Expression Of Novel Genes Research Articles

Unusual microbial xylanases from insect guts.

Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms. In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths). Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity. Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered. Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases. Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted beta-1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products. From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed.

Feasibility of global gene expression analysis in testicular biopsies from infertile men.

Numerous studies have documented the use of microarray analysis to identify patterns of global gene expression that distinguish normal development from that of the diseased state. Yet, there are no reports that compare global gene expression in the fertile and infertile human testis. Here, we report an initial study of global gene expression in testicular biopsies from several men with different infertility phenotypes. We found that microarray analysis of small biopsy samples was suitable for profiling expression of genes known to function in germ cell development and also identified expression of novel genes. Since it is now common for infertile men with spermatogenic failure to use intracytoplasmic sperm injection (ICSI) to achieve biological paternity, we hypothesize that molecular screening of testicular biopsies with microarrays may be suitable: (1) to categorize the molecular phenoytpes of infertile testes in a manner similar to standard morphologic analysis and (2) to initiate larger studies of gene expression in the infertile testes that may identify genetic signatures from biopsies that allow prediction of outcomes.

Metabolic engineering of β-lactam production

Metabolic engineering has become a rational alternative to classical strain improvement in optimisation of β-lactam production. In metabolic engineering directed genetic modification are introduced to improve the cellular properties of the production strains. This has resulted in substantial increases in the existing β-lactam production processes. Furthermore, pathway extension, by heterologous expression of novel genes in well-characterised strains, has led to introduction of new fermentation processes that replace environmentally damaging chemical methods. This minireview discusses the recent developments in metabolic engineering and the applications of this approach for improving β-lactam production.

Freeze-induced expression of a novel gene, fr47, in the liver of the freeze-tolerant wood frog, Rana sylvatica

The ability to endure the freezing of body fluids is well developed as an adaptation for winter survival in several species of woodland frogs. Recently, the mechanisms supporting natural freeze tolerance have been shown to include the expression of novel genes. One such novel gene, fr47, codes for a 390-amino acid protein present in the livers of freeze-tolerant anurans ( Rana sylvatica, Pseudacris crucifer, Hyla versicolor) but not in freeze-intolerant species ( Rana pipiens, Scaphiopus couchii). Regulatory influences on gene and protein expression were investigated using R. sylvatica. Northern blot analysis showed that transcript levels were increased following 24 h of freezing (5.1-fold), 24 h of anoxia exposure (6.4-fold), or the loss of 20% of total body water (2.7-fold). Immunoblotting with anti-FR47 antibody indicated that protein levels increased during freezing and thawing, but decreased somewhat during anoxia or dehydration exposure, although rebounding during recovery. These results suggest that (i) FR47 function is important for freeze survival, and (ii) that control at the protein level may be exerted posttranscriptionally. Finally, assessment of putative signal transduction pathways regulating fr47 gene expression, via in vitro incubations of liver slices, indicated the involvement of a protein kinase C-mediated pathway.

Altered expression of novel genes in the cerebral cortex following experimental brain injury

Damage to the cerebral cortex results in neurological impairments such as motor, attention, memory and executive dysfunctions. To examine the molecular mechanisms contributing to these deficits, mRNA expression was profiled using high-density cDNA microarray hybridization after experimental cortical impact injury in mice. The mRNA levels at 2 h, 6 h, 24 h, 3 days and 14 days after injury were compared with those of control animals. This revealed 86 annotated genes and 24 expression sequence tags (ESTs) as being differentially expressed with a 1.5-fold or greater change. Quantitative real-time PCR analysis was used to independently verify these results for selected genes. Seven functional classes of genes were found to be altered following injury, including transcription factors, signal transduction genes and inflammatory proteins. While a few of these genes have been previously reported to be differentially regulated following injury, the most of the genes have not been previously implicated in traumatic brain injury (TBI) pathophysiology. For example, consistent with previous reports, the transcription factor c-jun and the neurotrophic factor bdnf mRNA levels were altered as a result of TBI. Among the novel genes, the mRNA levels for the high mobility group protein 1 ( hmg-1), the regulator of G-protein signaling 2 ( rgs-2), the transforming growth factor β inducible early growth response ( tieg), the inhibitor of DNA binding 3 ( id3), and the heterogeneous nuclear ribonucleoprotein H ( hnrnp h) were changed following injury. The functional significance of these genes in neurite outgrowth, neuronal regeneration, and plasticity following injury are discussed.

