Sequence of the F0-atpase proteolipid (atp9) gene from rice mitochondria.
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Amino Acid Sequence↗
Biomedical subjects
V Walbot
Publications and source records attributed to V Walbot.
Sequence of the rice mitochondrial gene for cytochrome oxidase subunit 3.
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Amino Acid Sequence↗
Sequence of the rice mitochondrial gene for apocytochrome b.
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Amino Acid Sequence↗
DNA methylation in the Alcohol dehydrogenase-1 gene of maize.
Using a battery of methylation-sensitive restriction enzymes, cytosine methylation at 23 sites in a 7.6 kb region surrounding the Alcohol dehydrogenase-1 (Adh1) gene was measured in DNA prepared from immature maize cobs. Both the 5' upstream region and the entire coding region were hypomethylated in the two alleles examined. Methylation in Adh1 is independent of changes in Mutator transposable element methylation. The role of DNA methylation in Adh1 gene regulation is discussed.
Alcohol Dehydrogenase↗
DNA methylation in eukaryotes: kinetics of demethylation and de novo methylation during the life cycle.
We present a model for the kinetics of methylation and demethylation of eukaryotic DNA; the model incorporates values for de novo methylation and the error rate of maintenance methylation. From the equations, an equilibrium is reached such that the proportion of sites which are newly methylated equals the proportion of sites which become demethylated in a cell generation. This equilibrium is empirically determined as the level of maintenance methylation. We then chose reasonable values for the parameters using maize and mice as model species. In general, if the genome is either hypermethylated or hypomethylated it will approach the equilibrium level of maintenance methylation asymptotically over time; events occurring just once per life cycle to suppress methylation can maintain a relatively hypomethylated state. Although the equations developed are used here as framework for evaluating events in the whole genome, they can also be used to evaluate the rates of methylation and demethylation in specific sites over time.
Animals↗
RNA pseudoknot domain of tobacco mosaic virus can functionally substitute for a poly(A) tail in plant and animal cells.
The genomes of many RNA viruses terminate in a tertiary structure similar to the L-conformation of tRNAs and this structure is recognized by many tRNA-specific enzymes such as aminoacyl-tRNA synthetase. Virtually the entire 3'-untranslated region (UTR) of tobacco mosaic virus (TMV) RNA is involved in an extended tertiary structure containing, in addition to a tRNA-like structure, a pseudoknot domain that lies immediately upstream. Although the functions of these structures are not well understood, they are essential to the virus. We demonstrate that the addition of the 204-base TMV 3'-untranslated region to foreign mRNA constructs can increase gene expression up to 100-fold compared to nonadenylated mRNA. The 3'-UTR of TMV was equal to or greater than a polyadenylated tail in enhancing gene expression in electroporated dicot and monocot protoplasts. The TMV 3'-UTR is functionally similar to a polyadenylated tail in that it increases mRNA stability and translation and must be positioned at the 3' terminus to function efficiently. Similar effects on expression were observed in Chinese hamster ovary cells, demonstrating that the sequence functions in a wide range of eukaryotes. When the extended tertiary structure was dissected, the upstream pseudoknot domain was found to be largely responsible for increasing expression. The inclusion of the tRNA-like structure, however, was important for full regulation.
Animals↗
Bronze-2 gene of maize: reconstruction of a wild-type allele and analysis of transcription and splicing.
The maize Bronze-2 (Bz2) gene, whose product acts late in the anthocyanin biosynthetic pathway, has been cloned and its transcript has been mapped. We have developed a general procedure for reconstructing wild-type alleles from transposable element-induced mutants. An existing transposon-containing clone, bz2::mu1 [McLaughlin, M., and Walbot, V. (1987). Genetics 117, 771-776], was modified by replacing the region of bz2::mu1 containing the transposon with the corresponding polymerase chain reaction-amplified sequence from the progenitor allele that has no Mu insertion. Particle gun delivery of the reconstructed Bz2 gene to embryonic scutellar tissue lacking a functional Bz2 gene complemented the bz2 mutant phenotype, as demonstrated by the production of purple spots. Having cloned the wild-type allele, we then analyzed the Bz2 transcript, whose features include an 82-nucleotide 5'-untranslated leader, one small intron (78 base pairs) within the coding region, and multiple polyadenylation sites. Four Mutator transposon insertions that eliminate gene function were mapped within the 850-nucleotide transcription unit. We found that variable levels of unspliced Bz2 RNA are present in purple husk tissue; this finding may indicate that the expression of Bz2 is regulated in part at the level of transcript processing.
