Construction and characterization of two bacterial artificial chromosome libraries of pea (Pisum sativum L.) for the isolation of economically important genes.

Pea (Pisum sativum L.) has a genome of about 4 Gb that appears to share conserved synteny with model legumes having genomes of 0.2-0.4 Gb despite extensive intergenic expansion. Pea plant inventory (PI) accession 269818 has been used to introgress genetic diversity into the cultivated germplasm pool. The aim here was to develop pea bacterial artificial chromosome (BAC) libraries that would enable the isolation of genes involved in plant disease resistance or control of economically important traits. The BAC libraries encompassed about 3.2 haploid genome equivalents consisting of partially HindIII-digested DNA fragments with a mean size of 105 kb that were inserted in 1 of 2 vectors. The low-copy oriT-based T-DNA vector (pCLD04541) library contained 55 680 clones. The single-copy oriS-based vector (pIndigoBAC-5) library contained 65 280 clones. Colony hybridization of a universal chloroplast probe indicated that about 1% of clones in the libraries were of chloroplast origin. The presence of about 0.1% empty vectors was inferred by white/blue colony plate counts. The usefulness of the libraries was tested by 2 replicated methods. First, high-density filters were probed with low copy number sequences. Second, BAC plate-pool DNA was used successfully to PCR amplify 7 of 9 published pea resistance gene analogs (RGAs) and several other low copy number pea sequences. Individual BAC clones encoding specific sequences were identified. Therefore, the HindIII BAC libraries of pea, based on germplasm accession PI 269818, will be useful for the isolation of genes underlying disease resistance and other economically important traits.     

Functional PAK-2 knockout and replacement with a caspase cleavage-deficient mutant in mice reveals differential requirements of full-length PAK-2 and caspase-activated PAK-2p34

p21-Activated protein kinase 2 (PAK-2) has both anti- and pro-apoptotic functions depending on its mechanism of activation. Activation of full-length PAK-2 by the monomeric GTPases Cdc42 or Rac stimulates cell survival, whereas caspase activation of PAK-2 to the PAK-2p34 fragment is involved in the apoptotic response. In this study we use functional knockout of PAK-2 and gene replacement with the caspase cleavage-deficient PAK-2D212N mutant to differentiate the biological functions of full-length PAK-2 and caspase-activated PAK-2p34. Knockout of PAK-2 results in embryonic lethality at early stages before organ development, whereas replacement with the caspase cleavage-deficient PAK-2D212N results in viable and healthy mice, indicating that early embryonic lethality is caused by deficiency of full-length PAK-2 rather than lack of caspase activation to the PAK-2p34 fragment. However, deficiency of caspase activation of PAK-2 decreased spontaneous cell death of primary mouse embryonic fibroblasts and increased cell growth at high cell density. In contrast, stress-induced cell death by treatment with the anti-cancer drug cisplatin was not reduced by deficiency of caspase activation of PAK-2, but switched from an apoptotic to a nonapoptotic, caspase-independent mechanism. Homozygous PAK-2D212N primary mouse embryonic fibroblasts that lack the ability to generate the proapoptotic PAK-2p34 show less activation of the effector caspase 3, 6, and 7, indicating that caspase activation of PAK-2 amplifies the apoptotic response through a positive feedback loop resulting in more activation of effector caspases.     

Predicting in vitro drug sensitivity using Random Forests

MOTIVATION: Panels of cell lines such as the NCI-60 have long been used to test drug candidates for their ability to inhibit proliferation. Predictive models of in vitro drug sensitivity have previously been constructed using gene expression signatures generated from gene expression microarrays. These statistical models allow the prediction of drug response for cell lines not in the original NCI-60. We improve on existing techniques by developing a novel multistep algorithm that builds regression models of drug response using Random Forest, an ensemble approach based on classification and regression trees (CART). RESULTS: This method proved successful in predicting drug response for both a panel of 19 Breast Cancer and 7 Glioma cell lines, outperformed other methods based on differential gene expression, and has general utility for any application that seeks to relate gene expression data to a continuous output variable. Implementation: Software was written in the R language and will be available together with associated gene expression and drug response data as the package ivDrug at http://r-forge.r-project.org.     

Prediction of Associations between microRNAs and Gene Expression in Glioma Biology

Despite progress in the determination of miR interactions, their regulatory role in cancer is only beginning to be unraveled. Utilizing gene expression data from 27 glioblastoma samples we found that the mere knowledge of physical interactions between specific mRNAs and miRs can be used to determine associated regulatory interactions, allowing us to identify 626 associated interactions, involving 128 miRs that putatively modulate the expression of 246 mRNAs. Experimentally determining the expression of miRs, we found an over-representation of over(under)-expressed miRs with various predicted mRNA target sequences. Such significantly associated miRs that putatively bind over-expressed genes strongly tend to have binding sites nearby the 3'UTR of the corresponding mRNAs, suggesting that the presence of the miRs near the translation stop site may be a factor in their regulatory ability. Our analysis predicted a significant association between miR-128 and the protein kinase WEE1, which we subsequently validated experimentally by showing that the over-expression of the naturally under-expressed miR-128 in glioma cells resulted in the inhibition of WEE1 in glioblastoma cells.     

