Home About us Contact
|
Home About us Contact | ||
|
|
||
Gene Disruption (gene + disruption)
Kinds of Gene Disruption Selected AbstractsGeneration of Novel Landomycins M and O through Targeted Gene DisruptionCHEMBIOCHEM, Issue 4 2005Andriy Luzhetskyy Dr. Abstract Two genes from Streptomyces cyanogenous S136 that encode the reductase LanZ4 and the hydroxylase LanZ5, which are involved in landomycin A biosynthesis, were characterized by targeted gene inactivation. Analyses of the corresponding mutants as well as complementation experiments have allowed us to show that LanZ4 and LanZ5 are responsible for the unique C-11-hydroxylation that occurs during landomycin biosynthesis. Compounds accumulated by the lanZ4/Z5 mutants are the previously described landomycin F and the new landomycins M and O. [source] The characterization of functions involved in the establishment and maturation of Klebsiella pneumoniae in vitro biofilm reveals dual roles for surface exopolysaccharidesENVIRONMENTAL MICROBIOLOGY, Issue 3 2008Damien Balestrino Summary The ability to form biofilm is seen as an increasingly important colonization strategy among both pathogenic and environmental Klebsiella pneumoniae strains. The aim of the present study was to identify abiotic surface colonization factors of K. pneumoniae using different models at different phases of biofilm development. A 2200 K. pneumoniae mutant library previously obtained by signature-tagged mutagenesis was screened in static and dynamic culture models to detect clones impaired at early and/or mature stages of biofilm formation. A total of 28 mutants were affected during late phases of biofilm formation, whereas 16 mutants displayed early adhesion defect. These mutants corresponded to genes involved in potential cellular and DNA metabolism pathways and to membrane transport functions. Eight mutants were deficient in capsule or LPS production. Gene disruption and microscopic analyses showed that LPS is involved in initial adhesion on both glass and polyvinyl-chloride and the capsule required for the appropriate initial coverage of substratum and the construction of mature biofilm architecture. These results give new insight into the bacterial factors sequentially associated with the ability to colonize an abiotic surface and reveal the dual roles played by surface exopolysaccharides during K. pneumoniae biofilm formation. [source] Regulatory factor X4 variant 3: A transcription factor involved in brain development and disease,JOURNAL OF NEUROSCIENCE RESEARCH, Issue 16 2007Donghui Zhang Abstract Regulatory factor X4 variant 3 (RFX4_v3) is a recently identified transcription factor specifically expressed in the brain. Gene disruption in mice demonstrated that interruption of a single allele (heterozygous, +/,) prevented formation of the subcommissural organ (SCO), resulting in congenital hydrocephalus, whereas interruption of two alleles (homozygous, ,/,) caused fatal failure of dorsal midline brain structure formation. These mutagenesis studies implicated RFX4_v3 in early brain development as well as the genesis of the SCO. Rfx4_v3 deficiency presumably causes abnormalities in brain by altering the expression levels of many genes that are crucial for brain morphogenesis, such as the signaling components in the Wnt, bone morphogenetic protein, and retinoic acid pathways. RFX4_v3 might affect these critical signaling pathways in brain development. Cx3cl1, a chemokine gene highly expressed in brain, was identified as a direct target for RFX4_v3, indicating that RFX4_v3 possesses trans -acting activity to stimulate gene expression. Rfx4_v3 is highly expressed in the suprachiasmatic nucleus and might be involved in regulating the circadian clock. One haplotype in RFX4_v3 gene is linked to a higher risk of bipolar disorder, suggesting that this protein might contribute to the pathogenesis of the disease. This Mini-Review describes our current knowledge about RFX4_v3, an important protein that appears to be involved in many aspects of brain development and disease. © 2007 Wiley-Liss, Inc. [source] The rpf gene of Micrococcus luteus encodes an essential secreted growth factorMOLECULAR MICROBIOLOGY, Issue 3 2002Galina V. Mukamolova Summary Micrococcus luteus secretes a small protein called Rpf, which has autocrine and paracrine signalling functions and is required for the resuscitation of dormant cells. Originally isolated from the supernatant of actively growing cultures, Rpf was also detected on the surface of actively growing bacteria. Most molecules may be sequestered non-productively at the cell surface, as a truncated form of the protein, encompassing only the ,Rpf domain' is fully active. The C-terminal LysM module, which probably mediates binding to the cell envelope, is not required for biological activity. Rpf was essential for growth of M. luteus. Washed cells, inoculated at low density into a minimal medium, could not grow in its absence. Moreover, the incorporation of anti-Rpf antibodies into the culture medium at the time of inoculation also prevented bacterial growth. We were unable to inactivate rpf using a disrupted form of the gene, in which most of the coding sequence was replaced with a selectable thiostrepton resistance marker. Gene