Comparative Gene Expression Studies Research Articles

How to build a fruit: Transcriptomics of a novel fruit type in the Brassiceae.

Comparative gene expression studies are invaluable for predicting how existing genetic pathways may be modified or redeployed to produce novel and variable phenotypes. Fruits are ecologically important organs because of their impact on plant fitness and seed dispersal, modifications in which results in morphological variation across species. A novel fruit type in the Brassicaceae known as heteroarthrocarpy enables distinct dispersal methods in a single fruit through segmentation via a lateral joint and variable dehiscence at maturity. Given the close relationship to Arabidopsis, species that exhibit heteroarthrocarpy are powerful models to elucidate how differences in gene expression of a fruit patterning pathway may result in novel fruit types. Transcriptomes of distal, joint, and proximal regions from Erucaria erucarioides and Cakile lanceolata were analyzed to elucidate within fruit and between species differences in whole transcriptome, gene ontology, and fruit patterning expression profiles. Whole transcriptome expression profiles vary between fruit regions in patterns that are consistent with fruit anatomy. These transcriptomic variances do not correlate with changes in gene ontology, as they remain generally stable within and between both species. Upstream regulators in the fruit patterning pathway, FILAMENTOUS FLOWER and YABBY3, are expressed in the distal and proximal regions of E. erucarioides, but not in the joint, implicating alterations in the pathway in heteroarthrocarpic fruits. Downstream gene, INDEHISCENT, is significantly upregulated in the abscissing joint region of C. lanceolata, which suggests repurposing of valve margin genes for novel joint disarticulation in an otherwise indehiscent fruit. In summary, these data are consistent with modifications in fruit patterning genes producing heteroarthrocarpic fruits through different components of the pathway relative to other indehiscent, non-heteroarthrocarpic, species within the family. Our understanding of fruit development in Arabidopsis is now extended to atypical siliques within the Brassicaceae, facilitating future studies on seed shattering in important Brassicaceous crops and pernicious weeds.

C3aR signaling and gliosis in response to neurodevelopmental damage in the cerebellum

BackgroundConditional ablation of the Smarca5 gene in mice severely impairs the postnatal growth of the cerebellum and causes an ataxic phenotype. Comparative gene expression studies indicated that complement-related proteins were upregulated in the cerebellum of Smarca5 mutant mice. Complement proteins play critical roles within innate immune signaling pathways and, in the brain, are produced by glial cells under both normal and pathological conditions. The C3 complement protein-derived signaling peptide, C3a, has been implicated in contributing to both tissue damage and repair in conditions such as multiple sclerosis and stroke. Here, we investigated whether C3a receptor (C3aR) signaling promoted damage or repair in the developing cerebellum of Smarca5 mutant mice.MethodsBrain and cerebellum lysates from single Smarca5 conditional knockout (Smarca5 cKO) mice, C3aR1 KO mice, or double mutant mice were used for qRT-PCR and immunoblotting to assess the contribution of C3aR to the Smarca5 cKO brain pathology. Immunohistochemistry was used to characterize alterations to astroglia and phagocyte cells in the developing cerebellum of each of the genotypes.ResultsC3aR signaling was observed to limit gliosis and promote granule neuron survival during postnatal cerebellar development. In Smarca5 cKO mice, disorganized astroglia with increased GFAP expression develops concurrently with cerebellar granule neuron loss and phagocyte invasion over the first 10 days following birth. Potential ligand precursors of C3aR—VGF and C3—were found to have upregulated expression and/or altered processing during this time. Phagocytes (microglia and macrophages) in both the control and Smarca5 mutant mice were the only cells observed to express C3aR. Loss of C3aR in the Smarca5 cKO cerebellum resulted in increased numbers of apoptotic cells and early phagocyte invasion into the external granule cell layer, as well as an exacerbated disorganization of the Bergmann glia. The loss of C3aR expression also attenuated an increase in the expression of the efferocytosis-related protein, MerTK, whose transcript was upregulated ~ 2.5-fold in the Smarca5 mutant cerebellum at P10.ConclusionsThis data indicates that C3aR can play an important role in limiting astrogliosis and regulating phagocyte phenotypes following developmental cell loss in the brain.

