Biomedical subjects

V Walbot

Publications and source records attributed to V Walbot.

At least 109 records · Page 6Linked to original sources

PubMed1988

Heat Inducible Expression of a Chimeric Maize hsp70CAT Gene in Maize Protoplasts.

The response of maize (Zea mays L.) protoplasts to high temperature stress was investigated. After isolation and electroporation, protoplasts were preincubated for 12 hours at 26 degrees C then incubated for 6 hours at elevated temperatures. The pattern of polypeptides synthesized by these protoplasts during the last hour was monitored by in vivo labeling with (35)S-methionine. Incubation at 40 degrees and 42 degrees C resulted in the synthesis of polypeptides not detectable at 26 degrees C. Introduction of a chimeric maize heat shock protein 70 promoter-chloramphenicol acetyltransferase coding region gene into protoplasts via electroporation resulted in the temperature-dependent induction of chloramphenicol acetyltransferase activity with maximal activity at 40 degrees C. In the same protoplasts, a second chimeric gene, in which the firefly luciferase coding region was under the control of the 35S promoter from cauliflower mosaic virus, did not show an increase in expression after incubation at higher temperatures. Maize protoplasts provide a system to study molecular responses to high temperature stress.

Journal Article↗

PubMed1987

Expression of mRNA electroporated into plant and animal cells.

A general method to introduce RNA molecules into plant protoplasts and animal cells is described. This technique utilizes the ability of electric pulses of high field strength to form pores in biomembranes. RNA molecules containing the coding region for the bacterial enzyme chloramphenicol acetyltransferase (CAT) were used as a model system. The presence of CAT activity as a result of the in vivo translation of the introduced RNA is entirely dependent on the presence of a 5' cap and greatly increased by the presence of a poly A tail at the 3' end. The introduction of RNA into eukaryotic cells has broad applicability both as an assay for the uptake of nucleic acids into cells independent of transcriptional activity and as a tool to study eukaryotic mRNA translation.

Acetyltransferases↗

PubMed1987

Visual assays of transformation in plant cells.

We discuss the utility of visual assays for the expression of genes introduced into plant cells. Such assays are valuable for both transient and stable gene expression studies. We review the properties of three visual assays that are already in use or under development phase for maize and other cereal crops. These assays depend on the expression of beta-galactosidase, luciferase, or structural genes required for anthocyanin pigment biosynthesis.

Anthocyanins↗

PubMed1987

S2 episome of maize mitochondria encodes a 130-kilodalton protein found in male sterile and fertile plants.

The mitochondrial genome of the S-type male-sterile cytoplasm of maize contains two linear episomes, S1 (6397 base pairs) and S2 (5453 base pairs). The S2 episome contains two large unidentified open reading frames, URF1 (3512 base pairs) and URF2 (1017 base pairs). We have demonstrated that a polypeptide with an apparent molecular mass of 130 kDa is the gene product of URF1. This polypeptide was first detected in Coomassie blue-stained protein gels of cms-S (where cms = cytoplasmic male sterile) but not in those of cms-T, cms-C, or normal mitochondrial proteins. The protein product of a translational fusion containing the 5' end of Escherichia coli lacZ and an internal segment from URF1 of S2 was recognized by antisera raised against the 130-kDa variant polypeptide. The mitochondria of fertile F(1) hybrids of cms-S x Ky21 (the male parent carrying nuclear fertility restoration genes) contain as much of the 130-kDa protein as is found in cms-S mitochondria of sterile plants. Spontaneous fertile cytoplasmic revertants from cms-S in a WF9 nuclear background also synthesized the 130-kDa polypeptide. Therefore, the mere presence or absence of the URF1 gene product of S2 does not determine the fertility status of maize plants, because male sterile and male fertile (nuclear restored and revertant) plants can contain equivalent amounts of the 130-kDa polypeptide.

Journal Article↗

PubMed1987

Isolation and characterization of a 1.7-kb transposable element from a mutator line of maize.

We have cloned and sequenced a 1.7-kb Mu element from a Mutator line of maize and compared its structure to Mu1, a 1.4-kb element. With the exception of a 385-bp block of DNA present in the 1.7-kb element, these transposable elements are structurally similar, sharing terminally inverted and internal direct repeated sequences. Derivation of 1.4-kb elements from the 1.7-kb class via deletion of internal sequence is suggested by the finding that a portion of the extra DNA in Mu1.7 is part of a truncated direct repeat sequence in the 1.4-kb element. An abundant poly(A)+ RNA homologous to a portion of this extra DNA is present in several tissues of both Mutator and non-Mutator lines. Analysis of transcripts from an unstable mutant bronze 1 (bz) allele containing a Mu1.7 element inserted in an exon of the gene detects three species of poly(A)+ RNA that hybridize to a Bz1 (Bronze) gene probe: the largest contains the entire Mu1.7 element in the Bz1 gene transcript; another appears to be a spliced, chimeric transcript; the smallest is normal size Bz1 mRNA. The latter is most likely encoded by the normal-size alleles detected by Southern analysis of tissue expressing purple pigment, suggesting that normal gene function is restored by excision of the Mu1.7 element.

