Complex Pattern Of Gene Expression Research Articles

A Complex Pattern of Gene Expression in Tissue Affected by Viperid Snake Envenoming: The Emerging Role of Autophagy-Related Genes.

Viperid snake venoms induce severe tissue damage, characterized by the direct toxic action of venom components, i.e., phospholipases A2 (PLA2s) and metalloproteinases (SVMPs), concomitantly with the onset of endogenous inflammatory processes, in an intricate scenario of tissue alterations. Understanding the expression of relevant genes in muscle tissue will provide valuable insights into the undergoing pathological and inflammatory processes. In this study, we have used the Nanostring technology to evaluate the patterns of gene expression in mouse skeletal muscle 1 h, 6 h, and 24 h after injection of the venoms of Bothrops asper and Daboia russelii, two medically relevant species in Latin America and Asia, respectively, with somewhat different clinical manifestations. The dose of venoms injected (30 µg) induced local pathological effects and inflammation in muscle tissue. We focused our analysis on genes related to extracellular matrix (ECM) metabolism, immune system, programmed cell death, and autophagy. The results revealed a complex pattern of expression of genes. Regarding ECM metabolism and regulation, up-regulated genes included proteinase inhibitor Serpine 1, thrombospondin 1, collagens 1A1 and 4A1 (at 1 h in the case of B. asper), TIMP1, MMP-3 (at 24 h), and lysil oxidase (LOX). In contrast, collagen chains 5A3 and 5A1 were down-regulated, especially at 6 h. Transforming growth factor β (TGF-β) and several genes related to myofibroblast regulation were also up-regulated, which might be related to the development of fibrosis. Several genes related to cytokine and chemokine synthesis and regulation and NFκB signaling were also up-regulated. Our observations show a variable expression of genes associated with programmed cell death and autophagy, thus revealing a hitherto unknown role of autophagy in tissue affected by snake venoms. These results provide clues to understanding the complex pattern of gene expression in tissue affected by viperid snake venoms, which likely impacts the final pathophysiology of damaged tissue in envenomings.

Regulation of ssb Gene Expression in Escherichia coli

Bacterial SSB proteins, as well as their eukaryotic RPA analogues, are essential and ubiquitous. They avidly bind single-stranded DNA and regulate/coordinate its metabolism, hence enabling essential DNA processes such as replication, transcription, and repair. The prototypic Escherichia coli SSB protein is encoded by an ssb gene. Although the ssb gene promoters harbor an SOS box, multiple studies over several decades failed to elucidate whether ssb gene expression is inducible and SOS dependent. The SOS regulon is comprised of about 50 genes, whose transcription is coordinately induced under stress conditions. Using quantitative real-time PCR, we determined the ssb gene expression kinetics in UV- and γ-irradiated E. coli and revealed that ssb gene expression is elevated in irradiated cells in an SOS-dependent manner. Additionally, the expression of the sulA gene was determined to indicate the extent of SOS induction. In a mutant with a constitutively induced SOS regulon, the ssb gene was overexpressed in the absence of DNA damage. Furthermore, we measured ssb gene expression by droplet digital PCR during unaffected bacterial growth and revealed that ssb gene expression was equal in wild-type and SOS− bacteria, whereas sulA expression was higher in the former. This study thus reveals a complex pattern of ssb gene expression, which under stress conditions depends on the SOS regulon, whereas during normal bacterial growth it is unlinked to SOS induction. The E. coli ssb gene is SOS regulated in such a way that its basal expression is relatively high and can be increased only through stronger SOS induction. The remarkable SOS induction observed in undisturbed wild-type cells may challenge our notion of the physiological role of the SOS response in bacteria.