Identifying and Engineering Genes for Parthenogenesis in Plants.

Parthenogenesis is the spontaneous development of an embryo from an unfertilized egg cell. It naturally occurs in a variety of plant and animal species. In plants, parthenogenesis usually is found in combination with apomeiosis (the omission of meiosis) and pseudogamous or autonomous (with or without central cell fertilization) endosperm formation, together known as apomixis (clonal seed production). The initiation of embryogenesis in vivo and in vitro has high potential in plant breeding methods, particularly for the instant production of homozygous lines from haploid gametes [doubled haploids (DHs)], the maintenance of vigorous F1-hybrids through clonal seed production after combining it with apomeiosis, reverse breeding approaches, and for linking diploid and polyploid gene pools. Because of this large interest, efforts to identify gene(s) for parthenogenesis from natural apomicts have been undertaken by using map-based cloning strategies and comparative gene expression studies. In addition, engineering parthenogenesis in sexual model species has been investigated via mutagenesis and gain-of-function strategies. These efforts have started to pay off, particularly by the isolation of the PsASGR-BabyBoom-Like from apomictic Pennisetum, a gene proven to be transferable to and functional in sexual pearl millet, rice, and maize. This review aims to summarize the current knowledge on parthenogenesis, the possible gene candidates also outside the grasses, and the use of these genes in plant breeding protocols. It shows that parthenogenesis is able to inherit and function independently from apomeiosis and endosperm formation, is expressed and active in the egg cell, and can induce embryogenesis in polyploid, diploid as well as haploid egg cells in plants. It also shows the importance of genes involved in the suppression of transcription and modifications thereof at one hand, and in embryogenesis for which transcription is allowed or artificially overexpressed on the other, in parthenogenetic reproduction. Finally, it emphasizes the importance of functional endosperm to allow for successful embryo growth and viable seed production.

Ocimum metabolomics in response to abiotic stresses: Cold, flood, drought and salinity.

Ocimum tenuiflorum is a widely used medicinal plant since ancient times and still continues to be irreplaceable due to its properties. The plant has been explored chemically and pharmacologically, however, the molecular studies have been started lately. In an attempt to get a comprehensive overview of the abiotic stress response in O. tenuiflorum, de novo transcriptome sequencing of plant leaves under the cold, drought, flood and salinity stresses was carried out. A comparative differential gene expression (DGE) study was carried out between the common transcripts in each stress with respect to the control. KEGG pathway analysis and gene ontology (GO) enrichment studies exhibited several modifications in metabolic pathways as the result of four abiotic stresses. Besides this, a comparative metabolite profiling of stress and control samples was performed. Among the cold, drought, flood and salinity stresses, the plant was most susceptible to the cold stress. Severe treatments of all these abiotic stresses also decreased eugenol which is the main secondary metabolite present in the O. tenuiflorum plant. This investigation presents a comprehensive analysis of the abiotic stress effects in O. tenuiflorum. Current study provides an insight to the status of pathway genes’ expression that help synthesizing economically valuable phenylpropanoids and terpenoids related to the adaptation of the plant. This study identified several putative abiotic stress tolerant genes which can be utilized to either breed stress tolerant O. tenuiflorum through pyramiding or generating transgenic plants.

Comparative expression profiling reveals widespread coordinated evolution of gene expression across eukaryotes

Comparative studies of gene expression across species have revealed many important insights, but have also been limited by the number of species represented. Here we develop an approach to identify orthologs between highly diverged transcriptome assemblies, and apply this to 657 RNA-seq gene expression profiles from 309 diverse unicellular eukaryotes. We analyzed the resulting data for coevolutionary patterns, and identify several hundred protein complexes and pathways whose expression levels have evolved in a coordinated fashion across the trillions of generations separating these species, including many gene sets with little or no within-species co-expression across environmental or genetic perturbations. We also detect examples of adaptive evolution, for example of tRNA ligase levels to match genome-wide codon usage. In sum, we find that comparative studies from extremely diverse organisms can reveal new insights into the evolution of gene expression, including coordinated evolution of some of the most conserved protein complexes in eukaryotes.

