Differential gene expression and epiregulation of alpha zein gene copies in maize haplotypes

Multigenic traits are very common in plants and cause diversity. Nutritional quality is such a trait, and one of its factors is the composition and relative expression of storage protein genes. In maize, they represent a medium-size gene family distributed over several chromosomes and unlinked locations. Two inbreds, B73 and BSSS53, both from the Iowa Stiff Stock Synthetic collection, have been selected to analyze allelic and non-allelic variability in these regions that span between 80-500 kb of chromosomal DNA. Genes were copied to unlinked sites before and after allotetraploidization of maize, but before transposition enlarged intergenic regions in a haplotype-specific manner. Once genes are copied, expression of donor genes is reduced relative to new copies. Epigenetic regulation seems to contribute to silencing older copies, because some of them can be reactivated when endosperm is maintained as cultured cells, indicating that copy number variation might contribute to a reserve of gene copies. Bisulfite sequencing of the promoter region also shows different methylation patterns among gene clusters as well as differences between tissues, suggesting a possible position effect on regulatory mechanisms as a result of inserting copies at unlinked locations. The observations offer a potential paradigm for how different gene families evolve and the impact this has on their expression and regulation of their members.     

High-throughput sequencing of three Lemnoideae (duckweeds) chloroplast genomes from total DNA

Background

Chloroplast genomes provide a wealth of information for evolutionary and population genetic studies. Chloroplasts play a particularly important role in the adaption for aquatic plants because they float on water and their major surface is exposed continuously to sunlight. The subfamily of Lemnoideae represents such a collection of aquatic species that because of photosynthesis represents one of the fastest growing plant species on earth.

Methods

We sequenced the chloroplast genomes from three different genera of Lemnoideae, Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana by high-throughput DNA sequencing of genomic DNA using the SOLiD platform. Unfractionated total DNA contains high copies of plastid DNA so that sequences from the nucleus and mitochondria can easily be filtered computationally. Remaining sequence reads were assembled into contiguous sequences (contigs) using SOLiD software tools. Contigs were mapped to a reference genome of Lemna minor and gaps, selected by PCR, were sequenced on the ABI3730xl platform.

Conclusions

This combinatorial approach yielded whole genomic contiguous sequences in a cost-effective manner. Over 1,000-time coverage of chloroplast from total DNA were reached by the SOLiD platform in a single spot on a quadrant slide without purification. Comparative analysis indicated that the chloroplast genome was conserved in gene number and organization with respect to the reference genome of L. minor. However, higher nucleotide substitution, abundant deletions and insertions occurred in non-coding regions of these genomes, indicating a greater genomic dynamics than expected from the comparison of other related species in the Pooideae. Noticeably, there was no transition bias over transversion in Lemnoideae. The data should have immediate applications in evolutionary biology and plant taxonomy with increased resolution and statistical power.     

Divergent contractile and structural responses of the murine PKC-epsilon null pulmonary circulation to chronic hypoxia

Loss of PKC-epsilon limits the magnitude of acute hypoxic pulmonary vasoconstriction (HPV) in the mouse. Therefore, we hypothesized that loss of PKC-epsilon would decrease the contractile and/or structural response of the murine pulmonary circulation to chronic hypoxia (Hx). However, the pattern of lung vascular responses to chronic Hx may or may not be predicted by the acute HPV response. Adult PKC-epsilon wild-type (PKC-epsilon(+/+)), heterozygous null, and homozygous null (PKC-epsilon(-/-)) mice were exposed to normoxia or Hx for 5 wk. PKC-epsilon(-/-) mice actually had a greater increase in right ventricular (RV) systolic pressure, RV mass, and hematocrit in response to chronic Hx than PKC-epsilon(+/+) mice. In contrast to the augmented PA pressure and RV hypertrophy, pulmonary vascular remodeling was increased less than expected (i.e., equal to PKC-epsilon(+/+) mice) in both the proximal and distal PKC-epsilon(-/-) pulmonary vasculature. The contribution of increased vascular tone to this pulmonary hypertension (PHTN) was assessed by measuring the acute vasodilator response to nitric oxide (NO). Acute inhalation of NO reversed the increased PA pressure in hypoxic PKC-epsilon(-/-) mice, implying that the exaggerated PHTN may be due to a relative deficiency in nitric oxide synthase (NOS). Despite the higher PA pressure, chronic Hx stimulated less of an increase in lung endothelial (e) and inducible (i) NOS expression in PKC-epsilon(-/-) than PKC-epsilon(+/+) mice. In contrast, expression of nNOS in PKC-epsilon(+/+) mice decreased in response to chronic Hx, while lung levels in PKC-epsilon(-/-) mice remained unchanged. In summary, loss of PKC-epsilon results in increased vascular tone, but not pulmonary vascular remodeling in response to chronic Hx. Blunting of Hx-induced eNOS and iNOS expression may contribute to the increased vascular tone. PKC-epsilon appears to be an important signaling intermediate in the hypoxic regulation of each NOS isoform.     