disruption was possible in the presence of a second, functional, plasmid-located copy of rpf, but not in the presence of a rpf derivative whose protein product lacked the secretory signal sequence. As far as we are aware, Rpf is the first example of a truly secreted protein that is essential for bacterial growth. If the Rpf-like proteins elaborated by Mycobacterium tuberculosis and other mycobacteria prove similarly essential, interference with their proper functioning may offer novel opportunities for protecting against, and treating, tuberculosis and other mycobacterial disease. [source] Manipulating gene activity in Wnt1-expressing precursors of neural epithelial and neural crest cellsDEVELOPMENTAL DYNAMICS, Issue 1 2010Wei Hsu Abstract Targeted gene disruption or expression often encounters lethality. Conditional approaches, permitting manipulation at desired stages, are required to overcome this problem in order to analyze gene function in later developmental processes. Wnt1 has been shown to be expressed in neural crest precursors at the dorsal midline region. However, its expression was not detected in emigrated neural crest cells, the descendants of Wnt1-expressing precursors. We have developed mouse transgenic systems to manipulate gene activity in the Wnt1-expressing precursors and their derivatives by integrating the tetracycline-dependent activation and Cre-mediated recombination methods. A new Wnt1-rtTA strain, carrying rtTA under control of Wnt1 regulatory elements, has been created for gene manipulation in a spatiotemporal-specific fashion. Together with our previously developed Wnt1-Cre;R26STOPrtTA model, these systems permit conditional gene expression and ablation in pre-migratory and/or post-migratory neural crest cells. This study demonstrated the versatility of our mouse models to achieve gene manipulation in early neural development. Developmental Dynamics 239:338,345, 2010. © 2009 Wiley-Liss, Inc. [source] Sex differences in progesterone receptor immunoreactivity in neonatal mouse brain depend on estrogen receptor , expressionDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2001Christine K. Wagner Abstract Around the time of birth, male rats express higher levels of progesterone receptors in the medial preoptic nucleus (MPN) than female rats, suggesting that the MPN may be differentially sensitive to maternal hormones in developing males and females. Preliminary evidence suggests that this sex difference depends on the activation of estrogen receptors around birth. To test whether estrogen receptor alpha (ER,) is involved, we compared progesterone receptor immunoreactivity (PRir) in the brains of male and female neonatal mice that lacked a functional ER, gene or were wild type for the disrupted gene. We demonstrate that males express much higher levels of PRir in the MPN and the ventromedial nucleus of the neonatal mouse brain than females, and that PRir expression is dependent on the expression of ER, in these regions. In contrast, PRir levels in neocortex are not altered by ER, gene disruption. The results of this study suggest that the induction of PR via ER, may render specific regions of the developing male brain more sensitive to progesterone than the developing female brain, and may thereby underlie sexual differentiation of these regions. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 176,182, 2001 [source] Development of a novel quadruple auxotrophic host transformation system by argB gene disruption using adeA gene and exploiting adenine auxotrophy in Aspergillus oryzaeFEMS MICROBIOLOGY LETTERS, Issue 1 2004Feng Jie Jin Abstract We previously designed a triple auxotrophic host-vector system in Aspergillus oryzae by isolating red-colored adenine auxotrophic mutants upon UV mutagenesis of a double auxotrophic host (niaD,sC,). In the present study an effort to exploit this system and construct a novel quadruple auxotrophic host was made by disrupting the argB gene involved in arginine biosynthesis. The argB gene-disruption cassette was generated by fusion PCR, which required only two steps of PCR to insert the selectable marker, adeA, into the target argB gene. The chimeric DNA fragment was transformed into the triple auxotrophic strain (niaD,sC,adeA,) and the argB disruptants were obtained with a high rate of efficiency (approximately 40%). The argB disruptants were characterized by normal colony color and reversal of arginine auxotrophy by introduction of the wild-type argB gene. Quadruple auxotrophic strains (niaD,sC,,argB adeA, or niaD,sC,,argB adeB,) were subsequently isolated upon UV mutagenesis of the triple auxotrophic strain (niaD,sC,,argB) followed by screening of red-colored colonies for adenine auxotrophy. The results obtained showed that the adeA gene served as an efficient selection marker in developing a novel host-vector system with quadruple auxotrophy in A. oryzae, thus providing a powerful tool to breed multiple auxotrophic mutants from a deuteromycete wherein sexual crossing is impossible. [source] An unusual ,-ketoacyl:acyl carrier protein synthase and acyltransferase motifs in TaK, a putative protein required for biosynthesis of the antibiotic TA in Myxococcus xanthusFEMS MICROBIOLOGY LETTERS, Issue 2 2001Yossi