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DNA Fragments (dna + fragment)
Kinds of DNA Fragments Selected AbstractsDetection of bacterial DNA by PCR and reverse hybridization in the 16S rRNA gene with particular reference to neonatal septicemiaACTA PAEDIATRICA, Issue 2 2001S Shang Aim: The clinical diagnosis of sepsis is difficult, particularly in neonates. It is necessary to develop a rapid and reliable method for detecting bacteria in blood and cerebrospinal fluid (CSF) Polymerase chain reaction (PCR) and reverse hybridization of the 16S rRNA gene would permit fast and sensitive determination of the presence of bacteria and differentiate gram-positive bacteria from gram-negative ones in clinical specimens. Methods: We developed a pair of primers according to the gene encoding 16SrRNA found in all bacteria. DNA fragments from different bacterial species and from clinical samples were detected with PCR, and with reverse hybridization using a universal bacterial probe, a gram-positive probe and a gram-negative probe. Results: A 371 bp DNA fragment was amplified from 20 different bacterial species. No signal was observed when human DNA and viruses were used as templates. The sensitivity could be improved to 10T -12 g. All 26 culture-positive clinical samples (22 blood samples and 4 CSF samples) were positive with PCR. The gram-negative and gram-positive probes hybridized to clinical samples and to known bacterial controls, as predicted by Gram's stain characteristics. Conclusions: Our results suggest that the method of PCR and reverse hybridization is rapid, sensitive and specific in detecting bacterial infections. This finding may be significant in the clinical diagnosis of sepsis in neonates. [source] DNA sequence motifs conserved in endocrine promoters are essential for Pax4 expressionDEVELOPMENTAL DYNAMICS, Issue 4 2003Christopher Brink Abstract The paired box transcription factor Pax4 is required for maturation of insulin-producing ,- and somatostatin secreting ,-cells in the murine pancreas. It starts to be expressed in pancreatic precursors and later is restricted to ,- and ,-cells to finally be switched off after birth. A 0.9-kb genomic DNA fragment has been shown to mediate the Pax4 expression pattern. Transcription factors Pdx1 and NeuroD bind to this fragment at A2- and E1-sequence motifs. In this study, we downscale the size of this fragment to 409 bp. Another genomic fragment of 254 bp is still able to mediate the specificity, but not the strength of Pax4 expression. Deletion of the A2 and E1 elements results in loss or weakening of reporter gene expression. Because A2 and E1 elements are conserved in numerous pancreatic promoters, they might play a general role in regulating endocrine gene expression. Developmental Dynamics 228:617,622, 2003. © 2003 Wiley-Liss, Inc. [source] Selective expression of the small GTPase RhoB in the early developing mouse lensDEVELOPMENTAL DYNAMICS, Issue 3 2001Rupalatha Maddala Abstract This report describes the expression and distribution pattern of RhoB GTPase in the developing mouse lens. RhoB expression was confirmed by sequencing an reverse transcriptase-polymerase chain reaction,generated DNA fragment of RhoB. Immunohistochemical analysis of RhoB revealed expression in the lens vesicle (both anterior and posterior vesicle) at embryonic day (E) 11.5, and in the epithelium and primary fibers of the E14.5 lens. Compared with the neonatal stage (day 1), where RhoB is detected in the entire lens (epithelium, primary, and secondary fibers), expression of this protein is restricted to the epithelial and outer cortical secondary fibers in postnatal lenses (from day 7 to day18). Interestingly, in E11.5 and E14.5 lenses, RhoB is localized predominantly in the lens, but not detectable in the retina, cornea, or other ocular tissues. RhoB expression appears to be down-regulated in the postnatal lens with concomitant up-regulation in the retina and cornea, compared with earlier stages of development (eyes of E11.5, E14.5, and neonatal mice). This study reveals the selective expression of RhoB in the lens during early eye development and suggests a potential role for this small GTPase in cytoskeletal reorganization associated with lens epithelial cell elongation and differentiation. © 2001 Wiley-Liss, Inc. [source] Efficient and sensitive method of DNA silver staining in polyacrylamide gelsELECTROPHORESIS, Issue 1 2005Lujiang Qu Abstract DNA silver staining is widely used to detect DNA fragment in polyacrylamide gel with high sensitivity. Conventional procedures of the silver staining involve several steps, which take about 40 min to 2 h in