Recombinants isolated from most meiotic intragenic recombination experiments in maize, but not in yeast, are borne principally on crossover chromosomes. This excess of crossovers is not explained readily by the canonical double-strand break repair model of recombination, proposed to account for a large body of yeast data, which predicts that crossovers (COs) and noncrossovers (NCOs) should be recovered equally. An attempt has been made here to identify general rules governing the recovery of the CO and NCO classes of intragenic recombinants in maize. Recombination was analyzed in bz heterozygotes between a variety of mutations derived from the same or different progenitor alleles. The mutations include point mutations, transposon insertions, and transposon excision footprints. Consequently, the differences between the bz heteroalleles ranged from just two nucleotides to many nucleotides, indels, and insertions. In this article, allelic pairs differing at only two positions are referred to as dimorphic to distinguish them from polymorphic pairs, which differ at multiple positions. The present study has revealed the following effects at these bz heteroalleles: (1) recombination between polymorphic heteroalleles produces mostly CO chromosomes; (2) recombination between dimorphic heteroalleles produces both CO and NCO chromosomes, in ratios apparently dependent on the nature of the heteroalleles; and (3) in dimorphic heterozygotes, the two NCO classes are recovered in approximately equal numbers when the two mutations are point mutations but not when one or both mutations are insertions. These observations are discussed in light of a recent version of the double-strand break repair model of recombination that postulates separate pathways for the formation of CO and NCO products.

Functional analysis of chromosomal segments containing linked genes requires the insertion of contiguous genomic sequences from bacterial artificial chromosomes (BACs) into the genome. Therefore, we introduced a 90-kb large BAC clone carrying a 10-copy tandem array of kafirin storage protein genes from sorghum linkage group J, mixed with a selectable marker gene, directly into maize cells using the particle bombardment method. Transgenic plants were regenerated and seeds from eight different transgenic lines were produced. One such transgenic plant was selected that had the entire kafirin gene cluster on a single continuous DNA fragment spanning more than 45 kb integrated into its genome. When alcohol-soluble proteins from individual T2 and T3 seeds of this event were analyzed, significant levels of kafirin were found in addition to the endogenous zein storage proteins, demonstrating that the large exogenous DNA segment is stably integrated into the maize genome and expressed at high levels in subsequent generations. Therefore, we could provide a new utility of plant transformation by the particle bombardment method for functional genomics of multigene families and the modification of the nutritive quality of cereal grains. Despite a tandem array of highly homologous sequences at the transgenic locus, no gene silencing was observed, probably owing to the effects of co-transformed flanking sequences. The expression studies of the transgenic locus also revealed new features of storage protein gene promoters that differed from previous transient gene expression studies, thereby illustrating the significance of the concentration and configuration of DNA-protein interactions in the regulation of gene expression.

Helitrons are novel transposons discovered by bioinformatic analysis of eukaryotic genome sequences. They are believed to move by rolling circle (RC) replication because their predicted transposases are homologous to those of bacterial RC transposons. We report here evidence of somatic Helitron excision in maize, an unexpected finding suggesting that Helitrons can exhibit an excisive mode of transposition.

Plastids in Nicotiana tabacum are normally transmitted to the progeny by the maternal parent only. However, low-frequency paternal plastid transmission has been reported in crosses involving parents with an alien cytoplasm. Our objective was to determine whether paternal plastids are transmitted in crosses between parents with the normal cytoplasm. The transplastomic father lines carried a spectinomycin resistance (aadA) transgene incorporated in the plastid genome. The mother lines in the crosses were either (i) alloplasmic, with the Nicotiana undulata cytoplasm that confers cytoplasmic male sterility (CMS92) or (ii) normal, with the fertile N. tabacum cytoplasm. Here we report that plastids from the transplastomic father were transmitted in both cases at low (10(-4)-10(-5)) frequencies; therefore, rare paternal pollen transmission is not simply due to breakdown of normal controls caused by the alien cytoplasm. Furthermore, we have found that the entire plastid genome was transmitted by pollen rather than small plastid genome (ptDNA) fragments. Interestingly, the plants, which inherited paternal plastids, also carried paternal mitochondrial DNA, indicating cotransmission of plastids and mitochondria in the same pollen. The detection of rare paternal plastid transmission described here was facilitated by direct selection for the transplastomic spectinomycin resistance marker in tissue culture; therefore, recovery of rare paternal plastids in the germline is less likely to occur under field conditions.

