Molecular cloning and characterisation of a maize cDNA for a homologue of the large subunit of the eukaryotic initiation factor 3 (eIF3).

In order to identify genes that are specifically expressed in distinct cell populations of the maize root apex, we have constructed PCR-directed cDNA libraries from microdissected populations of cells, and screened them by differential hybridisation. A meristem-specific cDNA was isolated and characterised. This cDNA, termed ZmeIF3A, encodes a protein homologous to the large subunit of the eukaryotic translation Initiation Factor 3 (eIF3), which is an essential multi-protein complex for the initiation of protein synthesis. The ZmeIF3A protein is most similar to the yeast homologue RPG1, lacking the repeated C-terminal domain characteristic of its mammalian counterparts. However, despite this similarity, it fails to replace the RPG1 protein in complementation experiments on yeast mutants. Analysis of gene expression in situ showed that the ZmeIF3A transcript is expressed in the region of the root meristem surrounding the central stele. ZmeIF3A mRNA is also expressed in the young root, the male inflorescence, and the developing cob and seed. In maize, ZmeIF3A is encoded by one or two genomic sequences. This is the first report on the isolation and characterisation of a cDNA from higher plants that encodes a product homologous to a component of the eIF3 complex.

Annexin-like protein from Arabidopsis thaliana rescues delta oxyR mutant of Escherichia coli from H2O2 stress.

Reactive oxygen species are common causes of cellular damages in all aerobic organisms. In Escherichia coli, the oxyR gene product is a positive regulator of the oxyR regulon that is induced in response to H2O2 stress. To identify genes involved in counteracting oxidative stress in plants, we transformed a delta oxyR mutant of E. coli with an Arabidopsis thaliana cDNA library and selected for clones that restored the ability of the delta oxyR mutant to grow in the presence of H2O2. Using this approach, we isolated a cDNA that has strong homology with the annexin super-gene family. The complemented mutant showed higher catalase activity. mRNA expression of the annexin gene in A. thaliana was higher in roots as compared with other organs and was also increased when the plants were exposed to H2O2 stress or salicylic acid. Based on the results presented in this study, we propose a novel physiological role for annexin in counteracting H2O2 stress.

cDNA cloning and characterisation of a maize homologue of the MCM proteins required for the initiation of DNA replication.

A central question in cell cycle regulation is how DNA replication is initiated and executed only once in each cell cycle. The cell cycle-regulated assembly of specific initiation protein complexes at chromosomal origins appears to specify the initial sites and timing of DNA replication, and to restrict this process to only one round in the somatic cell cycle. Among the enzymes involved in origin activation, the MCM proteins play a conserved key role. In particular, MCM3 homologues have been shown to be components of the DNA replication licensing activity in yeast and vertebrates. In spite of our detailed knowledge of the regulation of the initiation of DNA synthesis in yeast, there is virtually no information available on the molecules involved in origin activation in higher plants. We have isolated a cDNA from maize root apices, termed ROA (Replication Origin Activator), encoding a protein which shares a high degree of homology with the MCM3 subfamily of MCM proteins. Analysis of gene organisation by Southern blotting shows 2-4 copies per haploid genome of closely related ROA sequences and the presence of further less related sequences in a multigene family. The steady-state levels of ROA mRNA are under developmental control, being relatively high in proliferative tissues such as the root apex, the developing cob and the coleoptile, and are strongly correlated with that of the histone H4 transcript. In situ hybridisation analysis in the root apex reveals that ROA mRNA expression is limited to specific subpopulations of cycling cells, which is typical of cell cycle-regulated expression. The isolation of nearly identical sequences from barley and Arabidopsis by the polymerase chain reaction indicates that MCM-related proteins are conserved in higher plants.

The resistance of cowpea seeds to bruchid beetles is not related to levels of cysteine proteinase inhibitors.

A cDNA encoding a cysteine proteinase inhibitor was isolated from a cDNA library prepared from developing seeds of an insect-resistant line of cowpea. The sequence of the encoded protein was homologous with those of other plant cysteine endoproteinase inhibitors, and with Type 2 cystatins from animals. Southern blot analyses indicated that small gene families were present in both resistant and susceptible lines of cowpea, while northern blot analyses showed similar levels of expression. It is concluded that the levels of expression of the inhibitor do not account for the differences in insect resistance of the two lines.

Molecular analyses of a barley multigene family homologous to the yeast protein kinase gene SNF1.