DNA-Binding Specificity of the ERF/AP2 Domain of Arabidopsis DREBs, Transcription Factors Involved in Dehydration- and Cold-Inducible Gene Expression

DRE/CRT is a cis-acting element that is involved in gene expression responsive to drought and low-temperature stress in higher plants. DREB1A/CBF3 and DREB2A are transcription factors that specifically bind to DRE/CRT in Arabidopsis. We precisely analyzed the DNA-binding specificity of DREBs. Both DREBs specifically bound to six nucleotides (A/GCCGAC) of DRE. However, these proteins had different binding specificities to the second or third nucleotides of DRE. Gel mobility shift assay using mutant DREB proteins showed that the two amino acids, valine and glutamic acid conserved in the ERF/AP2 domains, especially valine, have important roles in DNA-binding specificity. In the Arabidopsis genome, 145 DREB/ERF-related proteins are encoded. These proteins were classified into five groups—AP-2 subfamily, RAV subfamily, DREB subfamily, ERF subfamily, and others. The DREB subfamily included three novel DREB1A- and six DREB2A-related proteins. We analyzed expression of novel genes for these proteins and discuss their roles in stress-responsive gene expression.

Sensitivity of bovine blastocyst gene expression patterns to culture environments assessed by differential display RT-PCR

The use of culture media to support the development of preimplantation embryos to the blastocyst stage is often associated with detrimental effects on normal development. These effects have been uncovered largely by investigating the phenotypic abnormalities displayed by fetuses and newborns derived from cultured preimplantation embryos. Research to understand the impact of culture on the embryonic developmental programme has focused on embryo metabolism, gene expression and genomic imprinting. We have used differential display RT-PCR to examine culture influences on global transcript pools in bovine embryos. Others have examined culture influences on candidate "marker genes" in cultured murine, ovine and bovine embryos. These studies have demonstrated that culture conditions influence the amount of marker gene transcripts and downregulate or induce the expression of novel genes during early development. Optimized defined culture media maintain embryonic gene expression patterns closely resembling those displayed by embryos derived in vivo. Preimplantation mammalian embryos display an impressive capacity to respond to the pressures that suboptimal culture environments place upon them. However, this plasticity operates within a defined range of tolerances. Continued research using molecular techniques will lead to increased understanding of developmental mechanisms causing culture-related phenotypic abnormalities in post-implantation embryos.

Sensitivity of bovine blastocyst gene expression patterns to culture environments assessed by differential display RT-PCR

The use of culture media to support the development of preimplantation embryos to the blastocyst stage is often associated with detrimental effects on normal development. These effects have been uncovered largely by investigating the phenotypic abnormalities displayed by fetuses and newborns derived from cultured preimplantation embryos. Research to understand the impact of culture on the embryonic developmental programme has focused on embryo metabolism, gene expression and genomic imprinting. We have used differential display RT-PCR to examine culture influences on global transcript pools in bovine embryos. Others have examined culture influences on candidate "marker genes" in cultured murine, ovine and bovine embryos. These studies have demonstrated that culture conditions influence the amount of marker gene transcripts and downregulate or induce the expression of novel genes during early development. Optimized defined culture media maintain embryonic gene expression patterns closely resembling those displayed by embryos derived in vivo. Preimplantation mammalian embryos display an impressive capacity to respond to the pressures that suboptimal culture environments place upon them. However, this plasticity operates within a defined range of tolerances. Continued research using molecular techniques will lead to increased understanding of developmental mechanisms causing culture-related phenotypic abnormalities in post-implantation embryos.

