Biomedical subjects

C Damerval

Publications and source records attributed to C Damerval.

15 recordsLinked to original sources

PubMed2001

Quantification of individual zein isoforms resolved by two-dimensional electrophoresis: genetic variability in 45 maize inbred lines.

A two-dimensional (2-D) electrophoresis procedure was optimized to obtain well-resolved and reproducible patterns of zein polypeptides in maize. All zein isoforms obtained through zein-specific extraction were observed from whole meal extracted with a urea/Triton/2-mercaptoethanol solution. Loading the protein samples at the acidic side of the gradient, using an amino acid solution as catholyte and running for a short time period under high Vh reduced shrinking and instability at the basic side of the isoelectric focusing (IEF) gels. Good quality and reproducible 2-D patterns were thus obtained, allowing automatic spot quantification. A linear relationship between spot intensity and zein isoform amount was established for 20 of 22 zeins detected in a 5.5-8.5 pH range using colloidal Coomassie blue staining in one maize line. The analysis of 45 genetically diverse inbred lines allowed the detection of 59 isoforms belonging to the four classes of zeins, and revealed a large qualitative and quantitative variability of individual isoforms. The classical decrease in zein amount in o2 mutant genotype was observed, and could be quantified for every isoform. The improved technique will be useful to dissect the genetic control of zein expression in maize.

Alleles↗

PubMed1999

Proteomics for genetic and physiological studies in plants.

Proteomics is becoming a necessity in plant biology, as it is in medicine, zoology and microbiology, for deciphering the function and role of the genes that are or will be sequenced. In this review we focus on the various, mainly genetic, applications of the proteomic tools that have been developed in recent years: characterization of individuals or lines, estimation of genetic variability within and between populations, establishment of genetic distances that can be used in phylogenetic studies, characterization of mutants and localization of the genes encoding the revealed proteins. Improvements in specifically devoted software have permitted precise quantification of the variation in amounts of proteins, leading to the concept of "protein quantity loci" which, combined with the "quantitative trait loci" approach, results in testable hypotheses regarding the role of "candidate proteins" in the metabolism or phenotype under study. This new development is exemplified by the reaction of plants to drought, a trait of major agronomic interest. The accumulation of data regarding genomic and cDNA sequencing will be connected to the protein databases currently developed in plants.

Chromosome Mapping↗

PubMed1999

Quality protein maize: a biochemical study of enzymes involved in lysine metabolism.

Quality protein maize (QPM) varieties have been produced by the introduction of opaque-2 modifier genes. Two QPM varieties, BR451 and BR473, a wild type and an opaque-2 variety, have been used to study key enzymes controlling lysine metabolism in the endosperm during development. Aspartate kinase and homoserine dehydrogenase enzymes, which are involved in lysine and threonine biosynthesis, respectively, exhibited identical activity patterns during endosperm development, with a maximum specific activity at 16 days after pollination. The QPM varieties exhibited higher levels of aspartate kinase activity in the endosperm, suggesting an increased rate of lysine biosynthesis when compared to the opaque-2 and wild-type genotypes. Similar results were observed for the lysine ketoglutarate reductase and saccharopine dehydrogenase enzymes, which form a single bifunctional polypetide involved in endosperm lysine degradation. Both enzyme activities were strongly reduced in the opaque-2 maize variety when compared to the wild-type maize, whereas the QPM varieties exhibited even lower levels of lysine ketoglutarate reductase-saccharopine dehydrogenase activities when compared to the opaque-2 variety. The developmental pattern of enzyme activity showed a different profile when compared to the enzymes involved in lysine biosynthesis, with activity being detected only 12-16 days after pollination (DAP) and maximum activities approximately 24 DAP. These results also suggest that the modifier genes have intensified the effect of the opaque-2 mutation on lysine ketoglutarate reductase-saccharopine dehydrogenase. These alterations lead to an increase in soluble lysine in the endosperm of the QPM varieties when compared to the opaque-2 and wild type.

Aspartate Kinase↗

PubMed1998

Characterization of novel proteins affected by the o2 mutation and expressed during maize endosperm development.