total. To improve the efficiency of DNA silver staining, a more efficient protocol is developed in this study. The procedure comprises only four steps including impregnating, rinsing, developing, and stopping, and could be completed within 20 min. Nitric acid and ethanol in the silver-impregnation step of the new procedure eliminates the need for prior treatment of gels with a fixing solution and following rinse prior to impregnation with silver. The procedure has high sensitivity and long storage lifetime. The minimum detectable mass of DNA is 0.44 and 3.5 ng in denaturing and nondenaturing polyacrylamide gel, respectively. [source] Assigning Escherichia coli strains to phylogenetic groups: multi-locus sequence typing versus the PCR triplex methodENVIRONMENTAL MICROBIOLOGY, Issue 10 2008David M. Gordon Summary It is well recognized that Escherichia coli consists of a number of distinct phylo-groups and that strains of the different phylo-groups vary in their ecological niches, life-history characteristics and propensity to cause disease. Consequently, much can be learnt by assigning a strain of E. coli to one of the recognized phylo-groups. A triplex PCR-based method that enables strains of E. coli to be assigned to a phylo-group using a dichotomous key approach based on the presence or absence of two genes (chuA and yjaA) and an anonymous DNA fragment (TSPE4.C2) has been developed. However, the accuracy with which this method assigns strains to their correct phylo-group has not been adequately evaluated. Consequently, 662 strains of E. coli were characterized using a multi-locus sequence typing approach. Unsupervised population assignment algorithms were used to assign strains to phylo-groups based on the multi-locus sequence typing data. The analyses revealed that 85,90% of E. coli strains can be assigned to a phylo-group and that 80,85% of the phylo-group memberships assigned using the Clermont method are correct. However, the accuracy with which strains are assigned to the correct phylo-group depends on their Clermont genotype. For example, strains yielding a Clermont genotype consistent with phylo-groups B1 and B2 are assigned correctly 95% of the time. Strains failing to yield any PCR products using the Clermont method are seldom members of phylo-group A and strains with such a genotype should not be assigned to a phylo-group. [source] Identification and characterization of a novel transcriptional regulator, MatR, for malonate metabolism in Rhizobium leguminosarum bv. trifoliiFEBS JOURNAL, Issue 24 2000Hwan Young Lee A novel gene, matR, located upstream of matABC, transcribed in the opposite direction, and encoding a putative regulatory protein by sequence analysis was discovered from Rhizobium leguminosarum bv. trifolii. The matA, matB, and matC genes encode malonyl-CoA decarboxylase, malonyl-CoA synthetase, and a presumed malonate transporter, respectively. Together, these enzymes catalyze the uptake and conversion of malonate to acetyl-CoA. The deduced amino-acid sequence of matR showed sequence similarity with GntR from Bacillus subtilis in the N-terminal region encoding a helix-turn-helix domain. Electrophoretic mobility shift assay indicated that MatR bound to a fragment of DNA corresponding to the mat promoter region. The addition of malonate or methylmalonate increased the association of MatR and DNA fragment. DNase I footprinting assays identified a MatR binding site encompassing 66 nucleotides near the mat promoter. The mat operator region included an inverted repeat (TCTTGTA/TACACGA) centered ,46.5 relative to the transcription start site. Transcriptional assays, using the luciferase gene, revealed that MatR represses transcription from the mat promoter and malonate alleviates MatR-mediated repression effect on the expression of Pmat -luc+ reporter fusion. [source] Mice protected by oral immunization with Lactobacillus reuteri secreting fusion protein of Escherichia coli enterotoxin subunit proteinFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2007Chi-Ming Wu Abstract A green fluorescent protein (gfp) gene was ligated to the Lactobacillus reuteri -specific nisin-inducible expression-secretion vector pNIES, generating a pNIES-GFP vector capable of secreting the cloned gene as a GFP-fusion protein with fluorescent activity. To develop this system as a live vehicle carrying the heat-stable enterotoxin (ST) and heat-labile enterotoxin B (LTB) of the enterotoxigenic Escherichia coli (ETEC), a recombinant 5,- ST - LTB -3, DNA fragment was cloned into pNIES-GFP. The resulting L. reuteri/pNIES-GFP:STLTB system was found to possess the capability of adhering to the