Transcriptome of the synganglion in the tick Ixodes ricinus and evolution of the cys-loop ligand-gated ion channel family in ticks

BackgroundTicks represent a major health issue for humans and domesticated animals. Exploring the expression landscape of the tick’s central nervous system (CNS), known as the synganglion, would be an important step in understanding tick physiology and in managing tick-borne diseases, but studies on that topic are still relatively scarce. Neuron-specific genes like the cys-loop ligand-gated ion channels (cys-loop LGICs, or cysLGICs) are important pharmacological targets of acaricides. To date their sequence have not been well catalogued for ticks, and their phylogeny has not been fully studied.ResultsWe carried out the sequencing of transcriptomes of the I. ricinus synganglion, for adult ticks in different conditions (unfed males, unfed females, and partially-fed females). The de novo assembly of these transcriptomes allowed us to obtain a large collection of cys-loop LGICs sequences. A reference meta-transcriptome based on synganglion and whole body transcriptomes was then produced, showing high completeness and allowing differential expression analyses between synganglion and whole body. Many of the genes upregulated in the synganglion were associated with neurotransmission and/or localized in neurons or the synaptic membrane. As the first step of a functional study of cysLGICs, we cloned the predicted sequence of the resistance to dieldrin (RDL) subunit homolog, and functionally reconstituted the first GABA-gated receptor of Ixodes ricinus. A phylogenetic study was performed for the nicotinic acetylcholine receptors (nAChRs) and other cys-loop LGICs respectively, revealing tick-specific expansions of some types of receptors (especially for Histamine-like subunits and GluCls).ConclusionsWe established a large catalogue of genes preferentially expressed in the tick CNS, including the cysLGICs. We discovered tick-specific gene family expansion of some types of cysLGIC receptors, and a case of intragenic duplication, suggesting a complex pattern of gene expression among different copies or different alternative transcripts of tick neuro-receptors.

Gene Expression Pattern in Olive Tree Organs (Olea europaea L.).

The olive tree (Olea europaea L.) was one of the first plant species in history to be domesticated. Throughout olive domestication, gene expression has undergone drastic changes that may affect tissue/organ-specific genes. This is an RNA-seq study of the transcriptomic activity of different tissues/organs from adult olive tree cv. “Picual” under field conditions. This analysis unveiled 53,456 genes with expression in at least one tissue, 32,030 of which were expressed in all organs and 19,575 were found to be potential housekeeping genes. In addition, the specific expression pattern in each plant part was studied. The flower was clearly the organ with the most exclusively expressed genes, 3529, many of which were involved in reproduction. Many of these organ-specific genes are generally involved in regulatory activities and have a nuclear protein localization, except for leaves, where there are also many genes with a plastid localization. This was also observed in stems to a lesser extent. Moreover, pathogen defense and immunity pathways were highly represented in roots. These data show a complex pattern of gene expression in different organs, and provide relevant data about housekeeping and organ-specific genes in cultivated olive.

Bioinformatic analysis of FSH signaling pathways in granulosa cells

The canonical FSH signaling pathway governing granulosa cell maturation and differentiation requires activation of protein kinase A (PKA). However, PKA alone is not sufficient to reproduce the complex pattern of gene expression induced by FSH. We use bioinformatic promoter analysis of gene expression data to reveal novel key regulatory pathways in PKA-independent FSH signaling.

Identification of a Functionally Distinct Truncated BDNF mRNA Splice Variant and Protein in Trachemys scripta elegans

Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein.

Transcription Sites Are Developmentally Regulated during the Asexual Cycle of Plasmodium falciparum

Increasing evidence shows that the spatial organization of transcription is an important epigenetic factor in eukaryotic gene regulation. The malaria parasite Plasmodium falciparum shows a remarkably complex pattern of gene expression during the erythrocytic cycle, paradoxically contrasting with the relatively low number of putative transcription factors encoded by its genome. The spatial organization of nuclear subcompartments has been correlated with the regulation of virulence genes. Here, we investigate the nuclear architecture of transcription during the asexual cycle of malaria parasites. As in mammals, transcription is organized into discrete nucleoplasmic sites in P. falciparum, but in a strikingly lower number of foci. An automated analysis of 3D images shows that the number and intensity of transcription sites vary significantly between rings and trophozoites, although the nuclear volume remains constant. Transcription sites are spatially reorganized during the asexual cycle, with a higher proportion of foci located in the outermost nuclear region in rings, whereas in trophozoites, foci are evenly distributed throughout the nucleoplasm. As in higher eukaryotes, transcription sites are predominantly found in areas of low chromatin density. Immunofluorescence analysis shows that transcription sites form an exclusive nuclear compartment, different from the compartments defined by the silenced or active chromatin markers. In conclusion, these data suggest that transcription is spatially contained in discrete foci that are developmentally regulated during the asexual cycle of malaria parasites and located in areas of low chromatin density.