Identification of reference genes suitable for RT-qPCR studies of murine gastrulation and patterning

Quantitative reverse transcriptase PCR (RT-qPCR), a powerful and efficient means of rapidly comparing gene expression between experimental conditions, is routinely used as a phenotyping tool in developmental biology. The accurate comparison of gene expression across multiple embryonic stages requires normalisation to reference genes that have stable expression across the time points to be examined. As the embryo and its constituent tissues undergo rapid growth and differentiation during development, reference genes known to be stable across some time points cannot be assumed to be stable across all developmental stages. The immediate post-implantation events of gastrulation and patterning are characterised by a rapid expansion in cell number and increasing specialisation of cells. The optimal reference genes for comparative gene expression studies at these specific stages have not been experimentally identified. In this study, the expression of five commonly used reference genes (H2afz, Ubc, Actb, Tbp and Gapdh) was measured across murine gastrulation and patterning (6.5-9.5dpc) and analysed with the normalisation tools geNorm, Bestkeeper and Normfinder. The results, validated by RT-qPCR analysis of two genes with well-documented expression patterns across these stages, indicated the best strategy for RT-qPCR studies spanning murine gastrulation and patterning utilises the concurrent reference genes H2afz and Ubc.

Comparative Study of p40 Gene Expression of Mycoplasma Agalactiae Isolated from Iranian Provinces

Mycoplasma agalactiae pathogen is the main cause of agalactia in small ruminants and as such, results in some economic losses in Iran. Contagious agalactia disease should be regarded as a syndrome, caused by various Mycoplasmas which infect several organs. In the present study, 30 suspected samples from 11 provinces of Iran were isolated from mammary gland, joint and eyes of sheep and goats and subjected to genus and species detection via PCR technique for 2 genes. These Mycoplasma strains and three Iranian vaccinal strains of Mycoplasma agalactiae were submitted to sequence analysis of a surface lipoprotein called p40 protein. Based on a comparative study between Iranian strains and PG2 Spanish strain of Mycoplasma agalactiae, most Iranian strains presented 97% homology, whereas some strains showed 80-88% and three vaccinal strains were associated with 99% homology. According to PCR and bioinformatics analysis outcomes, 6 provinces of Iran were recognized as infection areas with different expressions of this effecter protein, suggesting that finding the individual characteristics of a particular effecter may require empirical testing for vaccination. Finally, the selected sequence of p40 gene was cloned into pGEMB1cloning vector and subsequently expressed in Escherichia coli by pET-22b+ expression plasmid under the control of the T7 promoter. The expression of this fusion protein was absorbed and confirmed by SDS-PAGE. The recombinant P40 protein was expressed with a molecular mass of 37 kDa on SDS-PAGE. The sera taken from rabbits infected with Mycoplasma agalactiae produced polyclonal antibody which was then used for westernblotting. The rabbits were bled 10 days after the booster immunization using cardiac puncture. Significantly, the use of recombinant specific antigens instead of other tools for diagnosis of a disease could improve the discrimination and separation of positive and negative animals in the area under investigation and therefore, it can be applied to control infected animals and reduce economic losses.

Comparative gene expression studies of candidate genes associated with defence response in ginger and mango ginger post inoculation with Ralstonia solanacearum

Bacterial wilt of ginger (Zingiber officinale Rosc.) causes serious economic losses in all major ginger growing areas of the world. The Indian mango ginger (Curcuma amada Roxb.) is found to be resistant to the disease. With an aim to decipher the factors involved in mango ginger resistance, tissue-specific expression of nine candidate genes viz., Ethylene Response Factor (ERF), HMG-CoA synthase (HMGS), HMG-CoA reductase (HMGR), ABC transporter, WRKY8 transcription factor, β-(1, 3)-glucanase, Callose synthase, Heat Shock Protein (HSP) and Mlo14 were carried out at different time intervals in leaf and rhizome tissues post pathogen inoculation using real-time PCR. The transcripts varied in the level of up and down-regulation, with a marked difference in the intensity and time of response. Compared to leaf tissues, the rhizome tissues of both the plants showed increased expression of all the transcripts except β-(1, 3)-glucanase and Mlo14. In both the resistant and susceptible plants β-(1, 3)-glucanase showed higher expression in the leaf tissues, whereas HSP and Callose synthase showed higher expression in rhizome tissues of ginger and mango ginger respectively. Compared to ginger, the rhizome tissues of mango ginger showed peak level expression at earlier hours. The rhizome of mango ginger may play an important role in preventing the entry of the pathogen through the soil. Traversing all these barriers if the pathogen is delivered directly to the xylem tissues, they can multiply profusely and induce bacterial wilt in mango ginger.