Complete exon sequencing of all known Usher syndrome genes greatly improves molecular diagnosis

Background

Usher syndrome (USH) combines sensorineural deafness with blindness. It is inherited in an autosomal recessive mode. Early diagnosis is critical for adapted educational and patient management choices, and for genetic counseling. To date, nine causative genes have been identified for the three clinical subtypes (USH1, USH2 and USH3). Current diagnostic strategies make use of a genotyping microarray that is based on the previously reported mutations. The purpose of this study was to design a more accurate molecular diagnosis tool.

Methods

We sequenced the 366 coding exons and flanking regions of the nine known USH genes, in 54 USH patients (27 USH1, 21 USH2 and 6 USH3).

Results

Biallelic mutations were detected in 39 patients (72%) and monoallelic mutations in an additional 10 patients (18.5%). In addition to biallelic mutations in one of the USH genes, presumably pathogenic mutations in another USH gene were detected in seven patients (13%), and another patient carried monoallelic mutations in three different USH genes. Notably, none of the USH3 patients carried detectable mutations in the only known USH3 gene, whereas they all carried mutations in USH2 genes. Most importantly, the currently used microarray would have detected only 30 of the 81 different mutations that we found, of which 39 (48%) were novel.

Conclusions

Based on these results, complete exon sequencing of the currently known USH genes stands as a definite improvement for molecular diagnosis of this disease, which is of utmost importance in the perspective of gene therapy.     

Epstein-Barr virus latent membrane protein 1 (LMP-1) half-life in epithelial cells is down-regulated by lytic LMP-1

This study examined the effect of naturally occurring Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) gene sequence variation on the LMP-1 half-life in epithelial cells. The LMP-1 half-life was not influenced by sequence variation in amino acids 250 to 307 or amino acids 343 to 352. The LMP-1 half-life was short when the amino acid encoded at position 129 was methionine, the initiation codon product of lytic LMP-1 (lyLMP-1). The mutation of amino acid 129 to isoleucine greatly increased the LMP-1 half-life. Expression of lyLMP-1 in trans down-regulated the LMP-1 half-life in a dose-dependent manner and restored a short-half-life phenotype to the mutated LMP-1 construct lacking the cis ability to express lyLMP-1. This observed dominant negative effect of lyLMP-1 expression on the LMP-1 half-life in epithelial cells in vitro may have implications for EBV epithelial oncogenesis in vivo.     

Thermal biology of the southernmost lizards in the world: Liolaemus sarmientoi and Liolaemus magellanicus from Patagonia,Argentina

We determined the efficiency of thermoregulation by the southernmost liolaemids Liolaemus sarmientoi and L. magellanicus from Patagonia, Argentina (51°S), by measuring body (T_b), microenvironmental, and operative temperatures in the field, and preferred body temperatures in the laboratory (T_pref). L. sarmientoi was found to be a poor thermoregulator, whereas L. magellanicus was deemed to be a constrained thermoconformer. Among all known species of Liolaemus, L. sarmientoi and L. magellanicus had the lowest T_b's when tested in the field; however, their T_pref's were similar to other liolaemids. These data suggest that these southernmost liolaemid species have not evolved appropriate thermoregulatory behaviors or made adequate physiological adaptations to face the extreme thermal challenges of their environment.     

A Neo-Sex Chromosome That Drives Postzygotic Sex Determination in the Hessian Fly (Mayetiola destructor)