Predicting response to paclitaxel/carboplatin-based therapy in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a systemic illness. The majority of newly diagnosed patients depend on systemic chemotherapy to improve outcome. Among the new chemotherapeutic agents recently tested, paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) has shown convincing single-agent activity in advanced NSCLC. The two initial phase II studies using paclitaxel alone showed a 1-year survival rate of 40%, comparable to that seen with combination regimens. Paclitaxel/carboplatin is one of several standard regimens for patients with stage IIIB to IV disease. It is as effective as any other new agent/platinum combination studied to date; it is easy to administer and well tolerated. Identification of the molecular and genetic events involved in each step of tumor progression seems to be crucial both to understanding lung cancer and for the development of new pharmacologic compounds that target specific cellular processes affecting growth and proliferation. An innovative strategy is to combine established chemotherapy with these new compounds. Resistance to available chemotherapy drugs is the major obstacle to effective chemotherapy. Genetic abnormalities could play a role in outlining some patterns of chemoresistance. Acquired resistance to paclitaxel can be mediated by several mechanisms, including overexpression of p-glycoprotein, altered expression of β-tubulin isotypes, intrinsic or acquired mutations in β-tubulin, and expression of novel genes. Beta-tubulin mutations were recently identified in 33% of 49 NSCLC patients, none of whom had an objective response to paclitaxel treatment. Cisplatin resistance is associated with several molecular alterations, including overexpression of metallothionein and the mRNA level of the excision repair cross-complementing ( ERCCI) gene. Early detection of circulating cancer cells in peripheral blood would enable more accurate lung cancer staging. Furthermore, sequential measurements of DNA concentration may be used to monitor the effects of therapy. Serum DNA can be used as a surrogate for detecting genetic abnormalities and as a potential guide for customizing treatment. We analyzed the presence of β-tubulin mutations in serum DNA from NSCLC patients and from healthy individuals. Beta-tubulin mutations were detected in 42% of the 131 patients and in none of the control group. Several clinical studies are proposed to develop more customized approaches in lung cancer.

Identification and expression of novel genes in osteoarthritis

Osteoarthritis (OA), the most common form of joint disease, represents a clinical classification of pathological conditions involving progressive degradation of articular cartilage and remodeling of subchondral bone. Genes whose products are involved in chondrogenesis and osteogenesis starting from the common progenitor cells, genes determining the terminal differentiation of chondrocytes and genes whose products trigger breakdown of the cartilaginous matrix are obvious candidates for therapeutic intervention. In this study we used human articular cartilage primary cells to conduct a series of gene expression profiling experiments. The cells were subjected to various treatments, which mimic OA initiation and development: IL-1β-FAD (FGF-2+Dexamethasone+Ascorbic acid) and mechanical stress. The gene expression profiles corresponding to various applied treatments were studied by microarray hybridization and analyzed by QBI's proprietary bioinformatics tools. The obtained gene expression patterns indicates that the chosen in vitro cell system accurately reflects the processes that occur in OA joints in vivo since many genes known to be markers of OA were identified by us as displaying the expected type of behavior. After performing the full analysis of hybridization results, a list of 254 genes was obtained. Among them are 210 known genes and 44 novel ones. Out of them 80 genes were further analyzed by in situ hybridization on sections obtained from OA patients and control group and by RT-PCR. 15 genes have shown promising results. Among them, genes whose expression was upregulated in chondrocytes located close to the eroded surface of the OA articular cartilage, in osteoprogenitor cells and in endothelial cells. Further studies are being conducted to analyze the role of these genes in OA.

Detection of novel gene expression in paraffin-embedded tissues by isotopic in situ hybridization in tissue microarrays.

