Molecular cloning of the a1 locus of Zea mays using the transposable elements En and Mu1

The a1 locus of Zea mays has been cloned using transposable elements as gene tags. The strategy was to make genomic libraries from maize stocks with a1 mutations induced either by En(Spm) or by Robertson's Mutator-system. These libraries were then screened with either Spm-I8 and En1, for the En-containing mutant, or with Mu1 for the Mu-induced mutation. There are many En and Mu1 hybridizing sequences present in the maize genome, however, by a process of cross-screening of the positives from the two libraries and by molecular analysis of the En-positive clones it was possible to identify clones in both libraries carrying all or part of the a1 gene.     

Nematodes associated with the rhizosphere of corn (Zea mays L.)

The research was carried out during 1999 in 8 different localities in Northern Italy. The nematodes were extracted from soil samples of rhizosphere of corn plants (Zea mays L.). The objective of the study was to investigate plant-parasite nematodes associated with maize. Some phytophagous genera are common pests of this crop and its yield-loss are often due to their high densities. In addition the nematode community was investigated for the genus composition, the trophic structure and its biodiversity. After the extraction from soil with a Bearmann funnel and Ludox centrifugation, nematodes were identified at genus level. They belonged to 22 families and 45 different genera. The genus Rhabditis, Pratylenchus, Helicotylenchus and Acrobeloides made up more than 70% of the total nematodes collected. The dominant trophic group was the bacterial feeders (61%) in particular Rhabditis, that was the most abundant and often the dominant one. Phytophagous represented in almost all fields more than 30% of the total nematodes. In all the examined sites biodiversity was quite low, being the H' values no more than 1.08. The data indicates a high level of disturbance. In some localities high densities of Helicotylenchus and Pratylenchus were found. While these nematodes have been identified as being potentially harmful for corn plants in our latitudes, especially in light soils, this research could give an indication for further monitoring studies regarding plant parasitic nematodes of corn crops. This data is particularly important considering that methyl bromide, often used in Italian agriculture against soil pathogens, has been banned since the beginning of 2005.     

The identification and characterization of two dominant r1 haplotype-specific inhibitors of aleurone color in Zea mays

We report the identification and characterization of two novel dominant inhibitors of aleurone color in Zea mays that interact with specific haplotypes of the r1 locus. One inhibitor locus, inr1 (inhibitor of r1 aleurone color 1), maps to the long arm of chromosome 10, distal to the TB-10L19 breakpoint and tightly linked to dull1, and the second inhibitor locus, inr2 (inhibitor of r1 aleurone color 2), maps to the long arm of chromosome 9. Dominant inhibitory alleles of inr1 and inr2 act by suppressing aleurone color conditioned by certain r1 haplotypes. Two haplotypes, R1-ch:Stadler and R1-Randolph, exhibit nearly complete suppression of aleurone color in the presence of inhibitory alleles of inr1 or inr2. Two members of the R1-d class of haplotypes, R1-d:Catspaw and R1-d:Arapaho, show partial suppression. Other haplotypes tested were not visibly affected. The response of r1 haplotypes to inhibitory inr1 and inr2 alleles provides another means of analyzing the complex behavior of the seed color components of r1 haplotypes. Possible mechanisms of action of inr1 and inr2 are discussed.     

Genome-wide analysis of immunophilin FKBP genes and expression patterns in Zea mays

The receptors for the immunosuppression drugs FK506 and rapamycin are called FKBPs (FK506-binding proteins). FKBPs comprise a large family; they are found in many species, including bacteria, fungi, animals, and plants. As a class of peptidyl-prolyl cis-trans isomerase enzymes, the FKBP genes have been the focus of recent studies on plant stress tolerance and immunology. We identified and analyzed gene families encoding these proteins in maize using computational and molecular biology approaches. Thirty genes were found to encode putative FKBPs according to their FK506-binding domain. The FKBP genes can be classified into single domain and multiple domain members based on the number of the domains. By analysis of the physical locations, the 30 FKBP genes were found to be widely distributed on 10 chromosomes. After analysis of the FKBP phylogenetic tree in the maize genome, we found that the 30 genes revealed two major clades. Gene duplication played a major role in the evolution of FKBP genes, which suggests that the FKBP genes in maize have a pattern significantly different from that of these genes in rice. Based on semi-quantitative RT-PCR, we found that the 30 FKBPs were expressed differently in various tissues in maize, which suggests that FKBP genes play different roles in each tissue. Several FKBPs were expressed at higher levels in roots, indicating that these genes in maize may have similar or overlapping functions.     

Identification of specific proteins and glycoproteins associated with membrane fractions isolated from Zea mays L. coleoptiles

The aim of this work was to identify proteins specific for plant cell membranes which could then be used as unique markers. A crude membrane fraction was isolated from corn coleoptiles and separated on non-linear sucrose density gradients. Separation of endoplasmic reticulum (NADH-cytochrome c reductase), mitochondria (cytochrome c oxidase), golgi (inosine diphosphatase), and plasma membranes (N-1-naphthylphthalamic acid-binding) was achieved. The membrane proteins from the gradient fractions were separated using sodium dodecyl sulphate-poly-acrylamide gel electrophoresis and the gels stained with coomassie blue or with concanavalin A/peroxidase to detect glycoproteins. Proteins specific for the various membranes were identified. Five proteins including two glycoproteins were plasma membrane markers. Protoplasts were isolated and iodinated using lactoperoxidase/glucose oxidase covalently attached to beads. Eleven iodinated proteins were found and three of these corresponded to proteins specifically associated with plasma membranes in the density gradients. Two methods for detecting Ca²⁺-binding proteins following sodium dodecylsulphate polyacrylamide gel electrophoresis were employed. The majority of such proteins were found in the endoplasmatic reticulum and one was specific for plasma membranes. In vitro and in vivo phosphorylation of membrane proteins was examined and the majority of proteins phosphorylated were glycoproteins. Two of the phosphorylated proteins (M_r=110,000 and 20,000) were also iodinated on protoplasts and may be part of the plasma membrane ATPases.Abbreviations ER endoplasmic reticulum - IDP inosine diphosphate - NPA N-1-naphthylphthalamic acid     

Sex- and age-dependent DNA methylation at the 17q12-q21 locus associated with childhood asthma

Chromosomal region 17q12-q21 is one of the best-replicated genome-wide association study (GWAS) hits and associated with childhood-onset asthma. However, the mechanism by which the genetic association is restricted to childhood-onset disease is unclear. During childhood, more boys than girls develop asthma. Therefore, we tested the hypothesis that the 17q12-q21 genetic association was sex-specific. Indeed, a TDT test showed that in the Saguenay-Lac-Saint-Jean familial collection, the 17q12-q21 association was significant among male, but not among female asthmatic subjects. We next hypothesized that the bias in the genetic association resulted from sex-specific and/or age-dependent DNA methylation at regulatory regions and determined the methylation profiles of five 17q12-q21 gene promoters using the bisulfite sequencing methylation assay. We identified a single regulatory region within the zona pellucida binding protein 2 (ZPBP2) gene, which showed statistically significant differences between males and females with respect to DNA methylation. DNA methylation also varied with age and was higher in adult males compared to boys. We have recently identified two functionally important polymorphisms, both within the ZPBP2 gene that influence expression levels of neighboring genes. Combined with the results of the present work, these data converge pointing to the same 5 kb region within the ZPBP2 gene as a critical region for both gene expression regulation and predisposition to asthma. Our data show that sex- and age-dependent DNA methylation may act as a modifier of genetic effects and influence the results of genetic association studies.