Alleles↗
Insertion of Mu1 elements in the first intron of the Adh1-S gene of maize results in novel RNA processing events.
Maize transposable elements, when inserted in or near genes, alter expression by several transcriptional and post-transcriptional mechanisms. Three independent, unstable insertions of the transposable element Mutator (Mu) into the first intron of the Alcohol dehydrogenase-1 (Adh1) gene have been shown to decrease expression [Strommer et al. (1982). Nature 300, 542-544]. We have developed an approach to elucidate the underlying molecular mechanisms responsible for the mutant phenotypes. Mu1 elements were inserted into Adh1-S intron 1 in vitro to create plasmid facsimiles of the mutant alleles. The Mu1 element was also inserted at novel positions within intron 1 to create new mutations. The Mu1/intron constructions were placed between the Adh1-S promoter/exon 1 segment and a reporter gene (firefly luciferase or beta-glucuronidase), and these chimeric gene constructs were tested in transient assays in maize protoplasts. When compared with the appropriate control, the Mu1 insertions decreased reporter gene expression to levels approximating the alcohol dehydrogenase enzyme activities observed for the Adh1-S mutants in vivo. The Mu1 insertions also showed a polarity effect with luciferase expression increasing as the insertions were placed nearer the 3' splice junction. In addition, Mu1 insertions within a different intron, actin intron 3, also significantly reduced luciferase expression, indicating that Mu1 insertions within introns are likely to diminish expression in many genes. The presence of the Mu1 sequences was correlated with decreased levels of steady-state luciferase transcript. Deletion analysis of the Mu1 element and RNase mapping indicate that the transposable element contains RNA processing signals in its central region that are largely responsible for the decrease in expression.
Actins↗
Molecular analysis of the linear 2.3 kb plasmid of maize mitochondria: apparent capture of tRNA genes.
The nucleotide sequence and transcription pattern of the linear 2.3 kb plasmid of maize mitochondria was analyzed in order to elucidate its possible function in the organelle. The plasmid has 170 bp inverted repeats at its termini composed, in turn, of shorter repetitive sequences. An open reading frame within the plasmid is transcribed and can potentially specify a 33 kD product. In addition the plasmid contains two tRNA genes homologous to chloroplast sequences; the tRNApro(CAA) and the tRNAtrp(UGG). Both of the tRNA genes of the plasmid are transcribed, but apparently only the tRNAtrp is processed to the correct size. These tRNA sequences are found in the main mitochondrial genome of all higher plants tested, and in most maize relatives. An exception is the close maize relative Northern teosinte in which the tRNAtrp gene is also carried on a plasmid. These results suggest that the 2.3 kb plasmid has acquired the tRNA sequences from the main mitochondrial DNA. It is possible that the plasmid-encoded tRNAtrp gene is essential for organelle function thereby ensuring the maintenance of the plasmid in the mitochondrion.
Base Sequence↗
Integrated R2 sequence in mitochondria of fertile B37N maize encodes and expresses a 130 kD polypeptide similar to that encoded by the S2 episome of S-type male sterile plants.
Expression of a 130 kDa protein from open reading frame 1 of the integrated form of the R2 mitochondrial plasmid in normal mitochondria of B37 and other inbred lines is described. The protein appears identical to that synthesized by the closely related S2 episome found in cytoplasmic male sterile maize of the S type. Protein was detected using antisera raised against a beta-galactosidase:ORF1 fusion product containing the most antigenic region of the ORF1 product. Detection of this protein is in contrast to previous reports that mitochondria of normal, male-fertile lines either do not contain this protein, or that there are 11 in-frame stop codons in the reading frame. The integrated R2 of B37N was cloned and this region sequenced, confirming that a continuous open reading frame existed. These results are discussed in relation to the possible role of the S-type episomes in causing cytoplasmic male sterility.
Amino Acid Sequence↗
Developmental and genetic aspects of Mutator excision in maize.
The regulation of excision of Mu elements of the Mutator transposable element family of maize is not well understood. We have used somatic instability of Mu receptor elements from the Bronze 1 and Bronze 2 loci to monitor the frequency and the timing of excision of Mu elements in several tissues. We show that spot size in the aleurone of a bz2::mu1 stock varies between one to approximately 256 cells. This indicates that excision events begin eight divisions prior to full aleurone differentiation and end after the last division of the aleurone. We show that excision is equally biased for late events in all other tissues studied. A locus on chromosome 5 has been identified that affects spot size, possibly by altering the timing of Mu excision. Using somatic excision as an assay of Mutator activity, we found that activity can change in small sectors of the tassel; however, there are no overall activity changes in the tassel during the period of pollen shedding. We also report the recovery of germinal revertants for the bz1::mu1 and bz2::mu1 alleles. One of these revertant alleles was characterized by Southern blot analysis and found to be similar to the progenitor of the mutable allele.