The effect of the o2 mutation on protein expression during grain development was examined by two-dimensional electrophoresis (2-D PAGE) in seven different pairs of near-isogenic maize lines. The aim was to identify a set of proteins that are consistently affected in mutants, and which could be the products of new genes that are direct or indirect targets of the transcriptional activator O2. The abundance of 36 polypeptides was found to be modified in the seven backgrounds. Seventeen polypeptides were present in greater amounts in wild types than in mutants, and most of these were affected early. The remaining polypeptides were expressed at higher levels in mutants than in the wild types and were generally affected later in development, suggesting that they might be products of indirect targets of O2. Products of known direct target genes such as zeins, b-32 protein and a pyruvate orthophospate dikinase were included in the first set of polypeptides. Microsequencing of internal stretches of 15 amino acids was performed for thirteen polypeptides and homologies with sequences stored in databases were found for nine of them. Enzymes belonging to various metabolic pathways were tentatively identified, most of which were not previously known to be affected by the o2 mutation. These results confirm that the O2 gene could act as a connecting regulatory gene for different pathways of grain metabolism.

Alleles↗

PubMed1997

High rates of polymorphism and recombination at the Opaque-2 locus in cultivated maize.

As a first step in the study of the functional consequences of molecular polymorphism at the Opaque-2 locus in maize, 1933 bp of the gene were sequenced in 21 inbred lines representative of the progenitors of cultivated varieties. High levels of polymorphism were found: 109 sites were variable in non-coding regions and 159 in protein-coding regions. Among the latter, 103 were nonsynonymous, resulting in 94 amino acid replacements in a 422-residue peptide. Moreover, 26 insertion/deletion polymorphisms were found, eight of them in coding regions. The high rate of polymorphism observed could indicate that the effective size of the ancestral population was very large, and/or that the O2 gene evolved under the influence of a high mutation rate or was subjected to some kind of balancing selection. The distribution of polymorphism within the sequence was not uniform. Silent polymorphisms were relatively frequent in exon 1 and rare in intron 5, whereas nonsynonymous polymorphisms were infrequent in the part of the sequence encoding the active domain of the transcription factor. The peptide sequence also showed a relatively higher level of resistance to amino acid replacements in this region. Sites in linkage disequilibrium were arranged mainly in three spatial patterns. This mosaic pattern can be related with an apparently large number of recombination events which might be a characteristic feature of this particular sample and/or imply that the O2 locus is a hot spot for recombination.

Base Sequence↗

PubMed1996

Two-dimensional protein patterns of Arabidopsis wild-type and auxin insensitive mutants, axr1, axr2, reveal interactions between drought and hormonal responses.

In order to detect gene products involved in Arabidopsis drought adaptive strategy, 2D-PAGE protein patterns of two auxin-insensitive mutants, axr1, axr2, differentially affected in specific drought responses, were compared to the wild-type Columbia ecotype, in well-watered and drought-stressed conditions. Coupled to computer analysis of polypeptide amounts, 2D-electrophoresis revealed subtle changes in protein expression induced by progressive drought stress and/or mutations affecting the auxin response pathway. The differential protein patterns of axr1 and axr2 were consistent with their contrasting drought responses. The specific leaf and root protein patterns of axr1 showed that this mutation disrupts drought responses related to auxin regulation. In particular, the near absence of drought rhizogenesis in axr1 was associated with a root protein pattern closer to the well-watered than to the water-stressed axr2 and Columbia wild-type root protein patterns. Also, the largely different effects of axr1 and axr2 mutations suggest that they affect different pathways in auxin response. Several sets of polypeptides, whose regulation was affected by drought and/or mutation, were thus detected. These polypeptides could play a role both in the auxin and the drought response pathways. Their identification, through microsequencing, should be most informative.

Arabidopsis↗

PubMed1995

Characterizing allelic proteins for genome mapping in maize.

Mapping cDNA probes in order to construct genetic linkage maps is becoming a widespread strategy for genome analysis and gene isolation, particularly in cultivated plant species. Nevertheless, almost all cDNAs reveal two or more unlinked loci, making it difficult to identify the gene(s) actually expressed. In a highly polymorphic species, such as maize, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) of proteins may circumvent this limitation. With the analysis of various segregating populations, we previously showed that the apparent position shifts of proteins on the 2-D gels are monogenic and codominant. In this paper we compared allelic proteins on the basis of their high performance liquid chromatography (HPLC) profile and partial amino acid sequences. In a sample of 20 position shifts, the allelic proteins appeared to be similar in all but one case, strongly suggesting that polymorphism of structural genes is involved. Thus 2-D PAGE could prove to be a useful tool for genome mapping: when a cDNA probe detects several loci, a position shift of the encoded protein will allow the identification of the gene translated in the organ considered.