Paitan Abstract The antibiotic TA of Myxococcus xanthus is produced by a type-I polyketide synthase mechanism. Previous studies have indicated that TA genes are clustered within a 36-kb region. The chemical structure of TA indicates the need for several post-modification steps, which are introduced to form the final bioactive molecule. These include three C -methylations, an O -methylation and a specific hydroxylation. In this study, we describe the genetic analysis of taK, encoding a specific polyketide ,-ketoacyl:acyl carrier protein synthase, which contains an unusual ,-ketoacyl synthase and acyltransferase motifs and is likely to be involved in antibiotic TA post-modification. Functional analysis of this ,-ketoacyl:acyl carrier protein synthase by specific gene disruption suggests that it is essential for the production of an active TA molecule. [source] Development of host and vector for high-efficiency transformation and gene disruption in Debaryomyces hanseniiFEMS YEAST RESEARCH, Issue 1 2009Anupriya Minhas Abstract Debaryomyces hansenii is one of the most osmotolerant and halotolerant yeasts. The molecular mechanisms underlying its extreme osmotolerance and halotolerance have drawn considerable attention in the recent past. However, progress in this regard has been limited due to lack of availability of a transformation system and molecular tools to study the functions of the genes in D. hansenii. Here, we have described the development of an efficient transformation system for D. hansenii that is based on a histidine auxotrophic recipient strain and the DhHIS4 gene as the selectable marker. By screening the D. hansenii genomic library, we have isolated several autonomous replication sequences that can be used for constructing a replicating vector. Moreover, our study is the first to demonstrate gene disruption in D. hansenii by homologous recombination. [source] Improved method for the PCR-based gene disruption in Saccharomyces cerevisiaeFEMS YEAST RESEARCH, Issue 2 2008Hiroshi Koyama Abstract The PCR-based gene disruption strategy originally devised by Baudin et al. is widely used for gene targeting in Saccharomyces cerevisiae. An advantage of this strategy is its simplicity in making targeting constructs. The efficiencies of the targeted disruption are highly variable from locus to locus, however, and often very low. In this report, a method for improving the gene deletion efficiency is described. [source] Use of the TRP1 auxotrophic marker for gene disruption and phenotypic analysis in yeast: a note of warningFEMS YEAST RESEARCH, Issue 1 2008Asier González Abstract The TRP1 marker has been commonly used for gene disruption experiments and subsequent phenotypic analysis. However, introduction of the TRP1 gene into a trp1 strain markedly affects growth under many conditions used for phenotypic profiling. Therefore, its use in the past should be revisited and utilization of this marker should be avoided in future analyses. [source] Novel mechanisms of gene disruption at the medulloblastoma isodicentric 17p11 breakpointGENES, CHROMOSOMES AND CANCER, Issue 2 2009Martin G. McCabe Isodicentric 17q is the most commonly reported chromosomal abnormality in medulloblastomas. Its frequency suggests that genes disrupted in medulloblastoma formation may play a role in tumorigenesis. We have previously identified two chromosome 17 breakpoint at a 1 Mb resolution. Our aims were to accurately map the position of these breakpoints and to identify mechanisms of gene disruption at this site. CGH with a custom tiling path genomic BAC array of chromosome 17 enriched with fosmids at the breakpoint regions was used to analyze a series of 45 medulloblastomas and three medulloblastoma-derived cell lines. In total, 17 of 45 medulloblastomas had an isodicentric 17q. Two breakpoint regions were identified and their positions were mapped. The array identified a more complex arrangement at the breakpoint than has been reported previously using lower resolution BAC arrays. The patterns observed indicated that dicentric chromosome formation occurs both via nonallelic homologous recombination between palindromically arranged low copy repeats (the previously accepted mechanism) and by recombination between nonidentical sequences. In addition, novel alternative structural alterations, a homozygous deletion and a duplication, were identified within the chromosome breakpoint region in two cases. At the resolution of the array, these structural alterations spanned the same genes as cases with dicentric 17q formation, implying that the disruption of genes at the chromosome breakpoint itself may be of greater biological significance than has previously been suspected. © 2008 Wiley-Liss, Inc. [source] A new complex rearrangement involving the ETV6, LOC115548, and MN1 genes in a case of acute myeloid leukemiaGENES, CHROMOSOMES AND CANCER, Issue 3 2004Elena Belloni A new complex rearrangement involving chromosome bands 5q13, 12p13, 22q11, and 3q12 was identified and characterized in a patient with acute myeloid leukemia. Fluorescence in situ hybridization showed the involvement of the ETV6 gene in 12p13. ETV6 primers were specifically designed for 3,- and 5,-RACE-PCR experiments, which led to the