Alleles↗
Identification in maize mitochondrial 26S rRNA of a short 5'-end sequence possibly involved in transcription initiation and processing.
By direct RNA mapping, we have identified the precise 5' end of the maize mitochondrial 26S rRNA. The 5' termini of the 26S rRNA are 17 and 18 nt downstream from the 5' end predicted by Dale et al. (1984) from homology between the maize rRNA and E. coli large subunit ribosomal RNA. In addition, we have discovered a larger 26S species, presumably a 26S precursor, and precisely mapped its 5' end. The maize mitochondrial rRNA genes are probably regulated by control regions that differ from those already characterized in other genomes: the maize mitochondrial 26S, 18S and 5S rRNAs lack sequences that resemble the promoter regions of genes from other mitochondria and bacteria. However, the mature and precursor rRNAs all contain a tetranucleotide, AAUC, at their 5' ends (AAAC in the 5S rRNA). The sequence is also seen in the same position in several other plant mitochondrial mRNAs. We propose therefore that AAUC is a transcription or processing signal which is possibly unique to plant mitochondria.
Base Sequence↗
Molecular and genetic characterization of Mu transposable elements in Zea mays: behavior in callus culture and regenerated plants.
Active Mutator lines of maize (Zea mays L.) have a high mutation rate and contain multiple hypomethylated 1.4-kb and 1.7-kb Mu transposable elements. Correlated with the inactivation of the Mutator system, these Mu elements cease to transpose and become more methylated. To determine whether the shock of tissue culture can affect Mutator activities, F1 progenies of outcrosses between active or inactive Mutator stocks and inbred line A188 were used to initiate embryogenic callus cultures. HinfI restriction digestion of genomic DNA isolated from 3-5-month-old cultures demonstrated that there is a very good correlation between the modification state of Mu elements in the cultures and the Mutator parent. Despite the dedifferentiation and rapid proliferation characteristic of tissue culture, the Mutator activity state is relatively stable during an extended tissue culture period. Cultures established from inactive Mutator lines were not reactivated; cultures established from active lines maintained a high Mu copy number, and most Mu elements remained unmodified. In contrast, weakly active Mutator parents gave rise to cultures in which Mu element modification could switch between low and high methylation during the culture period. Evidence for transposition was investigated with EcoRI digestion of genomic DNA isolated at different times during culture. The appearance of novel Mu-hybridizing fragments and a strong background hybridization are interpreted as evidence that transposition events occur during culture. Plants regenerated from such active cultures transmitted Mutator activity to their progeny.
Culture Techniques↗
Visualizing mRNA expression in plant protoplasts: factors influencing efficient mRNA uptake and translation.
In this paper we demonstrate that RNA sequences present upstream and downstream of a reporter gene coding region play an important role in determining the amount of protein produced from an mRNA. A translational enhancer, omega, derived from tobacco mosaic virus, when present at the 5'-end of beta-glucuronidase mRNA increased the efficiency of translation 16-fold to 18-fold in electroporated tobacco or carrot protoplasts, and threefold to 11-fold in maize or rice protoplasts. The presence of omega did not alter the half-life of the mRNA in vivo. We also demonstrate for the first time that a minimum polyadenylated tail length of 25 adenylate residues is sufficient to substantially increase the expression and half-life of the reporter mRNA in plants. When in vitro-produced mRNAs were synthesized such that extra sequence was added to the 3'-end of the poly(A) tail, however, the final level of expression was decreased up to 80%. Omega, the translational enhancer, and a poly(A) tail function independently of each other; their combined effect on translation, when both are present in an mRNA, is the multiplication of their individual effects. Histochemical analysis for the presence of beta-glucuronidase in tobacco established that virtually all viable cells receive mRNA during electroporation. Video image analysis of tobacco protoplasts electroporated with luciferase mRNA demonstrated that there is a wide range in the level of expression of this marker. Carrier RNA, when present during electroporation, had only a modest effect on increasing mRNA uptake. Reporter mRNA expression in electroporated protoplasts was directly proportional to the input mRNA up to at least 30 micrograms/ml.
Base Sequence↗
The ribosomal fraction mediates the translational enhancement associated with the 5'-leader of tobacco mosaic virus.