Evidence for a centrosome-attracting body like structure in germ-soma segregation during early development, in the urochordate Oikopleura dioica

BackgroundGerm cell formation has been investigated in sessile forms of tunicates. This process involves the release of a subset of maternal transcripts from the centrosome-attracting body (CAB) in the progenitor cells of the germ line. When germ-soma segregation is completed, CAB structures are missing from the newly formed primordial germ cells (PGCs). In free-swimming tunicates, knowledge about germ cell formation is lacking. In this investigation, comparative gene expression and electron microscopy studies were used to address germ cell formation in Oikopleura dioica (O. dioica).ResultsWe found that the RNA localization pattern of pumilio (pum1) is similar to the pattern described for a subset of maternal transcripts marking the posterior end of ascidian embryos. Transcripts marking the posterior end are called postplasmic or posterior-end mark (PEM) transcripts. We found no localization of vasa (vas) transcripts to any sub-region within the germ-line precursor cells. Expression of vas4 was detected in the newly formed PGCs. Electron microscopy studies confirmed the presence of structures with similar morphology to CAB. In the same cytoplasmic compartment, we also identified pum1 transcripts and an epitope recognized by an antibody to histone H3 phosphorylated on serine 28.ConclusionsOur findings support that a CAB-like structure participates in the segregation of maternal pum1 transcripts during germ-soma separation in O. dioica.

Dynamic evolution of regulatory element ensembles in primate CD4+ T cells.

How evolutionary changes at enhancers affect the transcription of target genes remains an important open question. Previous comparative studies of gene expression have largely measured the abundance of messenger RNA, which is affected by post-transcriptional regulatory processes, hence limiting inferences about the mechanisms underlying expression differences. Here, we directly measured nascent transcription in primate species, allowing us to separate transcription from post-transcriptional regulation. We used precision run-on and sequencing to map RNA polymerases in resting and activated CD4+ T cells in multiple human, chimpanzee and rhesus macaque individuals, with rodents as outgroups. We observed general conservation in coding and non-coding transcription, punctuated by numerous differences between species, particularly at distal enhancers and non-coding RNAs. Genes regulated by larger numbers of enhancers are more frequently transcribed at evolutionarily stable levels, despite reduced conservation at individual enhancers. Adaptive nucleotide substitutions are associated with lineage-specific transcription and at one locus, SGPP2, we predict and experimentally validate that multiple substitutions contribute to human-specific transcription. Collectively, our findings suggest a pervasive role for evolutionary compensation across ensembles of enhancers that jointly regulate target genes.

Genomewide identification of genes involved in the potato response to drought indicates functional evolutionary conservation with Arabidopsis plants.

SummaryPotato is one of the four most important food crop plants worldwide and is strongly affected by drought. The following two pairs of potato cultivars, which are related in ancestry but show different drought tolerances, were chosen for comparative gene expression studies: Gwiazda/Oberon and Tajfun/Owacja. Comparative RNA‐seq analyses of gene expression differences in the transcriptomes obtained from drought‐tolerant versus drought‐sensitive plants during water shortage conditions were performed. The 23 top‐ranking genes were selected, 22 of which are described here as novel potato drought‐responsive genes. Moreover, all but one of the potato genes selected have homologues in the Arabidopsis genome. Of the seven tested A. thaliana mutants with altered expression of the selected homologous genes, compared to the wild‐type Arabidopsis plants, six showed an improved tolerance to drought. These genes encode carbohydrate transporter, mitogen‐activated protein kinase kinase kinase 15 (MAPKKK15), serine carboxypeptidase‐like 19 protein (SCPL19), armadillo/beta‐catenin‐like repeat‐containing protein, high‐affinity nitrate transporter 2.7 and nonspecific lipid transfer protein type 2 (nsLPT). The evolutionary conservation of the functions of the selected genes in the plant response to drought confirms the importance of these identified potato genes in the ability of plants to cope with water shortage conditions. Knowledge regarding these gene functions can be used to generate potato cultivars that are resistant to unfavourable conditions. The approach used in this work and the obtained results allowed for the identification of new players in the plant response to drought.