Correlating altered gene expression patterns with particular disease states is a critical step in understanding disease processes and developing treatment strategies. Many thousands of novel gene sequences have recently been annotated in public and private databases and are now available for analysis. Tissue-specific expression patterns of these sequences can be evaluated physically on DNA arrays and other high throughput assays, or virtually by bioinformatics mining of expressed sequence tag (EST) databases. As a secondary screening tool, in situ hybridisation (ISH) not only confirms tissue specificity, but also reveals what is often valuable information about cell-type expression patterns of nov16l sequences. Due to their availability and long-term stability at room temperature, formalin-fixed paraffin-embedded clinical specimens provide an invaluable resource for evaluating expression patterns of novel human genes. We describe a high-throughput approach for identifying and quantifying the expression of novel genes in paraffin-embedded human tissues using isotopic in situ hybridisation and tissue microarrays (TMA).

Coordinate expression of novel genes during osteoblast differentiation.

To achieve new insights into the coordinate regulation of gene expression during osteoblast differentiation we utilized an approach involving global analysis of gene expression to obtain the identities of messenger RNAs (mRNAs) expressed using an established in vitro model of bone development. MC3T3-E1 osteoblast-like cells were induced to differentiate by the addition of beta-glycerophosphate (beta-GP) and ascorbic acid. RNA samples derived from induced and uninduced control MC3T3-E1 cells were used to prepare complementary DNA (cDNA) for serial analysis of gene expression (SAGE). A preliminary SAGE database was produced and used to prepare a hybridization array to further facilitate the characterization of changes in the expression levels of 92 of the SAGE-mRNA assignments after induction of osteoblast differentiation, specifically after 6 days and 14 days of ascorbate treatment. SAGE-array hybridization analysis revealed coordinate induction of a number of mRNAs including Rab24, calponin, and calcyclin. Levels of MSY-1, SH3P2, fibronectin, alpha-collagen, procollagen, and LAMPI mRNAs, present at day 6 postinduction, were markedly reduced by day 14 postinduction. A number of unanticipated and potentially important developmental genes were identified including the transforming growth factor beta (TGF-beta) superfamily member Lefty-1. Lefty-1 transcript and translation product were found to be induced during the course of MC3T3-E1 cell differentiation. We present evidence, using transient transfection and antibody neutralization approaches, that Lefty-1 modulates the induction of alkaline phosphatase (ALP) after treatment of MC3T3-E1 cells with ascorbate and beta-GP. These data should provide useful new information for future analysis of transcriptional events in osteoblast differentiation and mineralization.

Mechanical load stimulates expression of novel genesin vivoandin vitroin avian flexor tendon cells

Objective: Our experiments were designed to test the hypothesis that tendon cells might respond differently to applied strainin vitrothanin vivo.Design: We tested cells in whole tendons from exercised chickens and from isolated surface (TSC) and internal tendon (TIF)in vitrothat were subjected to mechanical strain. We hypothesized that tendon cells differentially express genes in response to mechanical loadingin vivoandin vitro.Methods: We utilized an in-vivo exercise model in which chickens were run on a treadmill in an acute loading regime for 1h 45min with the balance of time at rest to 6h total time. Gene expression was analyzed by a differential display technique. In addition, isolated avian flexor digitorum profundus TSC and TIF cells were subjected to cyclic stretching at 1Hz, 5% average elongation for 6h, ±PDGF-BB, IGF-I, TGF-β1, PTH, estrogen, PGE2, or no drug and/or no load. mRNA was then collected and samples were subjected to differential display analysis.Conclusions: Load with or without growth factor and hormone treatments induced expression of novel genes as well as some known genes that were novel to tendon cells. We conclude that the study of gene expression in mechanically loaded cellsin vivoandin vitrowill lead to the discovery of novel and important marker proteins that may yield clues to positive and negative cell strain responses that are protective under one set of conditions and destructive under another.