identification of the other two rearranged genes. The derivative chromosome 5 harbored a fusion of the ETV6 sequence with that of the LOC115548 gene. The two genes were placed in opposite orientation and did not encode a fusion protein. On the derivative chromosome 12, ETV6 was fused to the MN1 gene on chromosome 22. Also in this case, the insertion, within the MN1 sequence, of a portion of chromosome 3 prevented the formation of a fusion protein. Finally, the derivative chromosome 22 contained the 3, portions of both LOC115548 and MN1, and no fusion transcript with coding potential could be predicted. In conclusion, all chromosome breakpoints led to the truncation of the three involved genes in the absence of predicted fusion proteins. This study lends further support to the hypothesis that gene disruption resulting in either loss of function or haploinsufficiency may be relevant in acute myeloid leukemia pathogenesis. © 2004 Wiley-Liss, Inc. [source] Progress in understanding the biology of the human mutagen LINE-1,,HUMAN MUTATION, Issue 6 2007Daria V. Babushok Abstract Long interspersed nucleotide element (LINE)-1 retrotransposon (L1) has emerged as the largest contributor to mammalian genome mass, responsible for over 35% of the human genome. Differences in the number and activity levels of L1s contribute to interindividual variation in humans, both by affecting an individual's likelihood of acquiring new L1-mediated mutations, as well as by differentially modifying gene expression. Here, we report on recent progress in understanding L1 biology, with a focus on mechanisms of L1-mediated disease. We discuss known details of L1 lifecycle, including L1 structure, transcriptional regulation, and the mechanisms of translation and retrotransposition. Current views on cell type specificity, timing, and control of retrotransposition are put forth. Finally, we discuss the role of L1 as a mutagen, using the latest findings in L1 biology to illuminate molecular mechanisms of L1-mediated gene disruption. Hum Mutat 28(6), 527,539, 2007. Published 2007 Wiley-Liss, Inc. [source] Mouse models in non-alcoholic fatty liver disease and steatohepatitis researchINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 1 2006Quentin M. Anstee Summary Non-alcoholic fatty liver disease (NAFLD) represents a histological spectrum of liver disease associated with obesity, diabetes and insulin resistance that extends from isolated steatosis to steatohepatitis and cirrhosis. As well as being a potential cause of progressive liver disease in its own right, steatosis has been shown to be an important cofactor in the pathogenesis of many other liver diseases. Animal models of NAFLD may be divided into two broad categories: those caused by genetic mutation and those with an acquired phenotype produced by dietary or pharmacological manipulation. The literature contains numerous different mouse models that exhibit histological evidence of hepatic steatosis or, more variably, steatohepatitis; however, few replicate the entire human phenotype. The genetic leptin-deficient (ob/ob) or leptin-resistant (db/db) mouse and the dietary methionine/choline-deficient model are used in the majority of published research. More recently, targeted gene disruption and the use of supra-nutritional diets to induce NAFLD have gained greater prominence as researchers have attempted to bridge the phenotype gap between the available models and the human disease. Using the physiological processes that underlie the pathogenesis and progression of NAFLD as a framework, we review the literature describing currently available mouse models of NAFLD, highlight the strengths and weaknesses of established models and describe the key findings that have furthered the understanding of disease pathogenesis. [source] Possible Roles of Runx1 and Sox9 in Incipient Intramembranous Ossification,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2004Takashi Yamashiro DDS Abstract We evaluated the detailed expression patterns of Runx1 and Sox9 in various types of bone formation, and determined whether Runx1 expression was affected by Runx2 deficiency and Runx2 expression by Runx1 deficiency. Our results indicate that both Runx1 and Sox9 are intensely expressed in the future osteogenic cell compartment and in cartilage. The pattern of Runx1 and Sox9 expression suggests that both genes could potentially be involved in incipient intramembranous bone formation during craniofacial development. Introduction:Runx1, a gene essential for hematopoiesis, contains RUNX binding sites in its promoter region, suggesting possible cross-regulation with Runx2 and potential regulatory roles in bone development. On the other hand, Sox9 is essential for chondrogenesis, and haploinsufficiency of Sox9 leads to premature ossification of the skeletal system. In this study, we studied the possible roles of Runx1 and Sox9 in bone development. Materials and Methods:Runx1, Runx2/Osf2, and Sox9 expression was evaluated by in situ hybridization in the growing craniofacial bones of embryonic day (E)12,16 mice and in the endochondral bone-forming regions of embryonic and postnatal long bones. In addition, we evaluated Runx2/Osf2 expression in the growing face of Runx1 knockout mice