Two nonoverlapping autosomal inversions defined unusual neo-sex chromosomes in the Hessian fly (Mayetiola destructor). Like other neo-sex chromosomes, these were normally heterozygous, present only in one sex, and suppressed recombination around a sex-determining master switch. Their unusual properties originated from the anomalous Hessian fly sex determination system in which postzygotic chromosome elimination is used to establish the sex-determining karyotypes. This system permitted the evolution of a master switch (Chromosome maintenance, Cm) that acts maternally. All of the offspring of females that carry Cm-associated neo-sex chromosomes attain a female-determining somatic karyotype and develop as females. Thus, the chromosomes act as maternal effect neo-W''s, or W-prime (W′) chromosomes, where ZW′ females mate with ZZ males to engender female-producing (ZW′) and male-producing (ZZ) females in equal numbers. Genetic mapping and physical mapping identified the inversions. Their distribution was determined in nine populations. Experimental matings established the association of the inversions with Cm and measured their recombination suppression. The inversions are the functional equivalent of the sciarid X-prime chromosomes. We speculate that W′ chromosomes exist in a variety of species that produce unisexual broods.SEX chromosomes are usually classified as X, Y, Z, or W on the basis of their pattern of segregation and the gender of the heterogametic sex (Ohno 1967). However, when chromosome-based sex determination occurs postzygotically, the same nomenclature confounds important distinctions and may hide interesting evolutionary phenomena. The Hessian fly (Mayetiola destructor), a gall midge (Diptera: Cecidomyiidae) and an important insect pest of wheat, presents an excellent example (Stuart and Hatchett 1988, 1991). In this insect, all of the female gametes and all of the male gametes have the same number of X chromosomes (Figure 1A); no heterogametic sex exists. Nevertheless, Hessian fly sex determination is chromosome based; postzygotic chromosome elimination produces different X chromosome to autosome ratios in somatic cells (male A1A2X1X2/A1A2OO and female A1A2X1X2/A1A2X1X2, where A1 and A2 are the autosomes, X1 and X2 are the X chromosomes, and the paternally derived chromosomes follow the slash) (Stuart and Hatchett 1991; Marin and Baker 1998). Thus, Hessian fly “X” chromosomes are defined by their haploid condition in males, rather than by their segregation in the gametes.Open in a separate windowFigure 1.—Chromosome behavior and sex determination in the Hessian fly. (A) Syngamy (1) establishes the germ-line chromosome constitution: ∼32 maternally derived E chromosomes (represented as a single white chromosome) and both maternally derived (black) and paternally derived (gray) autosomes and X chromosomes. During embryogenesis, while the E chromosomes are eliminated, the paternally derived X chromosomes are either retained (2) or excluded (3) from the presumptive somatic cells. When the paternally derived X chromosomes are retained (2), a female-determining karyotype is established. When they are eliminated (3), a male-determining karyotype is established. Thelygenic mothers carry Cm (white arrow), which conditions all of their offspring to retain the X chromosomes. Recombination occurs during oogenesis (4). All ova contain a full complement of E chromosomes and a haploid complement of autosomes and X chromosomes. Chromosome elimination occurs during spermatogenesis (5). Sperm contain only the maternally derived autosomes and X chromosomes. (B) The segregation of Cm (white dot) on a Hessian fly autosome among monogenic families. Thelygenic females produce broods composed of equal numbers of thelygenic (Cm/−) and arrhenogenic (−/−) females (box 1). Arrhenogenic females produce males (box 2). (C) Matings between monogenic and amphigenic families. Cm (white dot) is dominant to the amphigenic-derived chromosomes (gray dot) and generates all-female offspring (box 3). Amphigenic-derived chromosomes are dominant to the arrhenogenic-derived chromosomes (no dot) and generate offspring of both sexes (box 4).An autosomal, dominant, genetic factor called Chromosome maintenance (Cm) complicates Hessian fly sex determination further (Stuart and Hatchett 1991). Cm has a maternal effect that acts upstream of X chromosome elimination during embryogenesis (Figure 1A). It prevents X chromosome elimination so that all of the offspring of Cm-bearing mothers obtain a female-determining karyotype. Cm-bearing females produce only female offspring and are therefore thelygenic. The absence of Cm usually has the opposite effect; all of the offspring of most Cm-lacking females obtain a male-determining karyotype. These Cm-lacking females produce only male offspring and are therefore arrhenogenic. Like a sex-determining master switch, Cm is usually heterozygous and present in only one sex (Figure 1B). Thus, thelygenic females (Cm/−) are “heterogametic,” as their Cm-containing gametes and Cm-lacking gametes produce thelygenic (Cm/−) and arrhenogenic (−/−) females in a 1:1 ratio. Collectively, thelygenic and arrhenogenic females are called monogenic because they produce unisexual families. However, some Hessian fly females produce broods of both sexes and are called amphigenic. No mating barrier between monogenic and amphigenic families exists (Figure 1C), but amphigenic females have always been found in lower abundance (Painter 1930; Gallun et al. 1961; Stuart and Hatchett 1991). In experimental matings, the inheritance of maternal phenotype was consistent with the segregation of three Cm alleles (Figure 1C): a dominant thelygenic allele, a hypomorphic amphigenic allele, and a null arrhenogenic allele (Stuart and Hatchett 1991).Here we report the genetic and physical mapping of Cm on Hessian fly autosome 1 (A1). Two nonoverlapping inversions were identified that segregated perfectly with Cm. The most distal inversion was present in all thelygenic females examined. The more proximal inversion extended recombination suppression. These observations suggested that successive inversions evolved to suppress recombination around Cm after it arose. The inversions therefore appear to have evolved in response to the forces that shaped vertebrate Y and W chromosomes (Charlesworth 1996; Graves and Shetty 2001; Rice and Chippindale 2001; Carvalho and Clark 2005). We therefore believe the inversion-bearing chromosomes may be classified as maternal effect neo-W''s.