Fusarium infection causes genotoxic disorders and antioxidant-based damages in Orobanche spp.

This study aims to evaluate the toxic effects of Fusarium oxysporum on root parasitic weed, Orobanche spp. Comparative genetic and gene expression studies were conducted on uninfected and fungus-infected orobanches. In genetic studies, isolated total DNA was amplified by RAPD PCR. Fragment properties were analysed by GTS test. According to the results, the fragment properties of control and Fusarium infected (experimental) groups varied widely; and it has been observed that Fusarium has genotoxic effects on the DNA of orobanches. In gene expression studies, the expression levels of genes encoding enzymes or proteins were associated with ROS damage and toxic effects, therefore, gene expressions of Mn-superoxide dismutase (SOD), Zn-superoxide dismutase (=SOD2, mitochondrial), glutamine synthetase (GS), heat shock protein gene (HSP70), BAX, Caspase-3 and BCL2 were significantly higher in the experimental group. In the light of obtained data, it was concluded that F. oxysporum (1) caused heavy ROS damage in Orobanche (2) induced significant irrevocable genotoxic effects on the DNA of Orobanche, (3) degraded protein metabolism and synthesis, and finally (4) triggered apoptosis. The results of this study can be a ground for further research on reducing the toxic effects of Fusarium on agricultural products, so that advancements in bio-herbicide technology may provide a sustainable agricultural production.

Cre/lox-Recombinase-Mediated Cassette Exchange for Reversible Site-Specific Genomic Targeting of the Disease Vector, Aedes aegypti

Site-specific genome modification (SSM) is an important tool for mosquito functional genomics and comparative gene expression studies, which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows for the creation of advanced transgenic strains for vector control programs. SSM circumvents the drawbacks of transposon-mediated transgenesis, where random transgene integration into the host genome results in insertional mutagenesis and variable position effects. We applied the Cre/lox recombinase-mediated cassette exchange (RMCE) system to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. In this context we created four target site lines for RMCE and evaluated their fitness costs. Cre-RMCE is functional in a two-step mechanism and with good efficiency in Ae. aegypti. The advantages of Cre-RMCE over existing site-specific modification systems for Ae. aegypti, phiC31-RMCE and CRISPR, originate in the preservation of the recombination sites, which 1) allows successive modifications and rapid expansion or adaptation of existing systems by repeated targeting of the same site; and 2) provides reversibility, thus allowing the excision of undesired sequences. Thereby, Cre-RMCE complements existing genomic modification tools, adding flexibility and versatility to vector genome targeting.

16-Dehydropregnenolone lowers serum cholesterol by up-regulation of CYP7A1 in hyperlipidemic male hamsters

16-Dehydropregnenolone (DHP) has been developed and patented as a promising antihyperlipidemic agent by CSIR-Central Drug Research Institute (CSIR-CDRI), India. Although DHP is implicated in controlling cholesterol homeostasis, the mechanism underlying its pharmacological effect in hyperlipidemic disease models is poorly understood. In the present study, we postulated that DHP lowers serum lipids through regulating the key hepatic genes accountable for cholesterol metabolism. The hypothesis was tested on golden Syrian hamsters fed with high-fat diet (HFD) following oral administration of DHP at a dose of 72mg/kg body weight for a period of one week. The serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and total bile acids (TBA) in feces were measured. Real time comparative gene expression studies were performed for CYP7A1, LXRα and PPARα level in liver tissue of hamsters. The results revealed that the DHP profoundly decreased the levels of serum TC, TG, LDL-C and atherogenic index (AI), whilst elevated the HDL-C/TC ratio. Besides, DHP exhibited an anti-hyperlipidemic effect in the HFD induced hyperlipidemic hamsters by means of: (1) up-regulating the gene expression of CYP7A1 encoded cholesterol 7α-hydroxylase, that promotes the catabolism of cholesterol to bile acid; (2) inducing the gene expression of transcription factors LXRα and PPARα; (3) increasing the TBA excretion through feces. Collectively, the findings presented confer the hypolipidemic activity of DHP via up-regulation of hepatic CYP7A1 pathway that promotes cholesterol-to-bile acid conversion and bile acid excretion.