Structure and evolution of the r/b chromosomal regions in rice, maize and sorghum

The r1 and b1 genes of maize, each derived from the chromosomes of two progenitors that hybridized >4.8 million years ago (MYA), have been a rich source for studying transposition, recombination, genomic imprinting, and paramutation. To provide a phylogenetic context to the genetic studies, we sequenced orthologous regions from maize and sorghum (>600 kb) surrounding these genes and compared them with the rice genome. This comparison showed that the homologous regions underwent complete or partial gene deletions, selective retention of orthologous genes, and insertion of nonorthologous genes. Phylogenetic analyses of the r/b genes revealed that the ancestral gene was amplified independently in different grass lineages, that rice experienced an intragenomic gene movement and parallel duplication, that the maize r1 and b1 genes are descendants of two divergent progenitors, and that the two paralogous r genes of sorghum are almost as old as the sorghum lineage. Such sequence mobility also extends to linked genes. The cisZOG genes are characterized by gene amplification in an ancestral grass, parallel duplications and deletions in different grass lineages, and movement to a nonorthologous position in maize. In addition to gene mobility, both maize and rice regions experienced recent transposition (<3 MYA).     

A novel MET-interacting protein shares high sequence similarity with RanBPM, but fails to stimulate MET-induced Ras/Erk signaling

MET is a receptor protein tyrosine kinase for hepatocyte growth factor, a multifunctional cytokine controlling cell growth, morphogenesis, and motility. In our previous study, RanBPM/RanBP9, whose name originated from its ability to interact with Ran, was identified as a MET-interacting protein. RanBPM/RanBP9 activates the Ras/Erk signaling pathway by serving as an adaptor protein of MET to recruit Sos. In this study, we identify a protein sharing a high amino acid sequence identity with RanBPM/RanBP9, especially in its SPRY domain, the region responsible for MET binding. This protein lacks the N-terminal poly-proline and poly-glutamine (Poly-PQ) stretch present in RanBPM/RanBP9 and has less homology with RanBPM/RanBP9 in its mid-region. We subsequently named this protein RanBP10 after demonstrating its interaction with Ran. We show that, like RanBPM/RanBP9, RanBP10 interacts with the tyrosine kinase domain of MET via its SPRY domain and these two proteins can compete with each other to bind to MET. Interestingly, unlike RanBPM/RanBP9, overexpression of RanBP10 cannot induce Erk1/2 phosphorylation and serum response element-luciferase (SRE-LUC) reporter gene expression. More importantly, co-transfection of RanBPM/RanBP9 and RanBP10 significantly represses SRE-LUC reporter gene expression induced by overexpression of RanBPM/RanBP9. Additional binding assays demonstrate that RanBP10 fails to interact with Sos, which explains its inability to activate the Ras/Erk pathway. Furthermore, we show that the N-terminus of RanBPM/RanBP9 with the Poly-PQ stretch is required for recruiting Sos and a truncated RanBPM/RanBP9 lacking this region fails to recruit Sos, indicating that the functional difference between RanBP10 and RanBPM/RanBP9 lies in their sequence difference in their N-termini.     