Base Sequence↗

PubMed1987

Cloning of a mutable bz2 allele of maize by transposon tagging and differential hybridization.

Sequences of Bronze2 (Bz2), a maize gene which is required for the synthesis of the purple pigment anthocyanin, have been cloned by combining the techniques of transposon tagging and differential hybridization. First, a mutable bz2 allele (bz2-mu1) was recovered from a Mutator line. The mutation was assumed to result from an insertion of the transposable element Mutator (Mu), which is found in 10-60 copies in Mutator lines. A library was prepared using DNA isolated from bz2-mul, and a small bank of Mu-specific candidate clones was selected. Because much is known about the genetic requirements for the synthesis of anthocyanin in different tissues, we were able to identify the bz2 clone based on its hybridization to RNA isolated from different bz2 mutants. Only one clone hybridized to RNA isolated from the husks of purple (Bz2, B, Pl) plants but not to RNA isolated from the husks of green (an-bz2-6923, b, pl) plants. Further confirmation was provided by the hybridization pattern of the clone on blots containing DNA from other bz2 mutants. We present a restriction map of the clone, indicating the location and orientation of the 1.4-kbp Mu insert. We identify the transcribed region, the direction of transcription and the location of the 1.4-kbp Mu insert in an independently isolated mutant, bz2-mu2.

Alleles↗

PubMed1987

Introns increase gene expression in cultured maize cells.

Using electroporation-mediated gene transfer, the gene encoding the Slow (S) migrating polypeptide of the maize (Zea mays L.) alcohol dehydrogenase-1 (Adh1) enzyme has been introduced stably and transiently into maize cells containing an endogenous Fast (F) ADH1 electromorph. In stable transformants an 11.5-kb fragment was sufficient to program normal S expression relative to the endogenous F allele. In transient assays, Adh1-S gene constructs lacking the 9 Adh1-S intervening sequences (introns) were expressed at levels 50- to 100-fold less than the intact gene; the presence of intron 1 alone restored levels of gene expression to those found with the intact gene. The last two introns also stimulate Adh1-S expression, but the level is threefold below that of the intact gene. The expression of a chimeric chloramphenicol acetyltransferase (CAT) gene utilizing the 5' promoter and 3' polyadenylation regions of the Adh1 gene was increased 100-fold by the addition of sequences containing the Adh1 intron 1. The Adh1 intron 1 sequences did not stimulate CAT expression when located outside the transcribed region. When located within the transcribed region, the Adh1 intron 1 region efficiently stimulated CAT expression only when located between the promoter and the CAT coding region. A construct containing the Adh1 intron 1 fragment produced 40-fold more mRNA than a construct containing an equivalent cDNA fragment. Both the Adh1 intron 1 and the intron from a second maize gene, Bronze1, stimulated expression from other promoters (cauliflower mosaic virus 35S and nopaline synthase) and of other coding regions (luciferase and neomycin phosphotransferase II) as well. These results indicated that introns increase both Adh1 and chimeric gene expression in maize and the optimal location for such an intron is near the 5' end of the mRNA.

Acetyltransferases↗

PubMed1986

DNA synthesis in purified maize mitochondria.

We have developed an in organelle DNA synthesis system using isolated mitochondria from maize. The organelles used in this assay are shown to be intact by a number of criteria. Both the high molecular weight components and the smaller plasmid-like components of the mitochondrial genome are used as templates; however, the plasmid-like elements are relatively more active as templates. The termini of the linear plasmids--S-1, S-2 and the 2.3 kbp plasmids--are more highly labelled than internal regions, probably as a result of filling in of gaps at the termini mediated by a DNA polymerase or to exonuclease degradation of the 3' OH termini, with subsequent filling in. Although most of the DNA synthesis observed in this system most likely results from this type of synthesis at DNA nicks or termini, a small amount of specific, potentially replication-associated, synthesis is also detected.

Cell-Free System↗

PubMed1986

Cloning and characterization of a linear 2.3 kb mitochondrial plasmid of maize.

A linear 2.3 kb DNA molecule found in maize mitochondria was cloned into pUC8. A natural deletion of this plasmid, found in cmsT and some N (fertile) types of maize plants, was mapped to one end of the plasmid. A minor sequence homology to S-2, another linear mitochondrial plasmid, was detected, as well as more significant sequence homology with chloroplast and maize nuclear DNA. Hybridization to teosinte mitochondrial DNA (mtDNA) revealed the presence of part of the maize plasmid in the high molecular weight mtDNA of the maize relatives. RNA dot hybridization indicates that the plasmid is transcribed in mitochondria. The termini of the 2.3 kb linear plasmid contain inverted repeated sequences; of the first 17 nucleotides of the termini, 16 are identical to the terminal inverted repeats of the linear S plasmids found in the mitochondria of cmsS maize plants.