Plastid DNA is absent in pollen or sperm cells of Arabidopsis thaliana. Accordingly, plastids and mitochondria, in a standard genetic cross, are transmitted to the seed progeny by the maternal parent only. Our objective was to test whether paternal plastids are transmitted by pollen as an exception. The maternal parent in our cross was a nuclear male sterile (ms1-1/ms1-1), spectinomycin-sensitive Ler plant. It was fertilized with pollen of a male fertile RLD-Spc1 plant carrying a plastid-encoded spectinomycin resistance mutation. Seedlings with paternal plastids were selected by spectinomycin resistance encoded in the paternal plastid DNA. Our data, in general, support maternal inheritance of plastids in A. thaliana. However, we report that paternal plastids are transmitted to the seed progeny in Arabidopsis at a low (3.9 x 10(-5)) frequency. This observation extends previous reports in Antirrhinum majus, Epilobium hirsutum, Nicotiana tabacum, Petunia hybrida, and the cereal crop Setaria italica to a cruciferous species suggesting that low-frequency paternal leakage of plastids via pollen may be universal in plants previously thought to exhibit strict maternal plastid inheritance. The genetic tools employed here will facilitate testing the effect of Arabidopsis nuclear mutations on plastid inheritance and allow for the design of mutant screens to identify nuclear genes controlling plastid inheritance.

Plastids and mitochondria, the DNA-containing cytoplasmic organelles, are maternally inherited in the majority of angiosperm species. Even in plants with strict maternal inheritance, exceptional paternal transmission of plastids has been observed. Our objective was to detect rare leakage of plastids via pollen in Nicotiana sylvestris and to determine if pollen transmission of plastids results in co-transmission of paternal mitochondria. As father plants, we used N. sylvestris plants with transgenic, selectable plastids and wild-type mitochondria. As mother plants, we used N. sylvestris plants with Nicotiana undulata cytoplasm, including the CMS-92 mitochondria that cause cytoplasmic male sterility (CMS) by homeotic transformation of the stamens. We report here exceptional paternal plastid DNA in approximately 0.002% of N. sylvestris seedlings. However, we did not detect paternal mitochondrial DNA in any of the six plastid-transmission lines, suggesting independent transmission of the cytoplasmic organelles via pollen. When we used fertile N. sylvestris as mothers, we obtained eight fertile plastid transmission lines, which did not transmit their plastids via pollen at higher frequencies than their fathers. We discuss the implications for transgene containment and plant evolutionary histories inferred from cytoplasmic phylogenies.

Glutamine-18 of the lac repressor (lacR) has been substituted by glycine, by serine, and by leucine. The specificities of wild-type lacR and of the three substituted lacR variants have been analyzed with respect to base pairs 5, 6, 7, 8, 9, and 10 of the lac operator (lacO). The data indicate that [Gly18]lacR, [Ser18]lacR, and [Leu18]lacR lose the ability to distinguish between the O+ base pair G . C and the Oc base pairs T . A and A . T at position 7 of lacO (KdOc/KdO+ approximately equal to 1). In contrast, the three substituted variants retain the ability to discriminate O+ from Oc at each other position, by factors of 9 to 37. Therefore, I propose that glutamine-18 contacts base pair 7 of lacO. These data suggest that the interaction between the helix-turn-helix motif and DNA may be very similar or identical in lacR and the catabolite gene activator protein.

Recent work has established that the Escherichia coli RNA polymerase alpha subunit consists of an amino-terminal domain containing determinants for interaction with the remainder of RNA polymerase, a carboxy-terminal domain containing determinants for interaction with DNA and interaction with transcriptional activator proteins, and a 13-36 amino acid unstructured and/or flexible linker. These findings suggest a simple, integrated model for the mechanism of involvement of alpha in promoter recognition and transcriptional activation.