Acetylcholine receptor subunit and P-glycoprotein transcription patterns in levamisole-susceptible and -resistant Haemonchus contortus

The mechanism of resistance to the anthelmintic levamisole in parasitic nematodes is poorly understood, although there is some evidence implicating changes in expression of nicotinic acetylcholine receptor (nAChR) subunit genes. Hence, in order to define levamisole resistance mechanisms in some Australian field-derived isolates of Haemonchus contortus we examined gene expression patterns and SNPs in nAChR subunit genes, as well as expression levels for P-glycoprotein (P-gp) and receptor ancillary protein genes, in various life stages of one levamisole-sensitive and three levamisole-resistant isolates of this species. Larvae of two isolates showed high-level resistance to levamisole (resistance ratios at the IC50>600) while the third isolate showed a degree of heterogeneity, with a resistance factor of only 1.1-fold at the IC50 alongside the presence of a resistant subpopulation. Transcription patterns for nAChR subunit genes showed a great degree of variability across the different life stages and isolates. The most consistent observation was the down-regulation of Hco-unc-63a in adults of all resistant isolates. Transcription of this gene was also reduced in the L3 stage of the two most resistant isolates, highlighting its potential as a resistance marker in the readily accessible free-living stages. There was down regulation of all four Hco-unc-29 paralogs in adults of one resistant isolate. There were no consistent changes in expression of P-gps or ancillary protein genes across the resistant isolates. The present study has demonstrated a complex pattern of nAChR subunit gene expression in H. contortus, and has highlighted several instances where reduced expression of subunit genes (Hco-unc-63a, Hco-unc-29) may be associated with the observed levamisole resistance. The data also suggests that it will be difficult to detect resistance using gene transcription-based methods on pooled larval samples from isolates containing only a resistant subpopulation due to the averaging of gene expression data across the whole population.

Is NEDD4-1 a negative regulator of phosphatase and tensin homolog in gastric carcinogenesis?

The expression of phosphatase and tensin homolog (PTEN), a tumor suppressor gene, is frequently down-regulated in gastric carcinomas due to mutation, loss of heterozygosity, and promoter hypermethylation. However, it is unknown if additional mechanisms may account for the down-regulation of PTEN expression. While neuronal precursor cell-expressed developmentally down-regulated 4-1 (NEDD4-1) is believed to be a potential dual regulator of PTEN, there are conflicting reports regarding their interaction. To gain further insight into the role of NEDD4-1 and its association with PTEN in gastric carcinoma development, we measured the protein expression of NEDD4-1 and PTEN in gastric mucosae with various pathological lesions and found that NEDD4-1 increased from normal gastric mucosa to intestinal metaplasia and decreased from dysplasia to gastric carcinoma. These changes did not correlate with PTEN expression changes during gastric carcinogenesis. Moreover, we found similar results in protein levels in the primary tumors and adjacent non-tumorous tissues. These results differ from a previous report showing that expression of NEDD4-1 is up-regulated in gastric carcinomas, and show a more complex pattern of NEDD4-1 gene expression during gastric carcinogenesis.

Progress in Understanding the Hemoglobin Switch

During fetal development and the first 6 months of neonatal life, a complex pattern of globin gene expression occurs called the hemoglobin switch (Figure 1). After a brief period of embryonic globin gene expression, the γ-globin chain of fetal hemoglobin (HbF, or α2γ2) is predominantly expressed, then is progressively replaced by the β-globin chain of adult hemoglobin (HbA, or α2β2).2 This process is clinically important because the persistence or reactivation of HbF production can be highly beneficial in attenuating the severity of the symptoms of inherited β-globin gene disorders, such as β-thalassemia . . .