Chromosome Deletion↗

PubMed1986

DNA modification of a maize transposable element correlates with loss of activity.

An unstable allele of the bronze 2 (bz2) locus was isolated from a Robertson's Mutator Zea mays line containing a family of active transposable Mu elements. This mutation is somatically unstable, resulting in numerous revertant purple sectors on a bronze kernel. By following the variegated kernel phenotype through two generations, several lineages have been identified that have a distorted transmission of the mutant phenotype (fewer variegated kernels are produced than expected). Southern blot analysis of Mu elements in these plants demonstrates a correlation between an inhibition of digestion of Mu elements by certain restriction enzymes and the loss of somatic reversion at the mutant allele. The DNA modification can occur in all the Mu elements in a plant within one generation; however, plants have been identified that contain both modified and unmodified elements, suggesting that the modification can occur in a progressive manner. We hypothesize that the DNA modification results in nonfunctional elements.

5-Methylcytosine↗

PubMed1986

Maize mitochondrial plasmid S-1 sequences share homology with chloroplast gene psbA.

The linear, 6397-base pair (bp), mitochondrial S-1 DNA molecule from maize contains a 420-bp segment that is homologous with the chloroplast gene (psbA) that codes for the quinone binding protein of photosystem II. This is the first report of a chloroplast sequence in a naturally occurring viral-like or plasmid DNA. The complete sequence of the S-1 chloroplast segment has been compared with homologous regions of six different chloroplast genes. The S-1 segment has diverged from the other genes both by length mutation and base substitution. Several of the length mutations are exact adjacent tandem duplications of 4 and 5 bp similar to "footprints" left after excision of transposable elements in maize nuclear DNA.

Base Sequence↗

PubMed1986

Evaluating quantitative variation in the genome of Zea mays.

Genomic diversity within the species Zea mays has been examined by measuring the variation in the repetitive component of the nuclear genome among North American inbred lines and varieties. This was done by preparing a set of clones of repetitive maize sequences that differ in function, molecular arrangement and multiplicity and then using these as probes for quantitative hybridization to DNA from various maize genotypes. The comparison showed that the majority of repeated sequences are markedly variable in copy number among the ten maize strains tested. The clone sample contained the rDNA and 5S genes, the major repeat of the chromosome knobs, sequences functioning as origins of DNA replication in yeast (ARS sequences) and randomly cloned sequences of unknown function and chromosomal location. The sequences ranged in reiteration frequency from 200 to greater than 10(5) copies and included both tandemly arrayed and dispersed repeats. The copy numbers were measured by hybridizing labeled cloned sequences to aliquots of high molecular weight genomic DNA that were applied to nitrocellulose filters through a slotted template (slot blotting). The hybridization signal on an autoradiogram occurred in a narrow band that could be scored reliably with a densitometer. This provided a rapid method of determining the abundance of particular repeated sequences in individual plants and plant populations. Using this technique, we found that the copy number of repeated sequences of all types generally varied among the strains by two- to threefold, although at least one sequence showed no detectable variation. In contrast to the variability found between strains, individuals within an inbred line or variety were found to be indistinguishable in terms of specific sequence multiplicity. Each genotype has a different pattern of copy numbers for the set of repeated sequence clones, and this pattern is characteristic of all individuals of a particular genotype. The data also show that the copy number of each sequence varies independently. No strains had uniformly high or low copy numbers for the entire set of probes.

DNA↗

PubMed1986

Stable non-mutator stocks of maize have sequences homologous to the Mu1 transposable element.

Mutator stocks of maize produce mutants at many loci at rates 20- to 50-fold above spontaneous levels. Current evidence suggests that this high mutation rate is mediated by an active transposable element system, Mu. Members of this transposable element family are found in approximately 10-60 copies in Mutator stocks. We report here an initial characterization of previously undetected sequences homologous to Mu elements in eight non-Mutator inbred lines and varieties of maize that have a normal low mutation rate. All stocks have approximately 40 copies of sequences homologous only to the terminal repeat and show weak homology to an internal probe. In addition, several of the stocks contain an intact Mu element. One intact Mu element and two terminal-specific clones have been isolated from one non-Mutator line, B37. The cloned sequences have been used to demonstrate that in genomic DNA the intact element, termed Mu1.4B37, is modified, such that restriction sites in its termini are not accessible to cleavage by the HinfI restriction enzyme. This modification is similar to that observed in Mutator lines that have lost activity. We hypothesize that the DNA modification of the Mu-like element may contribute to the lack of Mutator activity in B37.