mice gut, secreting GFP:STLTB product at 0.14 and 0.026 pgcell,1 under induced and noninduced conditions, respectively. Further analysis of the GFP:STLTB product confirmed its ganglioside-binding ability, LTB antigenicity and relative freedom from the ST-associated toxicity, making it suitable for use as an oral vaccine in mice. Oral inoculation of the L. reuteri/pNIES-GFP:STLTB culture in mice elicited significant (P<0.01) serum IgG and mucosal IgA antibodies against the STLTB antigen. These immunized mice were subsequently challenged with ETEC and showed full protection against the fluid influx response in the gut. This is the first report of using L. reuteri as a vaccine carrier to induce complete immunologic protection against ETEC. [source] Direct genotyping of the poplar leaf rust fungus, Melampsora medusae f. sp. deltoidae, using codominant PCR-SSCP markersFOREST PATHOLOGY, Issue 4 2005M. Bourassa Summary Two anonymous DNA markers that are revealed by single-strand conformational polymorphism (SSCP) analysis were developed for detection of polymorphisms in Melampsora medusae f. sp. deltoidae (Mmd). Mono-uredinial isolates of Mmd were first obtained, DNA was extracted from urediniospores and random amplified polymorphic DNA (RAPD) products of eight mono-uredinial isolates were separated on a SSCP gel to identify differences among them. Bands representing putative polymorphic loci among the eight isolates tested were excised from the SSCP gel and re-amplified by polymerase chain reaction (PCR), and then cloned and sequenced. A primer pair was designed to amplify a DNA fragment of a size suitable for SSCP analysis (<600 bp) for two out of three DNA fragments sequenced. Each set of primers amplified a PCR product for all eight isolates that were initially used to generate them and the resulting PCR products were analysed by SSCP. Polymorphisms among isolates were identified for both putative loci. The two primer pairs amplified a PCR product of the expected size on an additional 32 mono-uredinial isolates of Mmd tested. From the overall 40 mono-uredinial isolates tested, 5 and 11 alleles were detected, and 12 and 34 isolates showed to be heterozygous, as indicated by the presence of more than two bands on the SSCP gel, at loci A and B, respectively. The primer pairs were tested for specificity against 106 fungal isolates belonging to various taxa, including other rusts, and against DNA extracted from greenhouse-grown healthy poplar leaves. DNA amplification products of the expected size were obtained only when Mmd DNA was present. Optimization of PCR conditions with these two primer pairs allowed genotyping directly from single uredinia extracted from infected leaves, thus alleviating the need to culture the fungus to characterize individuals, hence making it possible to process large numbers of samples for population studies. Résumé Deux marqueurs génétiques anonymes, révélés par analyse SSCP (Single-Strand Conformational Polymorphism) ont été développés afin de détecter des polymorphismes génétiques chez le Melampsora medusae f. sp. deltoidae (Mmd). Dans un premier temps, des isolats mono-urédiniaux ont été obtenus, puis l'ADN a été extrait à partir des urédiniospores, les produits d'amplification RAPD (Random Amplified Polymorphic DNA) ont été générés à partir de huit de ces isolats mono-urédiniaux et les résultats d'amplification ont par la suite été séparés sur gel SSCP afin d'identifier des polymorphismes entre les isolats. Les bandes sur gel SSCP représentant des loci polymorphiques putatifs entre les isolats ont été prélevées du gel, ré-amplifiées par la technique d'amplification PCR (Polymerase Chain Reaction), clonées, puis séquencées. Pour deux fragments d'ADN séquencés sur un total de trois, une paire d'amorces a été développée afin de permettre l'amplification d'un fragment de taille adéquate pour analyse SSCP (<600 pb). Chaque paire d'amorces a produit un signal d'amplification positif pour chacun des huit isolats à l'origine de ces nouvelles amorces; les produits PCR ont ensuite été analysés par la technique SSCP. Les deux loci putatifs ont révélé des polymorphismes génétiques entre les isolats. Les deux paires d'amorces ont produit un fragment d'amplification de la taille attendue pour chacun des 32 isolats mono-urédiniaux supplémentaires testés. Des 40 isolats testés, 5 et 11 allèles ont été détectés, alors que 12 et 34 isolats se sont révélés hétérozygotes (tel qu'indiqué par la présence de plus de deux bandes sur gel SSCP) pour les loci A et B, respectivement. La spécificité des deux paires d'amorces a été testée à partir de 106 isolats fongiques appartenant à