High gene density is conserved at syntenic loci of small and large grass genomes.
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Comparative genomic analysis at the genetic-map level has shown extensive conservation of the gene order between the different grass genomes in many chromosomal regions. However, little is known about the gene organization in grass genomes at the microlevel. Comparison of gene-coding regions between maize, rice, and sorghum showed that the distance between the genes is correlated with the genome size. We have investigated the microcolinearity at Lrk gene loci in the genomes of four grass species: wheat, barley, maize, and rice. The Lrk genes, which encode receptor-like kinases, were found to be consistently associated with another type of receptor-like kinase (Tak) on chromosome groups 1 and 3 in Triticeae and on chromosomes homoeologous to Triticeae group 3 in the other grass genomes. On Triticeae chromosome group 1, Tak and Lrk together with genes putatively encoding NBS/LRR proteins form a cluster of genes possibly involved in signal transduction. Comparison of the gene composition at orthologous Lrk loci in wheat, barley, and rice revealed a maximal gene density of one gene per 4-5 kb, very similar to the gene density in Arabidopsis thaliana. We conclude that small and large grass genomes contain regions that are highly enriched in genes with very little or no repetitive DNA. The comparison of the gene organization suggested various genome rearrangements during the evolution of the different grass species. (+info)
A maize glycine-rich protein is synthesized in the lateral root cap and accumulates in the mucilage.
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The root cap functions in the perception of gravity, the protection of the root apical meristem, and facilitation of the passage of roots through the soil, but the genes involved in these functions are poorly understood. Here we report the isolation of a root-specific gene from the cap of maize (Zea mays L.) primary root by cDNA subtraction and differential screening. The gene zmGRP4 (Z. mays glycine rich protein 4) encodes a member of the glycine-rich proteins with a putative signal peptide at the amino terminus. The deduced molecular mass of mature zmGRP4 is 14.4 kD. In situ-hybridization analysis has shown zmGRP4 to be strongly expressed in the lateral root cap and weakly expressed in the root epidermis. A polyclonal antibody raised against recombinant zmGRP4 detected a protein of 36 kD in the insoluble protein fraction extracted from the root tip and the root proper, indicating posttranslational modification(s) of zmGRP4. Immunohistochemical analysis showed the accumulation of zmGRP4 in the mucilage that covers the root tip. These results indicate that lateral root-cap cells secrete modified zmGRP4 into the mucilage to which the protein may contribute to its characteristic physical properties. (+info)
Purification and characterization of the reconstitutively active citrate carrier from maize mitochondria.
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The citrate carrier from maize (Zea mays) shoot mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxyapatite and hydroxyapatite/celite in the presence of cardiolipin. SDS-gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 31 kD. When reconstituted into liposomes, the citrate carrier catalyzed a pyridoxal 5'-P-sensitive citrate/citrate exchange. It was purified 224-fold with a recovery of 50% and a protein yield of 0.22% with respect to the mitochondrial extract. In the reconstituted system the purified citrate carrier catalyzed a first-order reaction of citrate/citrate (0.065 min-1) or citrate/malate exchange (0.075 min-1). Among the various substrates and inhibitors tested, the reconstituted protein transported citrate, cis-aconitate, isocitrate, L-malate, succinate, malonate, glutarate, alpha-ketoglutarate, oxaloacetate, and alpha-ketoadipate and was inhibited by pyridoxal 5'-P, phenylisothiocyanate, mersalyl, and p-hydroxymercuribenzoate (but not N-ethylmaleimide), 1,2, 3-benzentricarboxylate, benzylmalonate, and butylmalonate. The activation energy of the citrate/citrate exchange was 66.5 kJ/mol between 10 degrees C and 35 degrees C; the half-saturation constant (Km) for citrate was 0.65 +/- 0.05 mM and the maximal rate (Vmax) of the citrate/citrate exchange was 13.0 +/- 1.0 micromol min-1 mg-1 protein at 25 degrees C. (+info)
A maize homolog of mammalian CENPC is a constitutive component of the inner kinetochore.
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Genes for three maize homologs (CenpcA, CenpcB, and CenpcC) of the conserved kinetochore assembly protein known as centromere protein C (CENPC) have been identified. The C-terminal portion of maize CENPC shares similarity with mammalian CENPC and its yeast homolog Mif2p over a 23-amino acid region known as region I. Immunolocalization experiments combined with three-dimensional light microscopy demonstrated that CENPC is a component of the kinetochore throughout interphase, mitosis, and meiosis. It is shown that sister kinetochore separation occurs in two discrete phases during meiosis. A partial separation of sister kinetochores occurs in prometaphase I, and a complete separation occurs in prometaphase II. CENPC is absent on structures known as neocentromeres that, in maize, demonstrate poleward movement but lack other important features of centromeres/kinetochores. CENPC and a previously identified centromeric DNA sequence interact closely but do not strictly colocalize on meiotic chromosomes. These and other data indicate that CENPC occupies an inner domain of the maize kinetochore. (+info)
Gnarley1 is a dominant mutation in the knox4 homeobox gene affecting cell shape and identity.