Alleles↗

PubMed1994

Quantification of silver-stained proteins resolved by two-dimensional electrophoresis: genetic variability as related to abundance and solubility in two maize lines.

Relative abundance and solubility of proteins from etiolated coleoptiles of maize were investigated using two-dimensional electrophoresis (2-D PAGE). Automatic quantification of silver-stained polypeptides on replicate 2-D gels made it possible to test the linearity of the relationship between spot integrated optical density and protein amount in the range from 15 micrograms to 135 micrograms per gel, in two inbred lines. A linear response was found for more than 60% of the spots in each genotype. When a linear response was found in both lines for a given spot, the slope values were similar in 94% of the cases, indicating the reliability of silver staining for polypeptide quantification. The parameters of the curves allowed the definition of protein classes of different abundances that could be compared for genetic variability between the two lines. From a comparison between standard 2-D patterns (trichloroacetic acid-acetone extracted proteins) and patterns obtained from Tris buffer extracted proteins, it appeared that 92% of the proteins visualized in the standard gels were soluble. No difference in genetic variability, either qualitative or quantitative, was evidenced between the various classes of abundance, or between soluble and insoluble proteins.

Analysis of Variance↗

PubMed1994

Quantitative trait loci underlying gene product variation: a novel perspective for analyzing regulation of genome expression.

A methodology to dissect the genetic architecture of quantitative variation of numerous gene products simultaneously is proposed. For each individual of a segregating progeny, proteins extracted from a given organ are separated using two-dimensional electrophoresis, and their amounts are estimated with a computer-assisted system for spot quantification. Provided a complete genetic map is available, statistical procedures allow determination of the number, effects and chromosomal locations of factors controlling the amounts of individual proteins. This approach was applied to anonymous proteins of etiolated coleoptiles of maize, in an F2 progeny between two distant lines. The genetic map included both restriction fragment length polymorphism and protein markers. Minimum estimates of one to five unlinked regulatory factors were found for 42 of the 72 proteins analyzed, with a large diversity of effects. Dominance and epistasis interactions were involved in the control of 38% and 14% of the 72 proteins, respectively. Such a methodology might help understanding the architecture of regulatory networks and the possible adaptive or phenotypic significance of the polymorphism of the genes involved.

Chromosome Mapping↗

PubMed1994

Mapping factors controlling genetic expression.

A methodology relying on two-dimensional (2D) electrophoresis and genetic mapping is proposed to dissect the genetic architecture of the quantitative control of gene products. Seventy-two anonymous polypeptide spots resolved using silver-stained high-resolution 2D gels of maize coleoptile extracts were automatically quantified for every individual of an F2 progeny between distant lines. A complete genetic linkage map, including 70 RFLP and 39 protein markers, allowed us to map Mendelian factors underlying quantitative variation for 42 polypeptides. One to 5 unlinked chromosomal regions were found to affect single polypeptides, 27% of which resulted in more than a doubling of the polypeptide spot intensity. Dominance was observed for half of the factors, with high amount dominant over low amount in most cases, which is consistent with the observation that the relative abundance of proteins in F1 hybrids is in average deviated to the high parental values. Epistatic interactions were shown to be involved in the control of 14% of the proteins. These features contrast sharply with the observations from 20 agromorphological traits measured in the same progeny, where limited substitution and dominance effects, and no epistatic interaction, were found.

Alleles↗

PubMed1993

Analysis of scaling methods to minimize experimental variations in two-dimensional electrophoresis quantitative data: application to the comparison of maize inbred lines.

The analysis of two-dimensional (2-D) electrophoresis quantitative data from a design involving 21 maize genotypes revealed a significant experimental variation. In order to minimize this variation, we investigated the possible causes and found that it was essentially due to global effects, affecting all the spots in a gel in a similar way, and occurring during the 2-D run/staining procedure. Three scaling methods to discard these experimental variations were analyzed: the linear scaling method, a method based on principal component analysis, and a combined method that unites the advantages of both of the former. Comparing these three methods, we found that they led to consistent results with regard to the factor under study, i.e. the genetic factor in our case. However, the combined scaling method was the most efficient in reducing experimental variations.

Data Interpretation, Statistical↗

PubMed1988

Genetic aspects of variation of protein amounts in maize and pea.