différents groupes taxonomiques, incluant d'autres rouilles, de même qu'à partir de l'ADN extrait de feuilles de peupliers cultivés en serre. Un signal d'amplification positif n'a été obtenu qu'en présence d'ADN du Mmd. Les conditions d'amplification PCR ont été optimisées pour les deux paires d'amorces développées afin de permettre le génotypage directement à partir d'urédinies individuelles prélevées sur des feuilles de peuplier infectées. La possibilité de génotyper directement des urédinies individuelles permet d'éviter l'obligation de cultiver le champignon pour génotyper les individus, ce qui représente un avantage important des marqueurs génétiques développés ici, puisqu'il devient dès lors possible de traiter un grand nombre d'échantillons lors de la réalisation d'études de populations. Zusammenfassung Zum Nachweis von Polymorphismen bei Melampsora medusae f. sp. deltoidae wurden zwei anonyme DNA Marker aus einer SSCP-Analyse entwickelt. Zunächst wurden Isolate aus einzelnen Uredinien gewonnen, die DNA wurde aus den Uredosporen extrahiert und polymorphe RAPD, Amplifikationsprodukte von acht Mono-Uredinium-Isolaten wurden auf einem SSCP-Gel getrennt, um Unterschiede zwischen ihnen nachzuweisen. Banden, die bei den acht geprüften Isolaten mögliche polymorphe Loci darstellten, wurden aus dem SSCP-Gel ausgeschnitten und mit PCR reamplifiziert, dann geklont und sequenziert. Für zwei von insgesamt drei sequenzierten DNA-Fragmenten wurde ein Primerpaar entwickelt, um ein in der Grösse für die SSCP-Analyse (<600 bp) geeignetes DNA-Fragment zu amplifizieren. Jedes Primerpaar amplifizierte bei allen acht ursprünglich für ihre Entwicklung verwendeten Isolaten ein PCR-Produkt, und diese wurden anschliessend mit SSCP analysiert. Für beide putativen Loci wurden bei den Isolaten Polymorphismen festgestellt. Die beiden Primerpaare amplifizierten ein PCR-Produkt der erwarteten Grösse bei allen 32 zusätzlich geprüften Mono-Uredinium-Isolaten des Pilzes. Bei den insgesamt 40 geprüften Mono-Uredinium-Isolaten wurden für die Loci A und B 5 bzw. 11 Allele gefunden, und 12 bzw. 34 Isolate erwiesen sich als heterozygot, was durch mehr als zwei Banden auf den SSCP-Gelen angezeigt wurde. Die Spezifität der Primerpaare wurden mit 106 Pilzisolaten aus verschiedenen Taxa geprüft, darunter andere Roste sowie DNA aus gesunden Pappelblättern aus Gewächshauskulturen. DNA-Amplifikationsprodukte der erwarteten Grösse wurden nur erhalten, wenn DNA von Melampsora medusae f. sp. deltoidae präsent war. Die PCR-Amplifikations-Bedingungen mit diesen beiden Primerpaaren wurde so optimiert, dass ein Genotyping direkt bei einzelnen von infizierten Blättern entnommenen Uredinien erfolgen kann und somit eine Pilzkultur zur Charakterisierung von Individuen entfällt. Dies ermöglicht grosse Probenzahlen in Populationsstudien. [source] Chromatin immunoprecipitation-mediated target identification proved aquaporin 5 is regulated directly by estrogen in the uterusGENES TO CELLS, Issue 10 2006Mika Kobayashi Estrogens play a central role in the reproduction of vertebrates and affect a variety of biological processes. The major target molecules of estrogens are nuclear estrogen receptors (ERs), which have been studied extensively at the molecular level. In contrast, our knowledge of the genes that are regulated directly by ERs remains limited, especially at the level of the whole organism rather than cultured cells. In order to identify genes that are regulated directly by ERs in vivo, we used estrogen treated mouse uterus and performed chromatin immunoprecipitation. Sequence analysis of a precipitated DNA fragment enabled alignment with the mouse genomic sequence and revealed that the promoter region of the gene encoding aquaporin 5 (AQP5) was precipitated with antibody against ER,. Quantitative PCR and DNA microarray analyses confirmed that AQP5 is activated soon after administration of estrogen. In addition, the promoter region of AQP5 contained a functional estrogen response element that was activated directly by estrogen. Although several AQP genes are expressed in the uterus, only direct activation of AQP5 could be detected following treatment with estrogen. This chromatin immunopreciptation-mediated target identification may be applicable to the study of other transcription factor networks. [source] Determination of haplotypes from single DNA molecules: a method for single-molecule barcoding,,HUMAN MUTATION, Issue 9 2007Ming Xiao Abstract Determining the haplotypes in a diploid individual is a major technical challenge in genetic studies of human complex traits. Here we report a method of molecular haplotyping by directly