Duplicate Gene Evolution Toward Multiple Fates at the <Emphasis Type="Italic">Drosophila melanogaster HIP</Emphasis>/<Emphasis Type="Italic">HIP-Replacement</Emphasis> Locus

Hsc/Hsp70-interacting protein (HIP) is a rapidly evolving Hsp70 cofactor. Analyses of multiple Drosophila species indicate that the HIP gene is duplicated only in D. melanogaster. The HIP region, in fact, contains seven distinctly evolving duplicated genes. The regional duplication occurred in two steps, fixed rapidly, and illustrates multiple modes of duplicate gene evolution. HIP and its duplicate HIP-R are adaptively evolving in a manner unique to the region: they exhibit elevated divergence from other drosophilids and low polymorphism within D. melanogaster. HIP and HIP-R are virtually identical, share polymorphisms, and are subject to gene conversion. In contrast, two other duplicate genes in the region, CG33221 and GP-CG32779, are pseudogenes, and the chimeric gene Crg1 is subject to balancing selection. HIP and HIP-R are evolving rapidly and adaptively; however, positive selection is not sufficient to explain the molecular evolution of the region as a whole. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Duplication of <Emphasis Type="Italic">AP1</Emphasis> within the <Emphasis Type="Italic">Spinacia oleracea</Emphasis> L. <Emphasis Type="Italic">AP1/FUL</Emphasis> clade is followed by rapid amino acid and regulatory evolution

The AP1/FUL clade of MADS box genes have undergone multiple duplication events among angiosperm species. While initially identified as having floral meristem identity and floral organ identity function in Arabidopsis, the role of AP1 homologs does not appear to be universally conserved even among eudicots. In comparison, the role of FRUITFULL has not been extensively explored in non-model species. We report on the isolation of three AP1/FUL genes from cultivated spinach, Spinacia oleracea L. Two genes, designated SpAPETALA1-1 (SpAP1-1) and SpAPETALA1-2 (SpAP1-2), cluster as paralogous genes within the Caryophyllales AP1 clade. They are highly differentiated in the 3′, carboxyl-end encoding region of the gene following the third amphipathic alpha-helix region, while still retaining some elements of a signature AP1 carboxyl motifs. In situ hybridization studies also demonstrate that the two paralogs have evolved different temporal and spatial expression patterns, and that neither gene is expressed in the developing sepal whorl, suggesting that the AP1 floral organ identity function is not conserved in spinach. The spinach FRUITFULL homolog, SpFRUITFULL (SpFUL), has retained the conserved motif and groups with Caryophyllales FRUITFULL homologs. SpFUL is expressed in leaf as well as in floral tissue, and shows strong expression late in flower development, particularly in the tapetal layer in males, and in the endothecium layer and stigma, in the females. The combined evidence of high rates of non-synonymous substitutions and differential expression patterns supports a scenario in which the AP1 homologs in the spinach AP1/FUL gene family have experienced rapid evolution following duplication. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A high-efficiency gene silencing in plants using two-hit asymmetrical artificial MicroRNAs

MicroRNAs (miRNAs) are small non-coding RNA molecules that play a crucial role in gene regulation. They are produced through an enzyme-guided process called dicing and have an asymmetrical structure with two nucleotide overhangs at the 3′ ends. Artificial microRNAs (amiRNAs or amiRs) are designed to mimic the structure of miRNAs and can be used to silence specific genes of interest. Traditionally, amiRNAs are designed based on an endogenous miRNA precursor with certain mismatches at specific positions to increase their efficiency. In this study, the authors modified the highly expressed miR168a in Arabidopsis thaliana by replacing the single miR168 stem-loop/duplex with tandem asymmetrical amiRNA duplexes that follow the statistical rules of miRNA secondary structures. These tandem amiRNA duplexes, called “two-hit” amiRNAs, were shown to have a higher efficiency in silencing GFP and endogenous PDS reporter genes compared to traditional “one-hit” amiRNAs. The authors also demonstrated the effectiveness of “two-hit” amiRNAs in silencing genes involved in miRNA, tasiRNA, and hormone signalling pathways, individually or in families. Importantly, “two-hit” amiRNAs were also able to over-express endogenous miRNAs for their functions. The authors compare “two-hit” amiRNA technology with CRISPR/Cas9 and provide a web-based amiRNA designer for easy design and wide application in plants and even animals.     

