codon is present at the DNA level, and the psbF mRNA here is not edited. To test if the psbF editing capacity is evolutionarily conserved, the tobacco psbF gene was modified to match the corresponding spinach sequence. The endogenous tobacco gene was replaced with the modified copy using biolistic transformation. We report here that the heterologous editing site remains unmodified in transplastomic tobacco plants. The lack of editing is associated with slower growth, lowered chlorophyll content and high chlorophyll fluorescence, a phenotype characteristic of photosynthetic mutants. This finding confirms that the editing of the psbF mRNA is an essential processing step for protein function and thus provides direct proof for the biological significance of plant organellar RNA editing. Given that a mutant phenotype is associated with the lack of editing, it seems likely that the evolutionary loss of the site-specific capacity for psbF editing was preceded by the mutation that eliminated the editing requirement.

Amino Acid Sequence↗

PubMed1994

Efficient targeting of foreign genes into the tobacco plastid genome.

The pPRV plasmids are vectors for targeted insertion of foreign genes into the tobacco plastid genome (ptDNA). The vectors are based on the pUC119 plasmid which replicates in E. coli but not in plastids. The spectinomycin resistance (aadA) gene and a multiple cloning site (MCS) are flanked by 1.8-kb and 1.2-kb ptDNA sequences. Biolistic delivery of vector DNA, followed by spectinomycin selection, yields plastid transformants at a reproducible frequency, approximately 1 transplastomic line per bombarded sample. The selected aadA gene and linked non-selectable genes cloned into the MCS are incorporated into the ptDNA by two homologous recombination events via the flanking ptDNA sequences. The transplastomes thus generated are stable, and are maternally transmitted to the seed progeny. The pPRV vector series targets insertions between the divergently transcribed trnV gene and the rps12/7 operon. The lack of readthrough transcription of appropriately oriented transgenes makes the vectors an ideal choice for the study of transgene promoter activity.

Base Sequence↗

PubMed1994

Extrachromosomal elements in tobacco plastids.

The plastid genome of higher plants is a circular double-stranded DNA molecule which is present in multiple identical copies. We report here an 868-bp plastid DNA minicircle, NICE1, that formed in tobacco (Nicotiana tabacum) plastids during transformation, as an unexpected product of homologous recombination. Such extrachromosomal elements are normally absent in plastids of higher plants. We have constructed shuttle plasmids containing NICE1 sequences which are maintained extrachromosomally when reintroduced into plastids by particle bombardment. Furthermore, recombination between homologous sequences in the shuttle plasmids and the main plastid genome occurs. Recombination products were characterized after recovery of the shuttle plasmids in Escherichia coli and of recombinant plastid genomes in the progeny of transformed plants. Our findings indicate that shuttle plasmids can be used to engineer plastid genes without concomitant integration of foreign DNA and to recover plastid mutations in E. coli.

Base Sequence↗