imaging multiple polymorphic sites on individual DNA molecules simultaneously. DNA fragments amplified by long-range PCR were labeled with fluorescent dyes at each polymorphic site using a modified gap-filled padlock probe ligation approach. The labeled DNA molecules were then stretched into linear form on a functionalized glass surface and imaged with multicolor total internal reflection fluorescence (TIRF) microscopy. By determining the colors and positions of the fluorescent labels with respect to the backbone at polymorphic sites, the haplotype can be inferred accurately, in a manner similar to reading a barcode, even when the DNA fragments are not fully labeled. The feasibility of this technology is demonstrated by the determination of the haplotype of a 9.3-kbp DNA fragment containing four SNPs. Hum Mutat 28(9), 913,921, 2007. Published 2007 Wiley-Liss, Inc. [source] An interferon-sensitive response element is involved in constitutive caspase-8 gene expression in neuroblastoma cellsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2007Alessandro De Ambrosis Abstract We previously identified a 1.2 Kb DNA element (P-1161/+16), 5, to caspase-8 exon-1, that acts as promoter in caspase-8-positive, but not in caspase-8-negative neuroblastoma (NB) cells. The P-1161/+16 DNA element regulates both constitutive and interferon IFN-,-inducible caspase-8 expression. Two GAS (IFN-activated sequence, STAT-1 binding site) and two ISRE (interferon sensitive response element, IRF binding site) were present in P-1161/+16. Deletion studies indicated that elements essential for promoter activity in NB cells were present in a 167 bp region 5, flanking exon-1 (P-151/+16), which contains an ISRE at position ,32. The transcription initiation site was mapped by 5, rapid amplification of cDNA end (RACE) at position ,20 from caspase-8 cDNA reference sequence. Disruption of the ISRE-32 indicated that it is required for both constitutive and IFN-,-inducible caspase-8 expression. IRF-1 and IRF-2 transcription factors bind to the (,151/+16) DNA fragment in vitro. Chromatin immunoprecipitation (ChIP) assays showed that IRF-1 and IRF-2 bind to the DNA region at the 5, of caspase-8 gene in NB cells, which show constitutive expression but not in caspase-8 negative cells. In these last cells, up-regulation of caspase-8 by IFN-, was associated to induction of IRF-1 and IRF-2 binding to caspase-8 promoter and increased histone acetylation. Moreover, RNA interference experiments also supported the involvement of IRF-1 and IRF-2 in constitutive caspase-8 expression in NB cells. © 2006 Wiley-Liss, Inc. [source] Elevated expression of bisecting N -acetylglucosaminyltransferase-III gene in a human fetal hepatocyte cell line by hepatitis B virusJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 12 2004JAE-KYOUNG SHIM Abstract Background and Aim:, UDP-N-acetylglucosamine: ,-D-mannoside ,-1,4 N-acetylglucosaminyltransferase III (GnT-III) is a key enzyme in N-glycan biosytnesis. Human GnT-III enzyme activity was found to be elevated in the serum of patients with hepatomas and liver cirrhosis and in hepatocellular carcinoma tissues. Therefore, to understand the relationship between the elevation in GnT-III activity and hepatitis B viral (HBV) hepartocarcinogenesis, we investigated GnT-III gene expression in the HBV-infected cells. Methods:, A cell line, HFH-T1, producing HBV was produced by natural infection of human fetal hepatocytes. A 170-bp band corresponding to the pre-S1 region of HBV was detected in the culture medium by polymerase chain reaction. Virions were also isolated from the culture medium by sucrose density gradient centrifugation. The synthesis of both ,-fetoprotein and albumin as an indicator that these cells were functional hepatocytes and the extent of differentiation was examined. Polymerase chain reaction and Western blot analysis using a monoclonal antibody, GT273, which was prepared using human aglycosyl recombinant GnT-III were used for HBV DNA and GnT-III detection. Results:, Two types of HBV-related particles were secreted into the culture medium; one was a Dane particle (40 nm in size) containing HBV DNA and the other was a subviral hepatitis B surface antigen particle (20 nm in size) that did not contain the viral genome. The secretion from the cell line was diminished by the number of passages and, thus, this cell was renamed as HFH-T2. A decreased level of the HBV was secreted from the cells after a rest period. HFH-T2 cells showed a weak staining for ,-fetoprotein and a moderate staining for albumin in the cytoplasm around the nucleus. High levels of a 0.7 kb DNA fragment originating from