Novel molecular markers of <Emphasis Type="Italic">Chlamydia pecorum</Emphasis> genetic diversity in the koala (<Emphasis Type="Italic">Phascolarctos cinereus</Emphasis>)

Background  

Chlamydia pecorum is an obligate intracellular bacterium and the causative agent of reproductive and ocular disease in several animal hosts including koalas, sheep, cattle and goats. C. pecorum strains detected in koalas are genetically diverse, raising interesting questions about the origin and transmission of this species within koala hosts. While the ompA gene remains the most widely-used target in C. pecorum typing studies, it is generally recognised that surface protein encoding genes are not suited for phylogenetic analysis and it is becoming increasingly apparent that the ompA gene locus is not congruent with the phylogeny of the C. pecorum genome. Using the recently sequenced C. pecorum genome sequence (E58), we analysed 10 genes, including ompA, to evaluate the use of ompA as a molecular marker in the study of koala C. pecorum genetic diversity.     

Multiple alleles at <Emphasis Type="Italic">Early flowering 1</Emphasis> locus making variation in the basic vegetative growth period in rice (<Emphasis Type="Italic">Oryza</Emphasis> <Emphasis Type="Italic">sativa</Emphasis> L.)

A recently established rice breeding program in low latitudes aims to develop varieties with extremely long basic vegetative growth (BVG) periods and weak photoperiod sensitivities. The Taiwanese japonica variety Taichung 65 (T65) harbors a recessive allele ef1 at the Ef1 (Early flowering 1) locus, thereby exhibiting an extremely long BVG period. The previous reported functional allele Ehd1 (Early heading date 1), located on chromosome 10, encodes a B-type response regulator, thereby shortening the BVG period, whereas its nonfunctional allele ehd1 greatly prolongs the BVG period. A conventional analysis using F₂ and F₃ populations and a subsequent CAPS analysis based on the amino acid sequences of Ehd1 and ehd1 showed that Ef1 and Ehd1 were at the same locus. The CAPS analysis also indicated that the Taiwanese japonica varieties with extremely long BVG periods all harbor ef1, but that ef1 does not exist among indica and japonica varieties in the low latitudes. Since ef1 has not been found in any japonica varieties outside Taiwan, this allele might have originated in Taiwan. Sequence analysis revealed that the mutant allele ef1-h, which prolongs the BVG period even more than ef1 does, harbors an mPing insertion in exon 2, which causes the complete loss of gene function. Our results indicate that both ef1 or ef1-h alleles can be used as new gene sources in developing rice varieties with extremely long BVG periods for low latitudes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Highly specific gene silencing by artificial microRNAs in Arabidopsis

Plant microRNAs (miRNAs) affect only a small number of targets with high sequence complementarity, while animal miRNAs usually have hundreds of targets with limited complementarity. We used artificial miRNAs (amiRNAs) to determine whether the narrow action spectrum of natural plant miRNAs reflects only intrinsic properties of the plant miRNA machinery or whether it is also due to past selection against natural miRNAs with broader specificity. amiRNAs were designed to target individual genes or groups of endogenous genes. Like natural miRNAs, they had varying numbers of target mismatches. Previously determined parameters of target selection for natural miRNAs could accurately predict direct targets of amiRNAs. The specificity of amiRNAs, as deduced from genome-wide expression profiling, was as high as that of natural plant miRNAs, supporting the notion that extensive base pairing with targets is required for plant miRNA function. amiRNAs make an effective tool for specific gene silencing in plants, especially when several related, but not identical, target genes need to be downregulated. We demonstrate that amiRNAs are also active when expressed under tissue-specific or inducible promoters, with limited nonautonomous effects. The design principles for amiRNAs have been generalized and integrated into a Web-based tool (http://wmd.weigelworld.org).     