at E12.5 and Runx1 expression in Runx2 knockout mice at E14.5. Results:Runx1 and Sox9 were expressed in cartilage, and the regions of expression expanded to the neighboring Runx2 -expressing osteogenic regions. Expression of both Runx1 and Sox9 was markedly downregulated on ossification. Runx1 and Sox9 expression was absent in the regions of endochondral bone formation and in actively modeling or remodeling bone tissues in the long bones as well as in ossified craniofacial bones. Runx2 expression was not affected by gene disruption of Runx1, whereas the expression domains of Runx1 were extended in Runx2,/, mice compared with wildtype mice. Conclusions:Runx1 and Sox9 are specifically expressed in the osteogenic cell compartments in the craniofacial bones and the bone collar of long bones, and this expression is downregulated on terminal differentiation of osteoblasts. Our results suggest that Runx1 may play a role in incipient intramembranous bone formation. [source] Nuclease sensitive element binding protein 1 gene disruption results in early embryonic lethalityJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2006Lin Fan Abstract Nuclease sensitive element binding protein 1 (NSEP1) is a member of the EFIA/NSEP1/YB-1 family of DNA-binding proteins whose members share a cold shock domain; it has also been termed DNA-binding protein B and Y box binding protein-1 because of its recognition of transcriptional regulatory elements. In addition, NSEP1 functions in the translational regulation of renin, ferritin, and interleukin 2 transcripts, and our laboratory has reported that it plays a role in the biosynthesis of selenium-containing proteins. To test the functional importance of NSEP1 in murine embryonic development, we have utilized a clone of ES cells in which the NSEP1 gene had been disrupted by integration of a plasmid gene-trapping vector into the seventh exon. Injection of these cells into C57BL/6 blastocysts resulted in 11 high percentage chimeric mice; crosses to wild type C57BL/6 mice generated 82 F1 agouti mice, indicating germ line transmission of the ES cell clone, but genotyping showed no evidence of the disrupted allele in any of these agouti offspring even though spermatozoa from four of five tested mice contained the targeted allele. Embryos harvested after timed matings of chimeric male mice demonstrated only the wildtype allele in 27 embryos tested at E7.5, E12.5, and E18.5. These results suggest that gene targeting of NSEP1 induces a lethal phenotype in early embryos, due to either haploinsufficiency of NSEP1 or formation of a dominant negative form of the protein. In either case, these data indicate the functional importance of the NSEP1 gene in murine early embryonic development. J. Cell. Biochem. © 2006 Wiley-Liss, Inc. [source] PbSR is synthesized in macrogametocytes and involved in formation of the malaria crystalloidsMOLECULAR MICROBIOLOGY, Issue 6 2008Victoria Carter Summary Crystalloids are transient organelles that form in developing malaria ookinetes and disappear after ookinete-to-oocyst transition. Their origins and functions remain poorly understood. The Plasmodium berghei scavenger receptor-like protein PbSR is essential for mosquito-to-host transmission of the parasite: PbSR knockout parasites produce normal numbers of oocysts that fail to form sporozoites, pointing to a role for PbSR in the oocyst during sporogony. Here, using fluorescent protein tagging and targeted gene disruption, we show that PbSR is synthesized in macrogametocytes, gets targeted to the crystalloids of developing ookinetes and is involved in crystalloid formation. While oocyst sporulation rates of PbSR knockout parasites are highly reduced in parasite-infected mosquitoes, sporulation rates in vitro are not adversely affected, supporting the view that mosquito factors could be involved in the PbSR loss-of-function phenotype. These findings are the first to identify a parasite protein involved with the crystalloid organelle, and suggest a novel protein-trafficking mechanism to deliver PbSR to the oocysts. [source] Studies on the aminopeptidase activities of Porphyromonas gingivalisMOLECULAR ORAL MICROBIOLOGY, Issue 4 2001D. Grenier Porphyromonas gingivalis is an asaccharolytic bacterium that requires nitrogen substrates as carbon and energy sources. The aims of this study were to investigate the aminopeptidase activities of P. gingivalis and to evaluate the effect of aminopeptidase inhibitors on bacterial growth. Only arginine aminopeptidase and dipeptidyl aminopeptidase IV activities were detected. Experimental evidence was obtained suggesting that the Arg-gingipains of P. gingivalis can function as both an endopeptidase and an aminopeptidase. Firstly, the arginine aminopeptidase activity was found to be inhibited by leupeptin, a well-known inhibitor of Arg-gingipain activity. Secondly, a preparation of Arg-gingipain activity could hydrolyze the chromogenic substrate for arginine aminopeptidase. Lastly, a mutant of P. gingivalis constructed via gene disruption by use of suicide plasmids and deficient in both Arg-gingipain A and B was also devoid of arginine aminopeptidase activity. To investigate the key role of aminopeptidase activities in growth of P. gingivalis, aminopeptidase