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V Walbot

Publications and source records attributed to V Walbot.

At least 37 records · Page 2Linked to original sources
PubMed1991

Genetic analysis of B-Peru, a regulatory gene in maize.

The B locus in maize is required for the accumulation of anthocyanin pigments. Numerous B alleles have been described: each determines a particular pattern of pigment synthesis with respect to the tissues that are pigmented and the time during development that pigment synthesis begins. We report here a genetic analysis of one B allele, B-Peru, which regulates synthesis of pigments in both kernel and plant tissues. We used stocks with active Mutator transposable elements to produce eight mutations in B-Peru. All eight alter pigment synthesis in all the kernel and plant tissues pigmented by B-Peru, suggesting that each mutation has disrupted a region of the gene required for expression in all tissues. Six of the mutations cause a colorless phenotype, while two cause a reduction in pigment in both kernel and plant tissues. Four of the mutations are unstable, and four are stable upon self-pollination. Multiple independent revertants were isolated from each unstable allele. DNA blot analysis demonstrated that all eight mutants are the result of insertions within an approximately 5-kb region that encodes the B-Peru transcript. One of the four unstable alleles contains a Mu element, Mu1.7. Two others contain insertions related to the Spm family of transposable elements. Thus, our Mutator stocks also contained active transposable elements from the unrelated Spm family. Our experiments suggest that the B-Peru allele is not complex, but contains a single coding region that regulates pigment synthesis in multiple tissues.

Alleles↗
PubMed1991

Analysis of a 120-kilobase mitochondrial chromosome in maize.

The organization of the mitochondrial genome in plants is not well understood. In maize mitochondrial DNA (mtDNA) several subgenomic circular molecules as well as an abundant fraction of linear molecules have been seen by electron microscopy. It has been hypothesized that the circular molecules are the genetic entities of the mitochondrial genome while the linear molecules correspond to randomly sheared mtDNA. A model has been proposed that explains the mechanism of generation of subgenomic circles (of a predictable size) by homologous recombination between pairs of large direct repeats found on a large (approximately 570 kb for the fertile (N) cytoplasm) master circle. So far the physical entities of the mitochondrial genome, as they exist in vivo, and the genes they carry, have not been identified. For this purpose, we used two gel systems (pulsed field gel electrophoresis and Eckhardt gels) designed to resolve large DNA. Large DNA was prepared from the Black Mexican Sweet (BMS) cultivar. We resolved several size classes of mtDNA circles and designate these as chromosomes. A 120 kb chromosome was mapped in detail. It is shown to contain the three ribosomal genes (rrn26, rrn18 and rrn5) plus two genes encoding subunits of cytochrome oxidase (Cox1 and Cox3); it appears to be colinear with the 570-kb master circle map of another fertile cytoplasm (B37N) except at the "breakpoints" required to form the 120-kb circle. The presence of the 120-kb chromosome could not have been predicted by homologous recombination through any of the known repetitive sequences nor is it a universal feature of normal maize mitochondria. It is present in mitochondria of BMS suspension cultures and seedlings, but is not detectable in seedlings of B37N. No master genome was detected in BMS.

Blotting, Southern↗
PubMed1991

Transcriptional and posttranscriptional regulation of maize mitochondrial gene expression.

Lysed maize mitochondria synthesize RNA in the presence of radioactive nucleoside triphosphates, and this assay was utilized to compare the rates of transcription of seven genes. The rates of incorporation varied over a 14-fold range, with the following rank order: 18S rRNA greater than 26S rRNA greater than atp1 greater than atp6 greater than atp9 greater than cob greater than cox3. The products of run-on transcription hybridized specifically to known transcribed regions and selectively to the antisense DNA strand; thus, the isolated run-on transcription system appears to be an accurate representation of endogenous transcription. Although there were small differences in gene copy abundance, these differences cannot account for the differences in apparent transcription rates; we conclude that promoter strength is the main determinant. Among the protein coding genes, incorporation was greatest for atp1. The most active transcription initiation site of this gene was characterized by hybridization with in vitro-capped RNA and by primer extension analyses. The DNA sequences at this and other transcription initiation sites that we have previously mapped were analyzed with respect to the apparent promoter strengths. We propose that two short sequence elements just upstream of initiation sites form at least a portion of the sequence requirements for a maize mitochondrial promoter. In addition to modulation at the level of transcription, steady-state abundance of protein-coding mRNAs varied over a 20-fold range and did not correlate with transcriptional activity. These observations suggest that posttranscriptional processes are important in the modulation of mRNA abundance.

Base Sequence↗
PubMed1990

Regulation of the timing of transposable element excision during maize development.

The ability of transposable elements (TEs) to insert into or excise out of a genetic locus can be regulated by genetic, environmental, and developmental factors. Tissue- or organ-specific activity of TEs is a frequent and well-characterized example of spatial, developmental regulation. Regulation of the timing of TE activity during ontogeny is less well understood. To analyze timing, TE-induced variegation was quantified in the aleurone of maize kernels, a tissue composed of only a single layer of cells, and sector sizes were assigned to specific cell divisions in aleurone development. Three TE families, Mu, Spm, and Ac/Ds, were studied at two genetic loci. It was found that the frequency of transposon excision changes drastically (up to 30-fold increase or equivalent decrease) during the proliferation of the aleurone. Moreover, these changes occur at the same cell divisions in all three TE families. These results suggest that the timing of TE excision during maize development can be controlled by the host.

Alleles↗
PubMed1990

Structural analysis of mature and dicistronic transcripts from the 18 S and 5 S ribosomal RNA genes of maize mitochondria.

Analysis of the 18 S and 5 S ribosomal RNA transcripts of maize mitochondria is described. The 5' and 3' ends of both mature rRNAs were defined by S1 nuclease protection analysis, which also showed that a small fraction of the total 18 S and 5 S rRNA population resides on common transcripts. The 5' termini of many of these RNAs are upstream from the mature 18 S 5' endpoint. Northern hybridization detected several high molecular weight RNAs that were homologous to the 5 S and 18 S genes and their flanking sequences. Because the two rRNAs share a number of transcripts, we propose that these genes are transcribed as one or more large dicistronic RNAs that are subsequently processed to mature ribosomal RNA molecules. A model for the processing of the large putative precursors is presented.

Base Sequence↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1989

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↗
PubMed1989

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↗
PubMed1989

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↗
PubMed1989

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↗
PubMed1989

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↗