Target site selection by the <Emphasis Type="Italic">mariner</Emphasis>-like element, <Emphasis Type="Italic">Mos1</Emphasis>

The eukaryotic transposon Mos1 is a class-II transposable element that moves using a “cut-and-paste” mechanism in which the transposase is the only protein factor required. The formation of the excision complex is well documented, but the integration step has so far received less investigation. Like all mariner-like elements, Mos1 was thought to integrate into a TA dinucleotide without displaying any other target selection preferences. We set out to synthesize what is currently known about Mos1 insertion sites, and to define the characteristics of Mos1 insertion sequences in vitro and in vivo. Statistical analysis can be used to identify the TA dinucleotides that are non-randomly targeted for transposon integration. In vitro, no specific feature determining target choice other than the requirement for a TA dinucleotide has been identified. In vivo, data were obtained from two previously reported integration hotspots: the bacterial cat gene and the Caenorhabditis elegans rDNA locus. Analysis of these insertion sites revealed a preference for TA dinucleotides that are included in TATA or TA × TA motifs, or located within AT-rich regions. Analysis of the physical properties of sequences obtained in vitro and in vivo do not help to explain Mos1 integration preferences, suggesting that other characteristics must be involved in Mos1 target choice.     

Antisense RNA decreases AP33 gene expression and cytoadherence by <Emphasis Type="Italic">T. vaginalis</Emphasis>

Background  

Host parasitism by Trichomonas vaginalis is complex. Adherence to vaginal epithelial cells (VECs) is mediated by surface proteins. We showed before that antisense down-regulation of expression of adhesin AP65 decreased amounts of protein, which lowered levels of T. vaginalis adherence to VECs. We now perform antisense down-regulation of expression of the ap33 gene to evaluate and confirm a role for AP33 in adherence by T. vaginalis. We also used an established transfection system for heterologous expression of AP33 in T. foetus as an additional confirmatory approach.     

MicroRNAs with analogous target complementarities perform with highly variable efficacies in Arabidopsis

In plants, the silencing efficacy of microRNAs (miRNAs) is thought to be predominantly determined by the degree of complementarity to their target genes. Here, silencing efficacy was determined for Arabidopsis miR159 and four artificial miRNAs (amiRNAs) that all target MYB33/MYB65 with analogous complementarities. As determined through complementation of a loss-of-function mir159 mutant, the amiRNAs displayed highly variable efficacies, none of which was as strong as endogenous miR159. This was despite amiRNA expression levels being many fold-higher than miR159 in wild-type. The results highlight the variable nature of miRNA silencing efficacy in plants, where it appears that factors additional to complementarity strongly impact silencing.     

Regions associated with repression of the barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) <Emphasis Type="Italic">VERNALIZATION1</Emphasis> gene are not required for cold induction

Activity of the VERNALIZATION1 (VRN1) gene is required for flowering in temperate cereals such as wheat and barley. In varieties that require prolonged exposure to cold to flower (vernalization), VRN1 is expressed at low levels and is induced by vernalization to trigger flowering. In other varieties, deletions or insertions in the first intron of the VRN1 gene are associated with increased VRN1 expression in the absence of cold treatment, reducing or eliminating the requirement for vernalization. To characterize natural variation in VRN1, the first intron of the barley (Hordeum vulgare) VRN1 gene (HvVRN1) was assayed for deletions or insertions in a collection of 1,000 barleys from diverse geographical regions. Ten alleles of HvVRN1 containing deletions or insertions in the first intron were identified, including three alleles that have not been described previously. Different HvVRN1 alleles were associated with differing levels of HvVRN1 expression in non-vernalized plants and with different flowering behaviour. Using overlapping deletions, we delineated regions in the HvVRN1 first intron that are associated with low levels of HvVRN1 expression in non-vernalized plants. Deletion of these intronic regions does not prevent induction of HvVRN1 by cold or the maintenance of increased HvVRN1 expression following cold treatment. We suggest that regions within the first intron of HvVRN1 are required to maintain low levels of HvVRN1 expression prior to winter but act independently of the regulatory mechanisms that mediate induction of HvVRN1 by cold during winter. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers 1179825, 1179833, 1179836, 1179858.     

Complex regulation and multiple developmental functions of <Emphasis Type="Italic">misfire</Emphasis>, the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>ferlin gene

Background  

Ferlins are membrane proteins with multiple C2 domains and proposed functions in Ca²⁺ mediated membrane-membrane interactions in animals. Caenorhabditis elegans has two ferlin genes, one of which is required for sperm function. Mammals have several ferlin genes and mutations in the human dysferlin (DYSF) and otoferlin (OTOF) genes result in muscular dystrophy and hearing loss, respectively. Drosophila melanogaster has a single ferlin gene called misfire (mfr). A previous study showed that a mfr mutation caused male sterility because of defects in fertilization. Here we analyze the expression and structure of the mfr gene and the consequences of multiple mutations to better understand the developmental function of ferlins.     