DNA Restriction Enzymes↗

PubMed1986

Inheritance of mutator activity in Zea mays as assayed by somatic instability of the bz2-mu1 allele.

Mutator lines of maize were originally defined by their high forward mutation rate, now known to be caused by the transposition of numerous Mu elements. A high frequency of somatic instability, seen as a fine purple spotting pattern on the aleurone tissue, is characteristic of Mu-induced mutable alleles of genes of the anthocyanin pathway. Loss of such somatic instability has been correlated with the de novo, specific modification of Mu element DNA. In this report the presence or loss of somatic instability at the bz2-mu1 allele has been monitored to investigate the inheritance of the Mutator phenomenon. The active state is labile and may become weakly active (low fraction of spotted kernel progeny) or totally inactive (no spotted kernel progeny) during either outcrossing to non-Mutator lines or on self-pollination. In contrast, the inactive state is relatively permanent with rare reactivation in subsequent crosses to non-Mutator lines. Cryptic bz2-mu1 alleles in weakly active lines can be efficiently reactivated to somatic instability when crossed with an active line. However, in reciprocal crosses of active and totally inactive individuals, strong maternal effects were observed on the inactivation of a somatically unstable bz2-mu1 allele and on the reactivation of cryptic bz2-mu1 alleles. In general, the activity state of the female parent determines the mutability of the progeny.

Alleles↗

PubMed1985

A deletion adjacent to the maize transposable element Mu-1 accompanies loss of Adh1 expression.

Insertion of the maize transposable element Mu-1 into the first intron of the alcohol dehydrogenase locus (Adh1) of maize produced mutant Adh1-S3034 with 40% of the wild-type level of protein and mRNA. Continued instability at this locus resulted in secondary mutations with lower levels of protein expression. One of these, Adh1-S3034a, has no detectable ADH1 expression. This paper describes the precise nature of the changes in the Adh1 gene that gave rise to the S3034a allele. The Mu-1 element is still present in the mutant, but Adh1 sequences immediately adjacent to the element are deleted. The deletion starts precisely at the Mu-1 insertion site and extends 74 bp leftward removing part of the first intron, the intron:exon junction and 2 bp of the eleventh amino acid codon in the first exon of the gene. Tests for reversion within the somatic tissue of plants show that mutant S3034a, unlike its progenitor, is stably null for ADH1 activity.

Alcohol Dehydrogenase↗

PubMed1985

Expression of genes transferred into monocot and dicot plant cells by electroporation.

We have developed a general method for electrically introducing DNA into plant cells. Gene transfer occurs when a high-voltage electric pulse is applied to a solution containing protoplasts and DNA. Carrot protoplasts were used as a model system to optimize gene-transfer efficiency, which was measured 24-48 hr after electroporation by the amount of chloramphenicol acetyltransferase activity resulting from the expression of the introduced chimeric plasmids. Gene-transfer efficiency increased with the DNA concentration and was affected by the amplitude and duration of the electric pulse as well as by the composition of the electroporation medium. Our optimized gene-transfer conditions were effective when applied to tobacco and maize protoplasts, demonstrating that the method is applicable to both monocot and dicot protoplasts.

Acetyltransferases↗

PubMed1985

Maize mitochondria synthesize organ-specific polypeptides.

We detected both quantitative and qualitative organ-specific differences in the total protein composition of mitochondria of maize. Labeling of isolated mitochondria from each organ demonstrated that a few protein differences are due to changes in the polypeptides synthesized by the organelle. The synthesis of developmental stage-specific mitochondrial polypeptides was found in the scutella of developing and germinating kernels. The approximately 13-kDa polypeptide synthesized by mitochondria from seedlings of the Texas (T) male-sterile cytoplasm was shown to be constitutively expressed in all organs of line B37T tested. Methomyl, an insecticide known to inhibit the growth of T sterile plants, was shown to be an effective inhibitor of protein synthesis in mitochondria from T plants.

Electrophoresis, Polyacrylamide Gel↗

PubMed1984

Mechanisms of cytoplasmic pH regulation in hypoxic maize root tips and its role in survival under hypoxia.

We show that a transient lactic fermentation provides the signal triggering ethanol production in hypoxic maize root tips. The signal is cytoplasmic pH. This interaction between lactic and ethanolic fermentation permits tight cytoplasmic pH regulation during hypoxia--cytoplasmic pH remaining near neutrality for several hours. Mutant roots unable to synthesize ethanol can neither regulate cytoplasmic pH nor maintain ATP levels during extended periods of hypoxia and, like vertebrate tissues, are less tolerant of hypoxia than normal maize. This indicates that cytoplasmic pH regulation is an important factor in survival under hypoxia.

Cytoplasm↗