Inferring regulatory change from gene expression: the confounding effects of tissue scaling.

Comparative studies of gene expression are often designed with the aim of identifying regulatory changes associated with phenotypic variation. In recent years, large-scale transcriptome sequencing methods have increasingly been applied to nonmodel organisms to ask important ecological or evolutionary questions. Although experimental design varies, many of these studies have been based on RNA libraries obtained from heterogeneous tissue samples, for example homogenized whole bodies. Comparisons between groups of samples that vary in tissue composition can introduce sufficient variation in RNA abundance to produce patterns of differential expression that are mistakenly interpreted as evidence of regulatory differences. Here, we present a simple model that demonstrates this effect. The model describes the relationship between transcript abundance and tissue composition in a two-tissue system, and how this relationship varies under different scaling relationships. Using a range of biologically realistic variables, including real biological examples, to parameterize the model we highlight the potentially severe influence of tissue scaling on relative transcript abundance. We use these results to identify key aspects of experimental design and analysis that can help to limit the influence of tissue scaling on the inference of regulatory difference from comparative studies of gene expression.

Evaluation of the efficacy of postmortem human bladder tissue as a normal comparator for case-controlled gene expression studies in urology.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a poorly understood disease with no absolute diagnostic marker. A molecular-based tool (biomarker) for IC/BPS diagnosis would have immediate clinical utility. We have generated a bank of bladder biopsy tissue from IC/BPS patients and require a control group for comparative gene expression studies. The objective of this pilot study was to investigate the feasibility of cadaveric bladder specimens as a viable source of control tissue. Cryopreserved cadaveric bladder specimens were obtained through the National Disease Research Interchange (NDRI) tissue repository. Decedent demographics, postmortem interval to autopsy, necropsy location and size were recorded. At least two punch biopsies were taken from each bladder sample and total RNA was extracted. Nucleic acid concentration and quality were measured as was the RNA integrity number (RIN). We purchased 15 necropsy bladder specimens that had been harvested from women postmortem and flash-frozen. For each bladder specimen, RNA was isolated from multiple sites for comparison both within and between individuals. Bioanalyzer results revealed severe degradation in the majority of samples as indicated by RINs ranging from "n/a" to 6.6, with most samples yielding RINs ≤2.5. Shorter postmortem interval did not correlate with an increase in RNA quantity or quality. Cadaver-derived bladder tissue from the NDRI does not routinely yield high-quality RNA needed for downstream gene expression applications, such as microarray and next-generation sequencing and, therefore, cannot be used as a reliable source for control samples.

Protocorms and Protocorm-Like Bodies Are Molecularly Distinct from Zygotic Embryonic Tissues in Phalaenopsis aphrodite.

The distinct reproductive program of orchids provides a unique evolutionary model with pollination-triggered ovule development and megasporogenesis, a modified embryogenesis program resulting in seeds with immature embryos, and mycorrhiza-induced seed germination. However, the molecular mechanisms that have evolved to establish these unparalleled developmental programs are largely unclear. Here, we conducted comparative studies of genome-wide gene expression of various reproductive tissues and captured the molecular events associated with distinct reproductive programs in Phalaenopsis aphrodite Importantly, our data provide evidence to demonstrate that protocorm-like body (PLB) regeneration (the clonal regeneration practice used in the orchid industry) does not follow the embryogenesis program. Instead, we propose that SHOOT MERISTEMLESS, a class I KNOTTED-LIKE HOMEOBOX gene, is likely to play a role in PLB regeneration. Our studies challenge the current understanding of the embryonic identity of PLBs. Taken together, the data obtained establish a fundamental framework for orchid reproductive development and provide a valuable new resource to enable the prediction of gene regulatory networks that is required for specialized developmental programs of orchid species.