Genomic sequences homologous to the yeast gene SNF1 have been isolated from barley (Hordeum vulgare) cv. Sunbar. SNF1 encodes a protein serine/threonine kinase required for the derepression of a number of genes, including SUC2 (invertase) in response to glucose deprivation. Southern blotting showed the presence of a family of related genes in barley and full-length sequences have been determined for two members of the family, one of which lacks an exon and is almost certainly non-functional. A partial sequence has been obtained for a third member of the family. The transcription start site of one of the genes has been determined by S1 nuclease protection. A transcript almost identical in sequence to the exons of one of the genes has been amplified from barley endosperm mRNA using the polymerase chain reaction. One of the full-length genomic sequences contains nine introns and 10 exons and the number and position of the introns in the second full-length sequence is identical except that it lacks exon 2. However, the length and sequence of the introns vary. Northern blot analyses indicated that related transcripts are present in aleurones, coleoptiles, endosperms, internodes, leaves, ovules, roots and root tips, with highest levels of expression in the aleurones and endosperms.

Restriction fragment analysis of the secalin loci of rye.

Analyses of wheat/rye addition lines by Southern blotting confirmed the presence of sequences related to the Sec 1, Sec 2, and Sec 3 loci on chromosomes 1R and 2R. Comparison of the 1R and 2R addition lines allowed the identification of gamma-secalin genes at Sec 1 and Sec 2, respectively, while omega-secalin and gamma-secalin genes at Sec 1 were discriminated by comparative hybridization with three probes: omega-secalin, total gamma-secalin, and 3' gamma-secalin. The high molecular weight (HMW) secalin genes at Sec 3 were identified using a homologous HMW subunit probe from wheat. Gene copy numbers were estimated as about 40-60 for omega-scalins, 5-10 for gamma-secalins, and 2 for HMW secalins. Comparison of individual plants of cv. Gazelle showed a high degree of polymorphism, particularly for sequences related to omega-secalins and HMW secalins.

Restriction fragment analysis of 'null' forms at the Gli-1 loci of bread and durum wheats.

Wheat accessions lacking some of the ω- and γ-gliadin components encoded by the Gli-1 loci on the short arm of chromosome 1D in bread wheat and chromosome 1A in durum wheat were studied by two-dimensional polyacrylamide gel electrophoresis and restriction fragment analysis. Digested genomic DNAs of 'normal' and 'null' forms were probed with a cDNA clone related to ω-/γ-gliadins and with a genomic clone encoding an LMW subunit of glutenin. The hybridisation patterns with the ω-/γ-gliadin probe were similar to those of cvs 'Chinese Spring' and 'Langdon' used as standards for bread and durum wheats, respectively, but several restriction fragments located on the 1D chromosome of bread wheat and the 1A chromosome of durum wheat were absent in the 'null' forms. In addition, specific LMW glutenin fragments encoded by the same chromosomes were also absent in the 'null' forms, suggesting that simultaneous deletions of blocks of genes for both ω-/γ-gliadins and LMW glutenins had occurred. Comparisons of the protein and RFLP patterns enabled some proteins to be mapped to specific restriction fragments.

Complementation of snf1, a mutation affecting global regulation of carbon metabolism in yeast, by a plant protein kinase cDNA.

A cDNA, cRKIN1, encoding a putative homologue of the yeast (Saccharomyces cerevisiae) SNF1-encoded protein-serine/threonine kinase, has been isolated from a library prepared from rye endosperm mRNA. Northern blot analysis demonstrated the presence of cRKIN1-related transcripts in developing endosperms but not in shoots, and Southern blot analysis showed the presence of a small gene family. SNF1 plays a central role in carbon catabolite repression in yeast and expression of the RKIN1 sequence in yeast snf1 mutants restored SNF1 function. This suggests that the RKIN1 protein has a role in the control of carbon metabolism in endosperms of rye.

Characterization and organization of gene families at the Gli-1 loci of bread and durum wheats by restriction fragment analysis.

Probes related to γ-gliadins and to the LMW subunits of glutenin were used to determine the complexity of the Gli-1 loci, by RFLP analysis of euploid and aneuploid lines of bread wheat cv Chinese Spring and durum wheat cv Langdon. The two probes hybridised to separate sets of fragments derived from chromosomes 1 A, 1 B and 1D. The fragments related to the LMW subunit probe had a total copy number in HindIII digests of about 35 in Chinese Spring and 17 in Langdon, with more fragments derived from chromosomes 1D. The fragments hybridising to the γ-gliadin probe could be divided into two classes, based on whether they hybridised to the whole probe at high stringency or to the 3' nonrepetitive region at moderate stringency. The fragments that failed to hybridise under these conditions were considered to be related to ω-gliadins. The fragments related to γ - and co-gliadins had total copy numbers of about 39 and 16, respectively, in HindIII digests of Chinese Spring, and about 24 and 12, respectively, in Langdon.