The omega sequence at the 5'-terminus of tobacco mosaic virus (TMV) RNA acts as a translational enhancer. The differential in omega-associated translational enhancement between the in vitro translation system derived from wheat germ (WG) and that from rabbit reticulocytes (MDL) was exploited to identify that lysate component which was responsible for a lysate's characteristic response to omega. Using fractionated MDL and WG lysates, which were reconstituted in various combinations, the high salt-washed ribosomal fraction was determined to be the responsive element in a lysate. Analysis of omega's ability to enhance translation was greatest at low mRNA and high ribosomal concentrations and to occur in the early phase of an in vitro translation assay. Translation of omega-containing CAT mRNA was more sensitive to the presence of micrococcal nuclease than CAT mRNA without an omega. In substitution experiments, WG ribosomes functioned at much reduced efficiency in MDL as did MDL ribosomes in WG lysate. The initiation factor-containing fraction of one system could not, as a whole, functionally replace that of the other and actually acted to inhibit translation in the heterologous system.
Acetyltransferases↗
Regulation of mutator activities in maize.
We discuss the properties of the Mutator (Mu) transposable element family of maize. We report the cloning of bz2-mu1, a mutable allele containing a 1.4-kb Mu element, using a combination of transposon tagging and tests for differential hybridization to northern and Southern blots. We report the sequence of this allele and the Mu element insertion, and propose a model for the structure of the Bz2 locus. We discuss the relationship between increased DNA modification of Mu elements and loss of somatic instability at bz2-mu1. To further explore this aspect of regulation of Mutator, we have used gene-specific probes to determine the level of modification at this locus in active and inactive Mutator lines. We have also utilized CsCl density gradients to estimate the overall level of DNA modification in active and inactive lines; we find that Mu elements in active lines are hypomethylated relative to other maize nuclear DNAs examined, and that in inactive lines the level of modification in Mu elements is similar to the genome as a whole. Utilizing gamma-irradiation, we have demonstrated that inactive lines can be reactivated; this reactivation is first noted as restitution of the spotted kernel phenotype characteristic of bz2-mu1 in active Mutator lines. Hybridization analysis of DNA from reactivated plants demonstrates that the Mu elements in general, and specifically the Mu element at bz2-mu1, have the lower level of DNA modification characteristic of active lines. These results are discussed in terms of the role and timing of DNA modification in regulating Mutator activities.
Base Sequence↗
Regulation of Mu element copy number in maize lines with an active or inactive Mutator transposable element system.
In the progeny of an active Mutator plant, the number of Mu elements increases on self-pollination and maintains the average parental Mu content on outcrossing to a non-Mutator line; both patterns of transmission require an increase in the absolute number of Mu elements from one generation to the next. The same average copy number of Mu elements is transmitted through the male and female, but there is wide variation in the absolute copy number among the progeny. In inactive Mutator plants-defined both by the loss of somatic instability at a reporter gene (bronze2-mu1) and by modification of the HinfI sites in the terminal inverted repeat sequences of Mu elements - the absolute copy number of Mu elements is fixed in the parent. Thus, in outcrosses Mu element number is halved, and on self-pollination Mu copy number is constant. Reactivation of somatic mutability at cryptic bz2-mu1 alleles in inactive individuals by crossing to an active line seems not to involve an increase in Mu element copy number transmitted by the inactive individual. These and other results suggest that increases in Mu copy number occur late in plant development or in the gametophyte rather than after fertilization.
DNA Transposable Elements↗
RNA processing and multiple transcription initiation sites result in transcript size heterogeneity in maize mitochondria.
Variation in the length of the 5' non-coding region of mitochondrial gene transcripts could result from multiple transcription initiation sites or post-transcriptional processing events. To distinguish between these possibilities, we have utilized the in vitro capping reaction catalyzed by guanylyl transferase to specifically label the 5' end of primary, unprocessed transcripts. Hybridization of in vitro capped mtRNA to immobilized DNA from the 5' flanking regions of 26 S, 18 S and 5 S rRNA genes and two protein-coding genes, ATP synthase subunit 9 (atp9) and apocytochrome b (cob), identified regions where transcription initiates. Single-strand specific RNase treatment of in vitro capped RNA hybridized to immobilized DNA containing the 5' flanking sequences from cob and atp9 suggests that these genes have multiple transcription initiation sites. Direct mapping of transcription initiation sites for the rRNA genes indicated that single major transcription initiation sites exist at approximately 180 and 230 nucleotides upstream from the mature 26 S and 18 + 5 S rRNA genes, respectively. Labeling of processed transcripts bearing a 5' hydroxyl moiety with T4 polynucleotide kinase and subsequent hybridization to the rRNA genes indicated that the mature forms of the rRNA are processed.
Base Sequence↗