ABI3 mediates dehydration stress recovery response in Arabidopsis thaliana by regulating expression of downstream genes

ABI3, originally discovered as a seed-specific transcription factor is now implicated to act beyond seed physiology, especially during abiotic stress. In non-seed plants, ABI3 is known to act in desiccation stress signaling. Here we show that ABI3 plays a role in dehydration stress response in Arabidopsis. ABI3 gene was upregulated during dehydration stress and its expression was maintained during subsequent stress recovery phases. Comparative gene expression studies in response to dehydration stress and stress recovery were done with genes which had potential ABI3 binding sites in their upstream regulatory regions. Such studies showed that several genes including known seed-specific factors like CRUCIFERIN1, CRUCIFERIN3 and LEA-group of genes like LEA76, LEA6, DEHYDRIN LEA and LEA-LIKE got upregulated in an ABI3-dependent manner, especially during the stress recovery phase. ABI3 got recruited to regions upstream to the transcription start site of these genes during dehydration stress response through direct or indirect DNA binding. Interestingly, ABI3 also binds to its own promoter region during such stress signaling. Nucleosomes covering potential ABI3 binding sites in the upstream sequences of the above-mentioned genes alter positions, and show increased H3 K9 acetylation during stress-induced transcription. ABI3 thus mediates dehydration stress signaling in Arabidopsis through regulation of a group of genes that play a role primarily during stress recovery phase.

Transposon-Based Reporter Marking Provides Functional Evidence for Intercellular Bridges in the Male Germline of Rabbits.

The Sleeping Beauty transposon system was established as a robust and efficient method for germline transgenesis in different mammalian species. The generation of transgenic mice, rats, rabbits and swine carrying an identical Venus reporter construct delivered by transposon-mediated gene transfer enables comparative studies of gene expression in these lines of mammalian models. Whereas comparable expression patterns of the Venus reporter were found in somatic tissues, preliminary studies suggested that a striking difference in reporter expression may exist in mature spermatozoa of these species. Here we clearly show the differential expression of Venus reporter protein during spermatogenesis of the two compared species, the laboratory rabbit and mice. We provide evidence for the functionality of intercellular bridges in the male germline and genotype-independent transgenic phenotype of rabbit spermatids. Our data suggest that the reporter rabbit line may be a suitable tool to identify molecular mechanisms in testicular development, and may contribute to develop better animal models for male infertility in men.

PD32-12 LONG-TERM FOLLOW-UP OF CIRCULATING TUMOR CELLS AS PREDICTORS FOR SURVIVAL IN MEN TREATED WITH ABIRATERONE ACETATE FOR CASTRATION RESISTANT PROSTATE CANCER FOLLOWING CHEMOTHERAPY

aRNAs from small numbers of PC3 cells spiked in normal blood and recovered using the CellSearch system. RESULTS: RNAs were successfully extracted from as few as 15 CTCs in blood samples. The relative expression levels of reference genes were maintained after RNA amplification. The integrity of the amplified RNA was demonstrated by RT-PCR analysis using primer sets that target the 50-end, middle, and 30-end of reference mRNAs. Besides epithelialand prostate-specific genes, Interleukin-6 was the only gene expressed in all CTCs analyzed. In this pilot study, we found partial concordance between gene expression patterns in CTCs and microdissected prostate cancer tissue from bone metastases of the same patients. CONCLUSIONS: aRNA amplification through in vitro transcription may serve as a useful method for gene analysis in small numbers of CTCs and tumor cells microdissected from BMBxs. To our knowledge, this is the first comparative study of gene expression in CTCs and BMBxs from individual mCRPC patients. Our exploratory study in this small cohort of patients suggests that molecular analysis of CTCs might reveal gene expression patterns that might be missed by analysis of single BMBx sites.