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Maize leaves have a stereotypical pattern of cell types organized into discrete domains. These domains are altered by mutations in knotted1 (kn1) and knox (for kn1-like homeobox) genes. Gnarley (Gn1) is a dominant maize mutant that exhibits many of the phenotypic characteristics of the kn1 family of mutants. Gn1 is unique because it changes parameters of cell growth in the basal-most region of the leaf, the sheath, resulting in dramatically altered sheath morphology. The strongly expressive allele Gn1-R also gives rise to a floral phenotype in which ectopic carpels form. Introgression studies showed that the severity of the Gn1-conferred phenotype is strongly influenced by genetic background. Gn1 maps to knox4, and knox4 is ectopically expressed in plants with the Gn1-conferred phenotype. Immunolocalization experiments showed that the KNOX protein accumulates at the base of Gn1 leaves in a pattern that is spatially and temporally correlated with appearance of the mutant phenotype. We further demonstrate that Gn1 is knox4 by correlating loss of the mutant phenotype with insertion of a Mutator transposon into knox4. (+info)
Molecular characterization of the maize Rp1-D rust resistance haplotype and its mutants.
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The Rp1-D gene for resistance to maize common rust (Puccinia sorghi) is a member of a complex locus (haplotype) composed of Rp1-D and approximately eight other gene homologs. The identity of Rp1-D was demonstrated by using two independent gene-tagging approaches with the transposons Mutator and Dissociation. PIC20, a disease resistance (R) gene analog probe previously mapped to the rp1 locus, detected insertion of Dissociation in an Rp1-D mutation and excision in three revertants. Independent libraries probed with the PIC20 or Mutator probes resulted in isolation of the same gene sequence. Rp1-D belongs to the nucleotide binding site, leucine-rich repeat class of R genes. However, unlike the rust resistance genes M and L6 from flax, the maize Rp1-D gene does not encode an N-terminal domain with similarity to the signal transduction domains of the Drosophila Toll protein and mammalian interleukin-1 receptor. Although the abundance of transcripts of genes from the rp1 complex changed with leaf age, there was no evidence of any change due to inoculation with avirulent or virulent rust biotypes. A set of 27 Rp1-D mutants displayed at least nine different deletions of Rp1-D gene family members that were consistent with unequal crossing-over events. One mutation (Rp1-D*-24) resulted in deletion of all but one gene family member. Other unique deletions were observed in the disease lesion mimic Rp1-D*-21 and the partially susceptible mutant Rp1-D*-5. Different rp1 specificities have distinct DNA fingerprints (haplotypes). Analysis of recombinants between rp1 specificities indicated that recombination had occurred within the rp1 gene complex. Similar analyses indicated that the rust R genes at the rp5 locus, 2 centimorgans distal to rp1, are not closely related to Rp1-D. (+info)
Component specificity for the thylakoidal Sec and Delta pH-dependent protein transport pathways.
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Prokaryotes and prokaryote-derived thylakoid membranes of chloroplasts share multiple, evolutionarily conserved pathways for protein export. These include the Sec, signal recognition particle (SRP), and Delta pH/Tat systems. Little is known regarding the thylakoid membrane components involved in these pathways. We isolated a cDNA clone to a novel component of the Delta pH pathway, Tha4, and prepared antibodies against pea Tha4, against maize Hcf106, a protein implicated in Delta pH pathway transport by genetic studies, and against cpSecY, the thylakoid homologue of the bacterial SecY translocon protein. These components were localized to the nonappressed thylakoid membranes. Tha4 and Hcf106 were present in approximately 10-fold excess over active translocation sites. Antibodies to either Tha4 or Hcf106 inhibited translocation of four known Delta pH pathway substrate proteins, but not of Sec pathway or SRP pathway substrates. This suggests that Tha4 and Hcf106 operate either in series or as subunits of a heteromultimeric complex. cpSecY antibodies inhibited translocation of Sec pathway substrates but not of Delta pH or SRP pathway substrates. These studies provide the first biochemical evidence that Tha4 and Hcf106 are specific components of the Delta pH pathway and provide one line of evidence that cpSecY is used specifically by the Sec pathway. (+info)
Targeted manipulation of maize genes in vivo using chimeric RNA/DNA oligonucleotides.
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Site-specific heritable mutations in maize genes were engineered by introducing chimeric RNA/DNA oligonucleotides. Two independent targets within the endogenous maize acetohydroxyacid synthase gene sequence were modified in a site-specific fashion, thereby conferring resistance to either imidazolinone or sulfonylurea herbicides. Similarly, an engineered green fluorescence protein transgene was site-specifically modified in vivo. Expression of the introduced inactive green fluorescence protein was restored, and plants containing the modified transgene were regenerated. Progeny analysis indicated Mendelian transmission of the converted transgene. The efficiency of gene conversion mediated by chimeric oligonucleotides in maize was estimated as 10(-4), which is 1-3 orders of magnitude higher than frequencies reported for gene targeting by homologous recombination in plants. The heritable changes in maize genes engineered by this approach create opportunities for basic studies of plant gene function and agricultural trait manipulation and also provide a system for studying mismatch repair mechanisms in maize. (+info)