Submolecular unfolding units of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> cytochrome <Emphasis Type="Italic">c</Emphasis>-551

Hydrogen exchange rates for backbone amide protons of oxidized Pseudomonas aeruginosa cytochrome c-551 (P. aeruginosa cytochrome c) have been measured in the presence of low concentrations of the denaturant guanidine hydrochloride. Analysis of the data has allowed identification of submolecular unfolding units known as foldons. The highest-energy foldon bears similarity to the proposed folding intermediate for P. aeruginosa cytochrome c. Parallels are seen to the foldons of the structurally homologous horse cytochrome c, although the heme axial methionine-bearing loop has greater local stability in P. aeruginosa cytochrome c, in accord with previous folding studies. Regions of low local stability are observed to correspond with regions that interact with redox partners, providing a link between foldon properties and function. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Involvement of methylation of group of miRNA genes in regulation of expression of <Emphasis Type="Italic">RAR-beta2</Emphasis> and <Emphasis Type="Italic">NKIRAS1</Emphasis> target genes in lung cancer

To date, there are more than 2000 known human miRNAs, each of which may be involved in the regulation of hundreds of protein-coding target genes. In turn, the methylation of CpG islands affects the miRNA gene expression. Our aim was to evaluate the role of methylation in the regulation of miRNA gene expression and, consequently, in the regulation of the expression of target genes in primary lung tumors. Using a common collection of samples of non-small-cell lung cancer, we have performed a comprehensive study, including an analysis of the methylation status and level of expression of some miRNA genes and their potential target genes on chromosome 3, i.e., RAR-beta2 and NKIRAS1. The increased frequency of methylation in lung tumors compared to histologically normal tissue was revealed for miR-9-1 and miR-34b/c genes with significant statistics (P < 0.05 by Fisher’s exact test) and for miR-9-3 and miR-193a was marginally significant (P < 0.1). A significant correlation was revealed between the changes in methylation and level of expression of miR-9-1 gene (P ≈ 5 × 10⁻¹² by Spearman) in lung tumors, which suggests the role of methylation in the regulation of expression of these miRNA genes. Furthermore, a statistically significant negative correlation (P ≈ 3 × 10⁻¹² to 5 × 10⁻¹³ by Spearman) was found between changes in the levels of expression of miR-9-1 and miR-17 and RAR-beta2 target genes, as well as between the changes in the level of expression of miR-17 and NKIRAS1 that were not previously analyzed. The inverse relationship between the levels of expression of miRNA genes and their target genes is consistent with the known mechanism of the suppression of the expression of protein-coding genes under the action of miRNA. For the first time, significant correlations (P ≈ 3 × 10⁻¹⁰ to 4 × 10⁻¹³ by Spearman) were shown between changes in the methylation status of miRNA genes (miR-9-1, miR-9-3, miR-34b/c, miR-193a) and the level of expression of the RAR-beta2 target gene and changes in the methylation status of miR-34b/c, and miR-193a and the level of expression of the NKIRAS1 target gene in the primary lung tumors, which suggests the possibility of indirect effects of the methylation of miRNA genes on the level of expression of target genes.     

Isolation of a <Emphasis Type="Italic">Ve</Emphasis> homolog, <Emphasis Type="Italic">mVe1</Emphasis>, and its relationship to verticillium wilt resistance in <Emphasis Type="Italic">Mentha longifolia</Emphasis> (L.) Huds

As a step toward greater understanding of the genetics of verticillium wilt resistance in plants, we report the sequencing of a candidate wilt resistance gene, mVe1, from the mint diploid model species, Mentha longifolia (Lamiaceae). mVe1 is a putative homolog of tomato (Solanum lycopersicum L.) verticillium wilt (Ve) resistance genes. The mVe1 gene has a coding region of 3,051 bp. The predicted mVe1 protein contains a leucine-rich repeat domain, a common feature of plant disease resistance proteins. We compared 13 mVe1 alleles from three mint species. These alleles shared 96.2–99.6% nucleotide identity. We analyzed four M. longifolia populations segregating with respect to mVe1 alleles and wilt resistance versus susceptibility and found one association between mVe1 genotype and wilt phenotype. We conclude that mVe1 may play a role in mint verticillium wilt resistance, but variation for resistance in our segregating progenies is likely polygenic. Therefore, further investigations of mVe1 and identification of additional candidate genes are both warranted.