GnT-III DNA were detected in HFH-T2 cells. Western blot analysis using a monoclonal antibody, GT273, whixh was prepared using human aglycosyl recombinant GnT-III showed a single band, corresponding to Mr 63 kDa, whereas aglycosyl GnT-III showed a band at Mr 53 kDa, with a molecular weight difference of about 10 kDa. This indicates that HFH-T2 cells express glycosylated GnT-III. GnT-III activities were 347.2 ± 53.6 pmol/mg of protein/h in HFH-T2, 276 ± 26.3 in Hep3B, 252.5 ± 23.3 in HepG2 and 30.7 ± 3.4 in NIH-3T3. GnT-III activity was higher in HFH-T2 cells than in the hepatoma cell lines, Hep3B and HepG2. Conclusion:, A human fetal hepatocyte cell line was transformed by infection with HBV and the cell line expressed high levels of GnT-III as the levels of secretion of HBV decreased. The decrease in HBV secretion from HFH-T2 cells could be due to a high level of expression of GnT-III. Such a cell line could be used to investigate relationships between HBV infection and glycosyltransferase gene expression. Furthermore, this cell line will be useful in future studies on the effect of the expression of GnT-III on other glycosyltransferase. [source] Structure of the Mouse Glutamate Decarboxylase 65 Gene and Its PromoterJOURNAL OF NEUROCHEMISTRY, Issue 4 2000Preferential Expression of Its Promoter in the GABAergic Neurons of Transgenic Mice Abstract: GABA is synthesized by glutamate decarboxylase (GAD), which has two forms, GAD65 and GAD67. To elucidate the molecular mechanisms of mouse GAD65 (mGAD65) gene expression, we isolated and characterized the mGAD65 gene. The mGAD65 gene was found to be divided into 16 exons and spread over 75 kb. The sequence of the first exon and the 5,-flanking region indicated the presence of potential neuron-specific cis -regulatory elements. We used transgenic mice to examine the expression pattern conferred by a 9.2-kb promoter-proximal DNA fragment of the mGAD65 gene fused to the bacterial lacZ reporter gene. Transgenic mice showed high ,-galactosidase activity specifically in brain and testis. They also showed characteristic patterns of transgene expression in olfactory bulb, cerebellar cortex, and spinal cord, a similar expression pattern to that of endogenous mGAD65. However, no transgene expression was observed in the ventral thalamus or hypothalamus, in which high mGAD65 gene expression levels have been observed. These results suggest that the 9.2-kb DNA fragment of the mGAD65 gene is associated with its tissue-specific expression and its targeted expression in GABAergic neurons of specific brain regions but that additional regulatory elements are necessary to obtain fully correct expression. [source] Oral malodorous compound causes apoptosis and genomic DNA damage in human gingival fibroblastsJOURNAL OF PERIODONTAL RESEARCH, Issue 4 2008K. Yaegaki Background and Objective:, Volatile sulfur compounds are the main cause of halitosis. Hydrogen sulfide is one of these volatile sulfur compounds and the principal malodorous compound in physiological halitosis. Periodontally pathogenic activities of hydrogen sulfide have been previously reported. Hydrogen sulfide induces apoptotic cell death in aorta smooth muscle cells and in other tissues. Apoptosis plays an important role in the onset and progress of periodontitis. The objective of this study was to determine whether hydrogen sulfide causes apoptosis in human gingival fibroblasts. Material and methods:, Necrotic cells were detected using a lactate dehydrogenase assay. Apoptosis was ascertained using a histone-complexed DNA fragment assay and flow cytometry. The level of caspase 3, a key enzyme in apoptotic signaling, was also measured, and the effects of hydrogen sulfide on reactive oxygen species and superoxide dismutase were assessed. DNA damage caused by hydrogen sulfide was examined by means of single-cell gel electrophoresis. Results:, After 72 h of incubation with 100 ng/mL of hydrogen sulfide, necrosis was found in less than 10% of human gingival fibroblasts, whereas apoptosis was significantly increased (p < 0.05). Superoxide dismutase activity was strongly inhibited, and reactive oxygen species production was enhanced, after 48 and 72 h of incubation. Caspase 3 activity was also increased after 72 h of incubation (p < 0.01). Tail length, percentage of DNA in tail, and tail moment, measured by single-cell gel electrophoresis, were also intensified after 72 h of incubation (p < 0.001). Conclusion:, Hydrogen sulfide caused apoptosis and DNA damage in human gingival fibroblasts. An increased level of reactive oxygen species stimulated by hydrogen sulfide may induce apoptosis and DNA strand