The biology of Bracon celer as a parasitoid of the olive fruit fly

A series of laboratory experiments was conducted on a colony of Bracon celer Szépligeti (Hymenoptera: Braconidae) reared on the olive fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae). Female B. celer preferentially probe and oviposit into olives containing late third-instar fly larvae. The parasitoid develops as a solitary, ectoparasitic idiobiont. Mean development time (oviposition to adult eclosion) at 22 °C was, for females, 36±1 (SE) days, and for males, 34±1 days. The mean longevity of adult female wasps when provided honey and water was significantly greater than when they were provided water alone, or nothing. The females produced an average of 9.7±7.2 progeny during their lifetimes, but production levels in the insectary colony suggested that this level of fecundity was artificially low and could be improved. The discrepancy may be a consequence of constraints on oviposition behavior imposed by the experimental design. The results are discussed with respect to insectary production methods and the potential use of B. celer as a biological control agent for olive fly in California.     

Retrotranspositions in orthologous regions of closely related grass species

Background  

Retrotransposons are commonly occurring eukaryotic transposable elements (TEs). Among these, long terminal repeat (LTR) retrotransposons are the most abundant TEs and can comprise 50–90% of the genome in higher plants. By comparing the orthologous chromosomal regions of closely related species, the effects of TEs on the evolution of plant genomes can be studied in detail.     

Structural and Functional Characterization of an Anesthetic Binding Site in the Second Cysteine-Rich Domain of Protein Kinase Cδ?

Elucidating the principles governing anesthetic-protein interactions requires structural determinations at high resolutions not yet achieved with ion channels. Protein kinase C (PKC) activity is modulated by general anesthetics. We solved the structure of the phorbol-binding domain (C1B) of PKCδ complexed with an ether (methoxymethylcycloprane) and with an alcohol (cyclopropylmethanol) at 1.36-Å resolution. The cyclopropane rings of both agents displace a single water molecule in a surface pocket adjacent to the phorbol-binding site, making van der Waals contacts with the backbone and/or side chains of residues Asn-237 to Ser-240. Surprisingly, two water molecules anchored in a hydrogen-bonded chain between Thr-242 and Lys-260 impart elasticity to one side of the binding pocket. The cyclopropane ring takes part in π-acceptor hydrogen bonds with the amide of Met-239. There is a crucial hydrogen bond between the oxygen atoms of the anesthetics and the hydroxyl of Tyr-236. A Tyr-236-Phe mutation results in loss of binding. Thus, both van der Waals interactions and hydrogen-bonding are essential for binding to occur. Ethanol failed to bind because it is too short to benefit from both interactions. Cyclopropylmethanol inhibited phorbol-ester-induced PKCδ activity, but failed to do so in PKCδ containing the Tyr-236-Phe mutation.     

Using evolutionary tools to facilitate the prediction and prevention of host‐based differentiation in biological control: a review and perspective

The unprecedented success of biological control (biocontrol) agents led some of the proponents of this technology to promote its use as a panacea for all pest problems. Following an accumulation of non‐target host interactions, because of generalist or new association introductions, techniques to help ensure classical biocontrol agent's success and reduce non‐target interactions were implemented. Even with these new measures in place, public and scientific mistrust and lack of consistency has resulted in increased regulation of biocontrol introductions. This has likely decreased the probability of effective, sustainable control measures being expeditiously implemented. With the current apprehension concerning the safety of biocontrol, we should incorporate the processes (adaptation, selection, etc.) and theoretical concepts of evolutionary biology to predict and enhance the effectiveness of biocontrol. The microevolutionary perspective that involves mutation, drift, selection and gene flow may be a crucial consideration in the realm of biocontrol. Here, we discuss how and why spatial and evolutionary models should be implemented into future risk assessment analyses of potential biocontrol agents. We suggest that it is necessary to re‐assess the approach that has developed over the past approximately 100 years of sustained releases and illuminate them in the context of an evolutionary timescale.