Identification of the lipoxygenase gene family from Vitis vinifera and biochemical characterisation of two 13-lipoxygenases expressed in grape berries of Sauvignon Blanc

Lipoxygenases (LOXs) are a group of non-haem iron-containing dioxygenases that catalyse oxygenation of polyunsaturated fatty acids (PUFAs) and lipids, and initiate the formation of biologically active compounds known as oxylipins. Several plant oxylipins comprise important flavours and aromas in food and beverages. Analysis of the grape (Vitis vinifera L.) genome revealed that the grape LOX family consists of 18 individual members. Phylogenetic analysis places all except one of the identified grape LOXs into either a type II 13-LOX cluster or the type I 9-LOX cluster. Four LOX genes (VvLOXA, VvLOXO, VvLOXC, VvLOXD), representative of the major LOX groupings observed in the phylogenetic analyses, were selected for analysis of patterns of transcript abundance in berry tissues. VvLOXA and VvLOXO represent putative 13-LOXs, while VvLOXC represents a putative 9-LOX. VvLOXD represents a unique LOX that differs significantly from other characterised plant LOXs. All four LOXs exhibited a complex pattern of gene expression. Across all developmental stages, VvLOXA was the most abundant LOX and was expressed predominantly in berry skins. The expression pattern of VvLOXC and -D are more evenly distributed between seeds, pulp and skin, while VvLOXO is mostly expressed in the seed. Mechanical wounding and infection of berries with Botrytis cinerea Pers.: Fr resulted in rapid accumulation of VvLOXC and -O transcripts. VvLOXA expression decreased in diseased berries. Biochemical analysis of VvLOXA and -O recombinant proteins confirmed that these LOX genes encode functional 13-LOXs that exhibit different pH and temperature optima. Both enzymes showed activity with linoleic, linolenic and arachidonic acids.

Distribution and Postnatal Development of Gpr54 Gene Expression in Mouse Brain and Gonadotropin-Releasing Hormone Neurons

Kisspeptin and G protein-coupled receptor 54 (GPR54) are now acknowledged to play essential roles in the neural regulation of fertility. Using a transgenic Gpr54 LacZ knock-in mouse model, this study aimed to provide 1) a detailed map of cells expressing Gpr54 in the mouse brain and 2) an analysis of Gpr54 expression in GnRH neurons across postnatal development. The highest density of Gpr54-expressing cells in the mouse central nervous system was found in the dentate gyrus of the hippocampus beginning on postnatal d 6 (P6). Abundant Gpr54 expression was also noted in the septum, rostral preoptic area (rPOA), anteroventral nucleus of the thalamus, posterior hypothalamus, periaqueductal grey, supramammillary and pontine nuclei, and dorsal cochlear nucleus. No Gpr54 expression was detected in the arcuate and rostral periventricular nuclei of the hypothalamus. Dual-labeling experiments showed that essentially all Gpr54-expressing cells in the rPOA were GnRH neurons. Analyses of mice at birth, P1, P5, P20, and P30 and as adults revealed a gradual increase in the percentage of GnRH neurons expressing Gpr54 from approximately 40% at birth through to approximately 70% from P20 onward. Whereas GnRH neurons located in the septum displayed a consistent increase across this time, GnRH neurons in the rPOA showed a sharp reduction in Gpr54 expression after birth (to approximately 10% at P5) before increasing to the 70% expression levels by P20. Together these findings provide an anatomical basis for the exploration of Gpr54 actions outside the reproductive axis and reveal a complex temporal and spatial pattern of Gpr54 gene expression in developing GnRH neurons.

Features of the ancestral bilaterian inferred from Platynereis dumerilii ParaHox genes

BackgroundThe ParaHox gene cluster is the evolutionary sister to the Hox cluster. Whilst the role of the Hox cluster in patterning the anterior-posterior axis of bilaterian animals is well established, and the organisation of vertebrate Hox clusters is intimately linked to gene regulation, much less is known about the more recently discovered ParaHox cluster. ParaHox gene clustering, and its relationship to expression, has only been described in deuterostomes. Conventional protostome models (Drosophila melanogaster and Caenorhabditis elegans) are secondarily derived with respect to ParaHox genes, suffering gene loss and cluster break-up.ResultsWe provide the first evidence for ParaHox gene clustering from a less-derived protostome animal, the annelid Platynereis dumerilii. Clustering of these genes is thus not a sole preserve of the deuterostome lineage within Bilateria. This protostome ParaHox cluster is not entirely intact however, with Pdu-Cdx being on the opposite end of the same chromosome arm from Pdu-Gsx and Pdu-Xlox. From the genomic sequence around the P. dumerilii ParaHox genes the neighbouring genes are identified, compared with other taxa, and the ancestral arrangement deduced.ConclusionWe relate the organisation of the ParaHox genes to their expression, and from comparisons with other taxa hypothesise that a relatively complex pattern of ParaHox gene expression existed in the protostome-deuterostome ancestor, which was secondarily simplified along several invertebrate lineages. Detailed comparisons of the gene content around the ParaHox genes enables the reconstruction of the genome surrounding the ParaHox cluster of the protostome-deuterostome ancestor, which existed over 550 million years ago.