inhibitors were incorporated in the culture medium prior to inoculation. Bestatin and actinonin were the only ones to inhibit growth of P. gingivalis. Their mechanism of growth inhibition appears to be different but does not involve inhibition of the two major aminopeptidase activities (arginine aminopeptidase and dipeptidyl aminopeptidase IV). [source] Involvement of trichothecenes in fusarioses of wheat, barley and maize evaluated by gene disruption of the trichodiene synthase (Tri5) gene in three field isolates of different chemotype and virulenceMOLECULAR PLANT PATHOLOGY, Issue 6 2006FRANK J. MAIER SUMMARY Fusarium graminearum is the main causative agent of Fusarium head blight on small grain cereals and of ear rot on maize. The disease leads to dramatic yield losses and to an accumulation of mycotoxins. The most dominant F. graminearum mycotoxins are the trichothecenes, with deoxynivalenol and nivalenol being the most prevalent derivatives. To investigate the involvement of trichothecenes in the virulence of the pathogen, the gene coding for the initial enzyme of the trichothecene pathway was disrupted in three field isolates, differing in chemotype and in virulence. From each isolate three individual disruption mutants were tested for their virulence on wheat, barley and maize. Despite the different initial virulence of the three wild-type progenitor strains on wheat, all disruption mutants caused disease symptoms on the inoculated spikelet, but the symptoms did not spread into other spikelets. On barley, the trichothecene deficient mutants showed no significant difference compared to the wild-type strains: all were equally aggressive. On maize, mutants derived from the NIV-producing strain caused less disease than their wild-type progenitor strain, while mutants derived from DON-producing strains caused the same level of disease as their progenitor strains. These data demonstrate that trichothecenes influence the virulence of F. graminearum in a highly complex manner, which is strongly host as well as moderately chemotype specific. [source] Importance of forkhead transcription factor Fkhl18 for development of testicular vasculatureMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2008Yuko Sato Abstract Forkhead transcription factors are characterized by a winged helix DNA binding domain, and the members of this family are classified into 20 subclasses by phylogenetic analyses. Fkhl18 is structurally unique, and is classified into FoxS subfamily. We found Fkhl18 expression in periendothelial cells of the developing mouse fetal testis. In an attempt to clarify its function, we generated mice with Fkhl18 gene disruption. Although KO mice developed normally and were fertile in both sexes, we frequently noticed unusual blood accumulation in the fetal testis. Electron microscopic analysis demonstrated frequent gaps, measuring 100,400 nm, in endothelial cells of blood vessels. These gaps probably represented ectopic apoptosis of testicular periendothelial cells, identified by caspase-3 expression, in KO fetuses. No apoptosis of endothelial cells was noted. Fkhl18 suppressed the transcriptional activity of FoxO3a and FoxO4. Considering that Fas ligand gene expression is activated by Foxs, the elevated activity of Foxs in the absence of Fkhl18 probably explains the marked apoptosis of periendothelial cells in Fkhl18 KO mice. Mol. Reprod. Dev. 75: 1361,1371, 2008. © 2008 Wiley-Liss, Inc. [source] Novel epididymis-specific mRNAs downregulated by HE6/Gpr64 receptor gene disruptionMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 5 2007Ben Davies Abstract Targeted disruption of the epididymis-specific HE6/Gpr64 receptor gene in mice led to male infertility. In order to characterize the phenotype at a molecular level, we compared the gene expression patterns of wild type (wt) versus knockout (KO) caput epididymides. The caput region of KO males, although morphologically normal, nevertheless showed an aberrant expression pattern. Combining micro array analysis, differential library screening, Northern blot analysis and quantitative RT-PCR, we found that the knockout of the HE6/Gpr64 receptor was mainly associated with the downregulation of genes specific to the initial segment. The list of KO downregulated transcripts comprised Enpp2/autotaxin, the lipocalins 8 and 9, the ,-defensin Defb42, cystatins 8 and 12, as well as the membrane proteins Adam (A Disintegrin And Metalloprotease) 28, claudin-10, EAAC1, and the novel Me9. Clusterin/ApoJ and osteopontin/Spp1 mRNAs, on the other hand, were upregulated in the KO tissues. The Me9 transcript was studied in further detail, and we report here a cluster of related epididymis-specific genes. Me9 is specifically expressed in the initial segment and is representative of a novel and highly conserved mammalian gene family. The family consists of single-exon genes only; intron-containing paralogs have not yet been ascertained. The cloned cDNA sequences predicted hydrophobic polytopic membrane proteins containing the DUF716 motif. Protein expression was shown in the rodent caput epididymidis but remained uncertain in primates. Mol. Reprod. Dev. 74: 539,553, 2007. © 2006 Wiley-Liss, Inc. [source] Zeocin resistance as a