Epidermal Growth Factor (EGF) is known for its role in promoting cell division and cellular differentiation in developing animals, but we know surprising little about what EGF does in vivo in mature adult animals. Here I review EGF signaling, emphasizing several recent studies that uncovered an unexpected role for EGF in promoting longevity and healthspan in mature adult C. elegans. EGF, acting through phospholipase Cγ and the IP3 receptor signaling, maintains pharyngeal and body wall muscle function in aging adults, and delays the accumulation of lipofuscin-enriched aging pigments within intestinal cells. EGF also acts through the Ras/ERK pathway to regulate protein homeostasis by promoting the expression of antioxidant genes, stimulating the activity of the Ubiquitin Proteasome System (UPS), and repressing the expression of small heat shock protein chaperones. The effects of EGF signaling on lifespan are largely independent of Insulin/IGF-like Signaling (IIS), as the effects of EGF signaling mutants on lifespan and heathspan are not affected by mutations in the DAF-2 insulin receptor or the DAF-16 FOXO transcription factor. Nevertheless, these two signal pathways have multiple points of overlap, coordination, and cross regulation. I propose that the IIS and EGF signaling pathways respond to environment and to developmental timing, respectively, so as to coordinate the appropriate physiological strategy that cells use to maintain protein homeostasis.

The plastids of higher plants have their own approximately 120-160-kb genome that is present in 1,000-10,000 copies per cell. Engineering of the plastid genome (ptDNA) is based on homologous recombination between the plastid genome and cloned ptDNA sequences in the vector. A uniform population of engineered ptDNA is obtained by selection for marker genes encoded in the vectors. Manipulations of ptDNA include (1) insertion of transgenes in intergenic regions; (2) posttransformation excision of marker genes to obtain marker-free plants; (3) gene knockouts and gene knockdowns, and (4) cotransformation with multiple plasmids to introduce nonselected genes without physical linkage to marker genes. Most experiments on plastome engineering have been carried out in the allotetraploid Nicotiana tabacum. We report here for the first time plastid transformation in Nicotiana sylvestris, a diploid ornamental species. We demonstrate that the protocols and vectors developed for plastid transformation in N. tabacum are directly applicable to N. sylvestris with the advantage that the N. sylvestris transplastomic lines are suitable for mutant screens.

We have engineered enhanced DNA-binding function into the a1 homeodomain by making changes in a loop distant from the DNA-binding surface. Comparison of the free and bound a1 structures suggested a mechanism linking van der Waals stacking changes in this loop to the ordering of a final turn in the DNA-binding helix of a1. Inspection of the protein sequence revealed striking differences in amino acid identity at positions 24 and 25 compared to related homeodomain proteins. These positions lie in the loop connecting helix-1 and helix-2, which is involved in heterodimerization with the alpha 2 protein. A series of single and double amino acid substitutions (a1-Q24R, a1-S25Y, a1-S25F and a1-Q24R/S25Y) were engineered, expressed and purified for biochemical and biophysical study. Calorimetric measurements and HSQC NMR spectra confirm that the engineered variants are folded and are equally or more stable than the wild-type a1 homeodomain. NMR analysis of a1-Q24R/S25Y demonstrates that the DNA recognition helix (helix-3) is extended by at least one turn as a result of the changes in the loop connecting helix-1 and helix-2. As shown by EMSA, the engineered variants bind DNA with enhanced affinity (16-fold) in the absence of the alpha 2 cofactor and the variant alpha 2/a1 heterodimers bind cognate DNA with specificity and affinity reflective of the enhanced a1 binding affinity. Importantly, in vivo assays demonstrate that the a1-Q24R/S25Y protein binds with fivefold greater affinity than wild-type a1 and is able to partially suppress defects in repression by alpha 2 mutants. As a result of these studies, we show how subtle differences in residues at a surface distant from the functional site code for a conformational switch that allows the a1 homeodomain to become active in DNA binding in association with its cofactor alpha 2.

We have investigated the methylation status of the alpha-tubulin genes, and the degree of accumulation of their mRNAs in endosperm, embryo and seedling tissues of Zea mays L. We have found that many of the alpha-tubulin genes are differentially demethylated in the endosperm relative to the embryo and seedling. However, only for tub alpha 2 and tub alpha 4 could a correlation between DNA demethylation and increased RNA accumulation be detected. By analyzing the inbred lines W64A and A69Y and their reciprocal crosses, we have also identified in the endosperm two alpha-tubulin genes, tub alpha 3 and tub alpha 4, that are differentially demethylated if transmitted by the maternal germline, but that remain hypermethylated when transmitted by the paternal germline.