Sustained hypoxia promotes the development of a pulmonary artery-specific chronic inflammatory microenvironment

Recent studies demonstrate that sustained hypoxia induces the robust accumulation of leukocytes and mesenchymal progenitor cells in pulmonary arteries (PAs). Since the factors orchestrating hypoxia-induced vascular inflammation are not well-defined, the goal of this study was to identify mediators potentially responsible for recruitment to and retention and differentiation of circulating cells within the hypoxic PA. We analyzed mRNA expression of 44 different chemokine/chemokine receptor, cytokine, adhesion, and growth and differentiation genes in PAs obtained via laser capture microdissection in adjacent lung parenchyma and in systemic arteries by RT-PCR at several time points of hypoxic exposure (1, 7, and 28 days) in Wistar-Kyoto rats. Analysis of inflammatory cell accumulation and protein expression of selected genes was concomitantly assessed by immunochemistry. We found that hypoxia induced progressive accumulation of monocytes and dendritic cells in the vessel wall with few T cells and no B cells or neutrophils. Upregulation of stromal cell-derived factor-1 (SDF-1), VEGF, growth-related oncogene protein-alpha (GRO-alpha), C5, ICAM-1, osteopontin (OPN), and transforming growth factor-beta (TGF-beta) preceded mononuclear cell influx. With time, a more complex pattern of gene expression developed with persistent upregulation of adhesion molecules (ICAM-1, VCAM-1, and OPN) and monocyte/fibrocyte growth and differentiation factors (TGF-beta, endothelin-1, and 5-lipoxygenase). On return to normoxia, expression of many genes (including SDF-1, monocyte chemoattractant protein-1, C5, ICAM-1, and TGF-beta) rapidly returned to control levels, changes that preceded the disappearance of monocytes and reversal of vascular remodeling. In conclusion, sustained hypoxia leads to the development of a complex, PA-specific, proinflammatory microenvironment capable of promoting recruitment, retention, and differentiation of circulating monocytic cell populations that contribute to vascular remodeling.

Light interacts with salt stress in regulating superoxide dismutase gene expression in Arabidopsis

Arabidopsis thaliana plants ( Col, Columbia, and N1438) were grown for 15 d under two light regimes providing different growth rates. The medium contained 0–85 mM NaCl. Shoot biomass and ion accumulation were measured. Superoxide dismutase (SOD) activity was assayed on gels, and the expression of six SOD genes was studied using real-time PCR. Mean growth rate was increased in high light (HL) regime as compared to low light (LL) regime. Parallely, most of SOD genes were overexpressed in Col and underexpressed in N1438 in response to HL. At 50 mM NaCl, plant growth was inhibited and a complex pattern of SOD gene expression was recorded. These responses varied according to the light regime, and differed between the accessions. This pattern, including the differences between the accessions, could be interpreted as a consequence of interaction between light and salt, hypothesizing that oxidative stress occurred when light energy input exceeded energy utilization when salt inhibited growth, and that oxidative stress induced overexpression of some SOD genes. Salt-induced excess Na + accumulation in leaves and limitation of K + provision to these organs might also participate in eliciting SOD genes’ response. Variability was observed between the two accessions for all these traits.