Regulation of Growth Factor Gene Expression in Degenerating Motoneurons of the Murine Mutantwobbler: A Cellular Patch-Sampling/RT–PCR Study

Motoneuronal degenerative diseases are characterized by their progressivity; once affected, the motoneurons remain in altered states during an intermediate phase of degeneration prior to their final disappearance. Whether this survival period coincides with active metabolic rearrangements in the affected neuron remains unknown. As a first step toward the elucidation of this question, we developed cDNA pooled samples obtained from degenerating and control motoneuron mRNA populations through cellular patch sampling and RT–PCR, using the murinewobblermutant as a model of spinal atrophy. Hybridization of the cDNA pools to various markers of intact or degenerating motoneurons allowed us to verify the cellular specificity of the patch sampling and indicated conservation of the original mRNA population complexity. Exploration of transcriptional alterations of genes encoding growth factors thought to be involved in motoneuronal development revealed that gene expression of the neurotrophin BDNF was induced in affected motoneurons, while expression of neurotrophin-3 was present in both neuronal types. Likewise, expression of a member of the epidermal growth factor (EGF) family, the neuregulin transcript sensory motor neuron-derived factor, was detected in both control and degenerating motoneurons, while transforming growth factor α, the functional homolog of EGF, was present only in the affected motoneurons. Immunohistochemical detection of corresponding proteins corroborated these observations. These results demonstrate that, during the course of their degeneration, motoneurons can initiate expression of novel genes which lead to the production of molecules endowed with trophic and/or differentiative properties for the neurons themselves and their glial environment. They also validate the use of the developed cDNA pooled samples for further exploration of transcriptional alterations taking place in degenerating motoneurons.

Contrasting patterns in the spread of two seed‐borne viruses in pea embryos

The temporal and spatial patterns of pea seed‐borne mosaic potyvirus (PSbMV) and pea early browning tobra‐virus (PEBV) accumulation in pea embryos were analyzed using in‐situ hybridization and immunohistochemistry. For PSbMV, which infects embryos after fertilization, the distribution changed as the embryo developed and some tissues remained free of virus infection. In contrast, embryos were uniformly infected with PEBV from the earliest stages of embryo development, and PEBV was detected in the egg cell and pollen grains, indicative of gametic transmission into the embryo. These observations suggest that gametically transmitted viruses may be appropriate as potential vectors for the ectopic and uniform expression of novel genes in embryonic tissues. Functional complementarity in the two processes of embryo invasion was tested following co‐inoculation with PSbMV and PEBV. Instead of complementation, interference in PSbMV seed transmission by PEBV was observed; PEBV seed transmission remained unaffected by PSbMV.

Expression of novel genes linked to the androgen-induced, proliferative shutoff in prostate cancer cells

Androgens control cell numbers in the prostate through three separate pathways: (a) inhibition of cell death, (b) induction of cell proliferation (Step-1) and (c) inhibition of cell proliferation (Step-2, proliferative shutoff). The mechanisms underlying these phenomena are incompletely understood. The human prostate carcinoma LNCaP variants express these pathways as follows: LNCaP-FGC express both steps, LNCaP-LNO expresses Step-2, LNCaP-TAC expresses Step-1, and LNCaP-TJA cells express neither step. These cells facilitated the search for mediators of the androgen-induced proliferative shutoff pathway. Androgen exposure for 24 h or longer induced an irreversible proliferative shutoff in LNCaP-FGC cells. The Wang and Brown approach for identifying differentially expressed mRNAs was used to search for mediators of Step-2. Ten unique inserts were identified and from those ten, three genes were further studied. The basal expression of these genes in shutoff-negative variants was not affected by androgen exposure. They were induced by androgens in shutoff-positive LNCaP variants and the androgen receptor-transfected, shutoff-positive, MCF7-AR1 cells. These genes were induced only in the range of androgen concentrations that elicited the shutoff response. Time course analysis showed that their induction precedes the commitment point by 12–18 h. In addition, they were expressed in the normal prostate during proliferative shutoff. These features suggest that the candidate genes have a role in the regulation cascade for proliferative shutoff.