breaks. [source] Identification and Molecular Characterization of ,Candidatus Phytoplasma mali' Isolates in North-western ItalyJOURNAL OF PHYTOPATHOLOGY, Issue 2 2010Paola Casati Abstract Apple proliferation (AP) is an important disease and is prevalent in several European countries. The causal agent of AP is ,Candidatus Phytoplasma mali' (,Ca. Phytoplasma mali'). In this work, isolates of ,Ca. Phytoplasma mali' were detected and characterized through polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses of 16S rRNA gene and non-ribosomal DNA fragment. The presence of three AP subtypes (AT-1, AT-2 and AP-15) was identified in 31 symptomatic apple trees and two samples each constituted by a pool of five insects, collected in north-western Italy, where AT-1 is a dominant subtype. Subsequent nucleotide sequence analysis of the PCR-amplified 1.8 kb (P1/P7) fragment, containing the 16S rDNA, the 16S,23S intergenic ribosomal region and the 5,-end of the 23S rDNA, revealed the presence of at least two phytoplasmal genetic lineages within the AT-1 subtype, designed AT-1a and AT-1b. Moreover, in silico single nucleotide polymorphism (SNP) analysis based on 16S rDNA sequence can differentiate AT-1 subtype from AT-2 and AP-15 subtypes. Our data showed a high degree of genetic diversity among ,Ca. Phytoplasma mali' population in north-western Italy and underlined the possible use of the 16S rDNA analysis for the identification and the geographical origin assignation of isolates of AP phytoplasma. Molecular markers on 16S rDNA, here identified, could be useful for studying the epidemiology of AP disease. [source] A food-grade site-directed mutagenesis system for Streptococcus thermophilus LMG 18311LETTERS IN APPLIED MICROBIOLOGY, Issue 3 2010T. Blomqvist Abstract Aims:, To develop a general method for site-directed mutagenesis in the dairy starter strain Streptococcus thermophilus LMG 18311 which does not depend on antibiotic-resistance genes or other selection markers for the identification of transformants. Methods and Results:, In a previous study, we demonstrated that Strep. thermophilus LMG 18311 can be made competent for natural genetic transformation by overexpression of the alternative sigma factor ComX. In the present study, we wanted to investigate whether the natural transformation mechanism of Strep. thermophilus LMG 18311 is efficient enough to make it feasible to perform site-directed mutagenesis in this strain without the use of a selection marker. Competent bacteria were mixed with a DNA fragment engineered to contain a nonsense and a frameshift mutation in the middle of the target gene (lacZ) and subsequently seeded on agar plates. By performing colony-lift hybridization using a digoxigenin-labelled oligonucleotide probe, we succeeded in identifying transformants containing the sought after mutation. Conclusions:, By exploiting the natural transformability of Strep. thermophilus LMG 18311 and standard molecular methods, we have demonstrated that the genome of this bacterium can be altered at preselected sites without introduction of any foreign DNA. Significance and Impact of the Study:, A food-grade site-directed mutagenesis system has been developed for Strep. thermophilus LMG 18311 that can be used by the dairy industry to construct starter strains with novel and/or improved properties. [source] A highly efficient gene-targeting system for Aspergillus parasiticusLETTERS IN APPLIED MICROBIOLOGY, Issue 5 2008P.-K. Chang Abstract Aims:, To establish a system that greatly increases the gene-targeting frequency in Aspergillus parasiticus. Methods and Results:, The ku70 gene, a gene of the nonhomologous end-joining (NHEJ) pathway, was replaced by the nitrate reductase gene (niaD) in A. parasiticus RHN1 that accumulates O -methylsterigmatocystin (OMST). The NHEJ-deficient strain, RH,ku70, produced conidia, sclerotia and OMST normally. It had identical sensitivity as RHN1 to the DNA-topoisomerase I complex inhibitor, camptothecin, and the DNA-damaging agent, melphalan. For targeting an aflatoxin biosynthetic pathway gene, adhA, partial restriction enzyme recognition sequences in its flanking regions were manipulated to create homologous ends for integration. Using a linearized DNA fragment that contained Aspergillus oryzae pyrithiamine resistance gene (ptr) marker the adhA -targeting frequency in RH,ku70 reached 96%. Conclusions:, The homologous recombination pathway is primarily responsible for repair of DNA damages in A. parasiticus. The NHEJ-deficient RH,ku70, easy creation of homologous ends for integration, and the ptr -based selection form a highly efficient gene-targeting