dominant selective marker for transformation and targeted gene deletions in Candida glabrataMYCOSES, Issue 6 2006Alex J. Alderton Summary Many of the genetic tools used to generate gene knockouts in Candida glabrata exploit auxotrophic markers but this is not suitable for use with clinical strains. Antibiotic resistance markers, however, allow one to target genes to be deleted without any prior genetic manipulation of clinical isolates. Such antibiotic selection markers have been widely reported for the manipulation of Saccharomyces cerevisiae. However, very few antibiotic resistance markers have been shown to be useful in C. glabrata. Here, we report the use of Zeocin resistance (ZeoR), encoded by the ble gene from Streptoalloteichus hindustanus, as a new positive selection marker for the genetic manipulation of C. glabrata including clinical strains that we show are significantly more sensitive to Zeocin than to G418. The potential of the ZeoR marker for targeted gene disruption in C. glabrata was confirmed by constructing deletions of the ADE2 in both a laboratory and a clinical strain of C. glabrata, using both short (90 bp) and long (400 bp) homology cassettes. [source] BCR gene disruption in a pilomyxoid astrocytomaNEUROPATHOLOGY, Issue 5 2006Bárbara Meléndez We report here a 4-month-old child with a large, solid enhancing mass involving predominantly the suprasellar and diencephalic regions, with extension of both hemispheres. The patient underwent partial resection of the mass by right temporal craniotomy. Histological diagnosis was of a low-grade glioma consistent with pilomyxoid astrocytoma. Cytogenetic analyses revealed an insertion on chromosome 17 that involved disruption of the BCR gene. This finding suggests a possible rearrangement of this gene that could act in a similar way to chronic myeloid leukemia with formation of a chimeric tyrosine kinase protein. This study may suggest the use of inhibitors of tyrosine kinase proteins as an alternative treatment approach in cases of refractory or disseminated pilocytic astrocytomas. [source] From malformations to molecular mechanisms in the male: three decades of research on endocrine disrupters,APMIS, Issue 4 2001John A. McLachlan For three decades, we have known that estrogens alter the development of the mammalian reproductive system in predictable ways. In mice exposed prenatally to diethylstilbestrol (DES) or other estrogens, the male offspring exhibit structural malformations including cryptorchidism, epididymal cysts and retained Mullerian ducts. The estrogen-associated alterations in the genital tract phenotype can be usefully considered as a model called Developmental Estrogenization Syndrome. While estrogen treatment during critical periods of morphogenesis of the male reproductive system has been associated with these changes, the mechanisms at the molecular level are still being discovered. Parallel findings on the hormones involved in Mullerian duct regression and testicular descent have helped guide research on the mechanisms of developmental estrogenization of the male. Cellular localization of molecular signals associated with key steps in genital tract development, use of mice with gene disruption, and knowledge of the mechanisms underlying persistent changes in gene expression are beginning to provide a blue print for both the physiological role and pathological effects of estrogens in reproductive tract development. Since many of the same biological principles underlie genital tract morphogenesis in mammals, one may expect some of the same changes in males of other species exposed to estrogen during the appropriate developmental periods. [source] Rac1 and Rac2 GTPases in haematopoiesisBIOESSAYS, Issue 3 2004Victoria J. Weston The highly homologous Rac1 and Rac2 GTPases are co-expressed in cells of haematopoietic origin and are likely to show some functional redundancy. While disruption of the Rac2 gene in mice has provided insight into some of its functions, Rac1 null mice are embryonic lethal and only recently has conditional gene disruption been possible. Consequently, two articles1,2 have recently elucidated some overlapping and unique key roles of Rac1 and Rac2 in haematopoietic processes including specialized roles in innate and humoral immunity. BioEssays 26:221,224, 2004. © 2004 Wiley Periodicals, Inc. [source] BAK and BAX deletion using zinc-finger nucleases yields apoptosis-resistant CHO cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Gregory J. Cost Abstract Anoxic and metabolic stresses in large-scale cell culture during biopharmaceutical production can induce apoptosis. Strategies designed to ameliorate the problem of apoptosis in cell culture have focused on mRNA knockdown of pro-apoptotic proteins and over-expression of anti-apoptotic ones. Apoptosis in cell culture involves mitochondrial permeabilization by the pro-apoptotic Bak and Bax proteins; activity of either protein is sufficient to permit apoptosis. We demonstrate here the complete and permanent elimination of both the Bak and Bax proteins in combination in Chinese hamster ovary (CHO) cells using zinc-finger nuclease-mediated gene disruption. Zinc-finger nuclease cleavage of BAX and BAK followed by inaccurate DNA repair resulted in knockout of both genes. Cells lacking Bax and Bak grow normally but fail to activate caspases in response to apoptotic stimuli. When grown using scale-down systems under conditions that mimic growth in large-scale bioreactors they are significantly more resistant to apoptosis induced by starvation, staurosporine, and sodium butyrate. When grown under starvation conditions, BAX - and BAK -deleted cells produce two- to fivefold more IgG than wild-type CHO cells. Under normal growth conditions in suspension culture in shake flasks, double-knockout cultures achieve equal or higher cell densities than unmodified wild-type cultures and reach viable cell densities relevant for large-scale industrial protein production. Biotechnol. Bioeng. 