Expression of novel genes during somatic embryogenesis of suspension-cultured carrot cells ( Daucus carota)

Summary During somatic embryogenesis of suspension cultured carrot cells several genes were identified that are either transcribed immediately after induction or in association with the typical morphogenic stages. They were identified via their cDNAs. For clone DC E1 a temporary accumulation of the specific mRNA between 4 and 24 h after induction was observed. The genes represented by clones DC A3 and DC B2 exhibit a maximum of expression at periods comprising the heart- and torpedo-shaped stages. For the mRNA of clone DC D2 a steady increase was monitored that started about 6 days after induction and reached a constant level after about 20 days. With regard to the nucleotide sequence and the deduced amino acid sequence DC A3 encodes a polypeptide with sequence motifs abundant in glycine and proline residues. The prominent feature of DC B2 is a putative bipartite nuclear location site, a signal sequence conferring the important of a protein into the cell nucleus. The predicted amino acid sequence of DC E1 displays significant homology with cytosolic ascorbate peroxidases.

The genomic response of tumor cells to hypoxia and reoxygenation. Differential activation of transcription factors AP-1 and NF-kappa B.

Hypoxia and reoxygenation are important pathophysiological conditions that occur during injury, ischemia, reperfusion and stroke. In tumors, hypoxia and oxidative stress are regarded as triggers for enhanced proliferation and metastasis. Hypoxia and reoxygenation exert part of their biological effects by inducing the expression of novel genes but very little is known about the transcription factors involved. Here, we have compared the behaviour of two redox-controlled factors, AP-1 and NF-kappa B, during hypoxia and reoxygenation. We report that the DNA-binding and transcriptional activity of transcription factor AP-1 is very strongly induced in a biphasic response when HeLa cells are exposed to reduced oxygen pressure. This induction required new AP-1 protein synthesis. Different members of the Jun/Fos family of transcription factors were found in the first and second maxima of activation. The pathogen-responsive, pre-existing transcription factor NF-kappa B was not activated under hypoxic conditions. However, a p50-p65 heterodimer of NF-kappa B was rapidly and strongly activated when HeLa cells were re-exposed to normal oxygen pressure. This explains the induction of NF-kappa B-controlled inflammatory cytokine genes during reperfusion of ischemic tissue. Our data suggest that the genomic response to hypoxia is primarily mediated by AP-1 while the inflammatory response to reoxygenation is mediated by NF-kappa B.

Differential expression of novel genes by bone marrow-derived macrophage populations

In the present study, we have constructed a subtraction cDNA library to identify novel genes induced by IFN-γ in GM-CSF-derived bone marrow macrophages (mø). Mø were treated with 50 U/ml IFN-γ for 40, 70 and 140 min to induce expression of early genes regulated by IFN-γ, and the mø were pooled. Poly(A) +RNA was prepared from both unactivated and IFN-γ-stimulated mø, and cDNA libraries were constructed in λZAP. Genes expressed in common by both mø populations were removed by subtraction using biotin-avidin precipitation of hybrid complexes. Further selection was performed by differential screening using cDNA prepared from mRNA of unactivated mø as a probe, followed by colony hybridization to remove sister clones. Of 17 clones from which sequence information was obtained, two appeared to be identical with the murine genes, C10 (clone GM2B1) and Mac-2 (clone GM2C4) and an additional two clones had high similarity to human cDNAs encoding proteins of unknown function. cDNAs containing sequences which did not match published sequences were used to probe Northern blots prepared from both unstimulated and IFN-γ-activated GM-CSF- and CSF-1-derived mø. Five clones (GM1A2, GM1B4, GM1F2, GM2A12 and GM2B8) showed enhanced transcript levels following IFN-γ treatment of GM-CSF-derived mø, but demonstrated high constitutive transcript levels in CSF-1-derived mø. In addition, C10 transcripts were constitutively expressed by GM-CSF-derived mø, but not by CSF-1-derived mø, even after activation by IFN-γ. These data suggest that much of the functional heterogeneity of GM-CSF- and CSF-1-derived mø resides in the differential expression of early genes specifically induced by IFN-γ.