Constitutively Active Protein Kinase A Qualitatively Mimics the Effects of Follicle-Stimulating Hormone on Granulosa Cell Differentiation

Activation of the protein kinase A (PKA) signaling system is necessary for FSH-induced granulosa cell differentiation, but it is not known whether activation of PKA is sufficient to account for the complex pattern of gene expression that occurs during this process. We addressed this question by infecting granulosa cells with a lentiviral vector that directs the expression of a constitutively active mutant of PKA (PKA-CQR) and compared the cellular responses to PKA-CQR with cells stimulated by FSH. Expression of PKA-CQR in undifferentiated granulosa cells resulted in the induction of both estrogen and progesterone production in the absence of cAMP. The stimulatory effects of both PKA-CQR and FSH on estrogen and progesterone production were suppressed by the PKA inhibitor H-89 and were mimicked by PKA-selective cAMP agonists. mRNA levels for P450scc and 3beta-HSD were induced to a similar extent by FSH and PKA-CQR, whereas mRNA levels for P450arom and the LHr were induced to a greater extent by FSH. Microarray analysis of gene expression profiles revealed that the majority of genes appeared to be comparably regulated by FSH and PKA-CQR but that some genes appear to be induced to a greater extent by FSH than by PKA-CQR. These results indicate that the PKA signaling pathway is sufficient to account for the induction of most genes (as identified by microarray analysis), including those of the progesterone biosynthetic pathway during granulosa cell differentiation. However, optimal induction of aromatase, the LHr, and other genes by FSH appears to require activation of additional signaling pathways.

Genetic strategies for dissecting mammalian and Drosophila voltage-dependent anion channel functions

Voltage-dependent anion channels (VDACs), also known as mitochondrial porins, are a family of small pore-forming proteins of the mitochondrial outer membrane that are found in all eukaryotes. VDACs are thought to play important roles in the regulated flux of metabolites between the cytosolic and mitochondrial compartments, in overall energy metabolism via interactions with cytosolic kinases, and a debated role in programmed cell death (apoptosis). The mammalian genome contains three VDAC loci termed Vdac1, Vdac2, and Vdac3, raising the question as to what function each isoform may be performing. Based upon expression studies of the mouse VDACs in yeast, biophysical differences can be identified but the physiologic significance of these differences remains unclear. Creation of "knockout" cell lines and mice that lack one or more VDAC isoforms has led to the characterization of distinct phenotypes that provide a different set of insights into function which must be interpreted in the context of complex physiologic systems. Functions in male reproduction, the central nervous system and glucose homeostasis have been identified and require a deeper and more mechanistic examination. Annotation of the genome sequence of Drosophila melanogaster has recently revealed three additional genes (CG17137, CG17139, CG17140) with homology to porin, the previously described gene that encodes the VDAC of D. melanogaster. Molecular analysis of these novel VDACs has revealed a complex pattern of gene organization and expression. Sequence comparisons with other insect VDAC homologs suggest that this gene family evolved through a mechanism of duplication and divergence from an ancestral VDAC gene during the radiation of the genus Drosophila. Striking similarities to mouse VDAC mutants can be found that emphasize the conservation of function over a long evolutionary time frame.

Disruption of Tcfap2c in Primordial Germ Cells using Prdm1-Cre Prevents Germ Cell Development

The formation of germ cells during embryonic development is driven by a complex pattern of gene expression. The role of transcription factor Tcfap2c in germ cell development has been demonstrated in conditional mutants using the epiblast-specific Sox2-Cre model. Traditional Tcfap2c mutant embryos die at embryonic day 7.5 (E7.5) due to extraembryonic lineage defects which prevents the investigation of embryonic germ cell development. The embryonic lethality of the Tcfap2c deletion is overcome in the Sox2-Cre model because Cre is primarily expressed in the epiblast. Mutant ovaries and testes from this model failed to produce germ cells as was demonstrated by the lack of germ cell specific markers Ddx4, Pou5f1 and Ifitm3 at E14.5. Similarly, immunohistochemistry at E18.5 failed to detect the germ cell specific marker NOBOX or the meiotic marker SYCP3 confirming a lack of germ cells in Tcfap2c mutant ovaries. However, the Sox2-Cre model has limitations for investigating germ cell development. The Tcfap2c Sox2-Cre mutant mice exhibit delayed development and die at birth. This lethality is likely due to the deletion of the gene throughout the embryo and perhaps a partial deletion of Tcfap2c in the placenta. Therefore, to test a more specific model we mated animals containing a Tcfap2c floxed allele to animals containing a Prdm1-Cre allele. Prdm1-Cre expression is detected in the primordial germ cell population at the time of germ cell specification around E7. In addition to the germ cells, Prdm1 expression has been detected in other tissues including the forebrain, forelimb and heart. Prdm1 null animals die at E10.5 due to severe hemorrhaging, which may be related to a defect in the labyrinth layer of the placenta, even though Prdm1 was not detected in extraembryonic structures by in situ hybridization. Therefore, we first tested this model for viability of mutants and then for germ cell production. Prdm1-Cre Tcfap2c mutants were born alive allowing ovaries and testes to be collected at 14 days post partum (dpp). The mutant ovaries and testes were noticeably reduced in size when compared to littermate controls. Histological analysis revealed that mutant ovaries lacked follicle structures and no oocytes were detected. Spermatogenesis is also affected in the mutant with testes displaying disorganized development and germ cell loss within the tubules at 14 dpp. These results validate the Prdm1-Cre Tcfap2c mutant model for investigating germ cell production from E7.0 to the adult and demonstrate a critical role for Tcfap2c in germ cell development. The next step is to use this model to investigate when and why germ cells fail in Tcfap2c mutant ovaries.