2010; 105: 330,340. © 2009 Wiley Periodicals, Inc. [source] Microbial aldo-keto reductasesFEMS MICROBIOLOGY LETTERS, Issue 2 2002Elizabeth M Ellis Abstract The aldo-keto reductases (AKR) are a superfamily of enzymes with diverse functions in the reduction of aldehydes and ketones. AKR enzymes are found in a wide range of microorganisms, and many open reading frames encoding related putative enzymes have been identified through genome sequencing projects. Established microbial members of the superfamily include the xylose reductases, 2,5-diketo- d -gluconic acid reductases and ,-keto ester reductases. The AKR enzymes share a common (,/,)8 structure, and conserved catalytic mechanism, although there is considerable variation in the substrate-binding pocket. The physiological function of many of these enzymes is unknown, but a variety of methods including gene disruptions, heterologous expression systems and expression profiling are being employed to deduce the roles of these enzymes in cell metabolism. Several microbial AKR are already being exploited in biotransformation reactions and there is potential for other novel members of this important superfamily to be identified, studied and utilized in this way. [source] Identification of gene disruptions for increased poly-3-hydroxybutyrate accumulation in Synechocystis PCC 6803BIOTECHNOLOGY PROGRESS, Issue 5 2009Keith E. J. Tyo Abstract Inverse metabolic engineering (IME) is a combinatorial approach for identifying genotypes associated with a particular phenotype of interest. In this study, gene disruptions that increase the biosynthesis of poly-3-hydroxybutyrate (PHB) in the photosynthetic bacterium Synechocystis PCC6803 were identified. A Synechocystis mutant library was constructed by homologous recombination between the Synechocystis genome and a mutagenized genomic plasmid library generated through transposon insertion. Using a fluorescence-activated cell sorting-based high throughput screen, high PHB accumulating mutants from the library grown in different nutrient conditions were isolated and characterized. While several mutants isolated from the screen had increased PHB accumulation, transposon insertions in only two ORFs could be linked to increased PHB production. Disruptions of sll0461, coding for gamma-glutamyl phosphate reductase (proA), and sll0565, a hypothetical protein, resulted in increased accumulation in standard growth media and acetate supplemented media. These genetic perturbations have increased PHB accumulation in Synechocystis and serve as markers for engineering increased polymer production in higher photosynthetic organisms. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] New Approaches for Validation of Lethal Phenotypes and Genetic Reversion in Helicobacter pyloriHELICOBACTER, Issue 1 2001Timothy K. McDaniel Background. Because of limited genetic tools for use in Helicobacter pylori, tests routinely applied in other bacteria for demonstrating a gene's role in viability and other phenotypes have not been applied to this organism. In a mutational study of putative response regulator genes, we aimed to develop such tools for H. pylori. Materials and Methods. We attempted to mutate five response regulator genes by allelic exchange insertional mutagenesis. For genes that yielded no viable mutants, a second copy of the gene was inserted into the chromosome via a suicide vector, and it was seen if providing the second copy would permit the gene's disruption. For genes that yielded mutants with selectable phenotypes, a strategy was developed for reversion whereby an intact copy of the gene is introduced to the organism by transformation with PCR products. Following this procedure, revertants were selected by phenotypic tests then tested for genetic reversion. Results. After failure to attain transformants upon attempted mutation of genes HP0166 and HP1365, we inserted a second copy of each gene within the H. pylori chromosome. In each case the second copy relieved the block of transformation. Mutation of genes HP0703 and HP1021 gave non-motile and small-colony phenotypes, respectively. Following transformation with PCR products containing intact copies of the genes, both phenotype and genotype had reverted following phenotypic selections. Conclusions. The methods used in this study provide new approaches for confirming suspected genotype/phenotype associations and should be widely applicable in the study of H. pylori. [source] | |||||||||||||||||||||||||||||||