Using high-resolution two-dimensional polyacrylamide gel electrophoresis we studied the polymorphism of protein amounts in some genotypes of maize and pea. This type of variability seems to be rather common and insensitive to environmental conditions, as attested by the comparison of the patterns of two maize lines harvested in two different years. A large-scale experiment involving 5 lines, 7 of their hybrids, and 6 organs (or physiological stages) of maize allowed us to examine numerous polypeptides regarding their genetic variability, their amount differences between organs and the inheritance of their abundance. Genetic and organ variations are not independent: polypeptides whose amount varies from one organ to another are, for the most part, genetically variable (59%), while the stable polypeptides are not often genetically variable (18%). We found a striking organ specificity for (i) the extent of quantitative variability (from 2.3-15.4% of the polypeptides), (ii) the occurrence and the type of variation for a given polypeptide (an intensity difference seen in an organ can disappear or even be reversed in another one), (iii) the kind of inheritance (additive/non-additive): combining the 6 organs and the 7 hybrids we found 101 cases of non-additivity (4% of the total) which concern as many as 72 different spots, that is to say that in most cases a polypeptide displaying nonadditivity in an organ seems to display additivity in the other ones. Moreover, for most of the polypeptides with nonadditive inheritance the hybrid spot presents an intensity similar to that of the most intense parental spot.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophoresis, Gel, Two-Dimensional↗

PubMed1988

Differential Two-Dimensional Protein Patterns as Related to Tissue Specificity and Water Conditions in Brassica napus var oleifera Root System.

Differential two-dimensional protein patterns as related to tissue specificity and water conditions were investigated within Brassica napus var oleifera root system. The different parts of the root system (tap root, lateral roots, and drought-induced short roots) were analyzed under various moisture regimes (regular watering at field capacity, progressive drought stress, and rewatering). Tissue specificity was evident from 25 differences in protein patterns (qualitative and quantitative) between well-watered lateral and tap roots. Twice as many polypeptides (52) were drought-affected and the response to the water stress was shown to be similar in both root types. In addition, more than half of the polypeptides detected as organ-specific were affected by drought. Based upon the trend of variation observed under drought and rehydration, three categories of polypeptides could be defined that might be differently involved in drought susceptibility or tolerance. A highly differentiated protein pattern characterized the drought-induced short roots. This pattern appeared as far from the watered as from the water-stressed normal roots. In particular, 13 unique polypeptides were detected which could be relevant to their adaptive morphogenesis and/or their specific drought tolerance induction. Upon rehydration, their polypeptide pattern and their specific morphology returned to a normal well-watered lateral root type.

Journal Article↗

PubMed1987

Drought-Induced Changes in Protein Patterns of Brassica napus var. oleifera Roots.

Drought-induced changes in two-dimensional silver stained protein patterns of Brassica napus L. var. oleifera M. root system were detected both at quantitative and qualitative levels. Particularly, 13 new polypeptides of low molecular weight were evidenced in the drought-stressed tap root, 12 of which were also present in the short tuberized roots, a specific drought-induced root type. The reversibility of these modifications, observed after 3 days rehydration, suggests that they might be involved in drought tolerance.

Journal Article↗

PubMedDate not supplied

QTL analysis of proteome and transcriptome variations for dissecting the genetic architecture of complex traits in maize.

In this review, we present some studies on genetic analysis of proteome and transcriptome variations, which exemplify new strategies for a better understanding of the molecular and genetic bases of complex traits. A large genetic variability was revealed at the proteome expression level, which raised the possibility to predict phenotypical performance on the basis of gene product variability. This approach yielded limited results, but could be re-newed by extensive identification of proteins now allowed by mass spectrometry. The dissection of the genetic basis of the variation of individual protein amounts proves very powerful to select 'candidate' proteins, physiologically relevant for a given phenotypical trait, as shown by a study on the effect of water stress in maize. In order to investigate factors of grain quality in maize, we selected a regulatory locus known to control the expression of several storage protein genes, Opaque-2, and investigated the relationships between variability in zein amount and composition and the molecular polymorphism at this locus. Moreover, a QTL analysis revealed that the variability in Opaque-2 transcript abundance was controlled by several polymorphic trans-acting regulators unlinked to the Opaque-2 structural gene. Such genetic approaches should represent additional tools for physiological analysis of the huge amounts of data generated by transcritome and proteome projects.

DNA-Binding Proteins↗