Gene Expression Profiling and Its Relevance to the Blood-Epididymal Barrier in the Human Epididymis1

The luminal environment along the epididymal duct is important for spermatozoal maturation. This environment is unique and created by the blood-epididymal barrier, which is formed by tight and adhering junctions. For the human epididymis, little information exists on the proteins that comprise these junctions. Our objectives were to assess the gene expression profiles in the different segments of the human epididymis and to identify the proteins that make up the blood-epididymal barrier. Using microarrays, we identified 2980 genes that were differentially expressed by at least 2-fold between the various segments. Of the many genes involved in diverse functions, were those that encoded adhesion proteins (cadherins and catenins) and tight junctional proteins (claudins [CLDN] and others). PCR analyses confirmed the microarray data. Immunolocalization of CLDNs 1, 3, 4, 8, and 10 revealed that the localization of CLDNs differed along the epididymis. In all three segments, CLDNs 1, 3, and 4 were localized to tight junctions, along the lateral margins of adjacent principal cells, and at the interface between basal and principal cells. CLDN8 was localized to tight junctions in all three segments, in addition to being localized in the caput along the lateral margins of principal cells, and in the corpus, at the interface between principal and basal cells. CLDN10, tight junction protein 1, and occludin were localized exclusively to tight junctions in all three epididymal segments. These data indicate that the epididymis displays a complex pattern of gene expression, which includes genes that are implicated in the formation of the blood-epididymal barrier, which suggests complex regulation of this barrier.

Positional Conservation of Clusters of Overlapping Promoter-Like Sequences in Enterobacterial Genomes

The selective mechanisms operating in regulatory regions of bacterial genomes are poorly understood. We have previously shown that, in most bacterial genomes, regulatory regions contain high densities of sigma70 promoter-like signals that are significantly above the densities detected in nonregulatory genomic regions. In order to investigate the molecular evolutionary forces that operate in bacterial regulatory regions and how they affect the observed redundancy of promoter-like signals, we have undertaken a comparative analysis across the completely sequenced genomes of enteric gamma-proteobacteria. This analysis detects significant positional conservation of promoter-like signal clusters across enterics, some times in spite of strong primary sequence divergence. This suggests that the conservation of the nature and exact position of specific nucleotides is not necessarily the priority of selection for maintaining the transcriptional function in these bacteria. We have further characterized the structural conservation of the regulatory regions of dnaQ and crp across all enterics. These two regions differ in essentiality and mode of regulation, the regulation of crp being more complex and involving interactions with several transcription factors. This results in substantially different modes of evolution, with the dnaQ region appearing to evolve under stronger purifying selection and the crp region showing the likely effects of stabilizing selection for a complex pattern of gene expression. The higher flexibility of the crp region is consistent with the observed less conservation of global regulators in evolution. Patterns of regulatory evolution are also found to be markedly different in endosymbiotic bacteria, in a manner consistent with regulatory regions suffering some level of degradation, as has been observed for many other characters in these genomes. Therefore, the mode of evolution of bacterial regulatory regions appears to be highly dependent on both the lifestyle of the bacterium and the specific regulatory requirements of different genes. In fact, in many bacteria, the mode of evolution of genes requiring significant physiological adaptability in expression levels may follow patterns similar to those operating in the more complex regulatory regions of eukaryotic genomes.