High valence states in manganese clusters are a key feature of the function of one of the most important catalysts found in nature, the water-oxidizing complex of photosystem II. We describe a detailed electrochemical investigation of two bio-inspired manganese-oxo complexes, [Mn4O4L6] (L = diphenylphosphinate (1) and bis(p-methoxyphenyl)phosphinate (2)), in solution, attached to an electrode surface and suspended within a Nafion film. These complexes contain a cubic [Mn4O4]6+ core stabilized by phosphinate ligands. They have previously been shown to be active and durable photocatalysts for the oxidation of water to dioxygen. A comparison of catalytic photocurrent generated by films deposited by two methods of electrode immobilization reveals that doping of the catalyst in Nafion results in higher photocurrent than was observed for a solid layer of cubane on an electrode surface. In dichloromethane solution, and under conditions of cyclic voltammetry, the one-electron oxidation processes 1/1+ and 2/2+ were found to be reversible and quasi-reversible, respectively. Some decomposition of 1+ and 2+ was detected on the longer timescale of bulk electrolysis. Both compounds also undergo a two-electron, chemically irreversible reduction in dichloromethane, with a mechanism that is dependent on scan rate and influenced by the presence of a proton donor. When immersed in aqueous electrolyte, the reduction process exhibits a limited level of chemical reversibility. These data provide insights into the catalytic operation of these molecules during photo-assisted electrolysis of water and highlight the importance of the strongly electron-donating ligand environment about the manganese ions in the ability of the cubanes to photocatalyze water oxidation at low overpotentials.

Caenorhabditis elegans RAB-10 functions in endocytic recycling in polarized cells, regulating basolateral cargo transport in the intestinal epithelia and postsynaptic cargo transport in interneurons. A similar role was found for mammalian Rab10 in MDCK cells, suggesting that a conserved mechanism regulates these related pathways in metazoans. In a yeast two-hybrid screen for binding partners of RAB-10 we identified EHBP-1, a calponin homology domain (CH) protein, whose mammalian homolog Ehbp1 was previously shown to function during endocytic transport of GLUT4 in adipocytes. In vivo we find that EHBP-1-GFP colocalizes with RFP-RAB-10 on endosomal structures of the intestine and interneurons and that ehbp-1 loss-of-function mutants share with rab-10 mutants specific endosome morphology and cargo localization defects. We also show that loss of EHBP-1 disrupts transport of membrane proteins to the plasma membrane of the nonpolarized germline cells, a defect that can be phenocopied by codepletion of RAB-10 and its closest paralog RAB-8. These results indicate that RAB-10 and EHBP-1 function together in many cell types and suggests that there are differences in the level of redundancy among Rab family members in polarized versus nonpolarized cells.

Epidermal growth factor (EGF) signalling regulates growth and differentiation. Here, we examine the function of EGF signalling in Caenorhabditis elegans lifespan. We find that EGF signalling regulates lifespan via the Ras-MAPK pathway and the PLZF transcription factors EOR-1 and EOR-2. As animals enter adulthood, EGF signalling upregulates the expression of genes involved in the ubiquitin proteasome system (UPS), including the Skp1-like protein SKR-5, while downregulating the expression of HSP16-type chaperones. Using reporters for global UPS activity, protein aggregation, and oxidative stress, we find that EGF signalling alters protein homoeostasis in adults by increasing UPS activity and polyubiquitination, while decreasing protein aggregation. We show that SKR-5 and the E3/E4 ligases that comprise the ubiquitin fusion degradation (UFD) complex are required for the increase in UPS activity observed in adults, and that animals that lack SKR-5 or the UFD have reduced lifespans and indications of oxidative stress. We propose that as animals enter fertile adulthood, EGF signalling switches the mechanism for maintaining protein homoeostasis from a chaperone-based approach to an approach involving protein elimination via augmented UPS activity.