system. It substantially reduces the time and workload necessary to obtain knockout strains for functional studies. Significance and Impact of the Study:, The developed system not only streamlines targeted gene replacement and disruption but also can be used to target specific chromosomal locations like promoters or intergenic regions. It will expedite the progresses in the functional genomic studies of A. parasiticus and Aspergilllus flavus. [source] PY54, a linear plasmid prophage of Yersinia enterocolitica with covalently closed endsMOLECULAR MICROBIOLOGY, Issue 4 2003Stefan Hertwig Summary PY54 is a temperate phage isolated from Yersinia enterocolitica. Lysogenic Yersinia strains harbour the PY54 prophage as a plasmid (pY54). The plasmid has the same size (46 kb) as the PY54 genome isolated from phage particles. By electron microscopy, restriction analysis and DNA sequencing, it was demonstrated that the phage and the plasmid DNAs are linear, circularly permuted molecules. Unusually for phages of Gram-negative bacteria, the phage genome has 3,-protruding ends. The linear plasmid pY54 has covalently closed ends forming telomere-like hairpins. The equivalent DNA sequence of the phage genome is a 42 bp perfect palindrome. Downstream from the palindrome, an open reading frame (ORF) was identified that revealed strong DNA homology to the telN gene of Escherichia coli phage N15 encoding a protelomerase. Similar to PY54, the N15 prophage is a linear plasmid with telomeres. The N15 protelomerase has cleaving/joining activity generating the telomeres by processing a 56 bp palindrome (telomere resolution site tel RL). To study the activity of the PY54 protein, the telN -like gene was cloned and expressed in E. coli. A 77 kDa protein was obtained and partially purified. The protein was found to process recombinant plasmids containing the 42 bp palindrome. Telomere resolution of plasmids under in vivo conditions was also investigated in Yersinia infected with PY54. Processing required a plasmid containing the palindrome as well as adjacent DNA sequences from the phage including an additional inverted repeat. Regions on the phage genome important for plasmid maintenance were defined by the construction of linear and circular miniplasmid derivatives of pY54, of which the smallest miniplasmid comprises a 4.5 kb DNA fragment of the plasmid prophage. [source] Xis protein of the conjugative transposon Tn916 plays dual opposing roles in transposon excisionMOLECULAR MICROBIOLOGY, Issue 6 2001Douglas Hinerfeld The binding of Tn916 Xis protein to its specific sites at the left and right ends of the transposon was compared using gel mobility shift assays. Xis formed two complexes with different electrophoretic mobilities with both right and left transposon ends. Complex II, with a reduced mobility, formed at higher concentrations of Xis and appeared at an eightfold lower Xis concentration with a DNA fragment from the left end of the transposon rather than with a DNA fragment from the right end of the transposon, indicating that Xis has a higher affinity for the left end of the transposon. Methylation interference was used to identify two G residues that were essential for binding of Xis to the right end of Tn916. Mutations in these residues reduced binding of Xis. In an in vivo assay, these mutations increased the frequency of excision of a minitransposon from a plasmid, indicating that binding of Xis at the right end of Tn916 inhibits transposon excision. A similar mutation in the specific binding site for Xis at the left end of the transposon did not reduce the affinity of Xis for the site but did perturb binding sufficiently to alter the pattern of protection by Xis from nuclease cleavage. This mutation reduced the level of transposon excision, indicating that binding of Xis to the left end of Tn916 is required for transposon excision. Thus, Xis is required for transposon excision and, at elevated concentrations, can also regulate this process. [source] Activation of enteropathogenic Escherichia coli (EPEC) LEE2 and LEE3 operons by LerMOLECULAR MICROBIOLOGY, Issue 4 2000Vanessa Sperandio Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing lesions (AE) on epithelial cells. The genes involved in the formation of the AE lesions are contained within a pathogenicity island named the locus of enterocyte effacement (LEE). The LEE comprises 41 open reading frames organized in five major operons: LEE1, LEE2, LEE3, LEE4 and tir. The first gene of the LEE1 operon encodes a transcription activator of the other LEE operons that is called the LEE-encoded regulator (Ler). The LEE2 and LEE3 operons are divergently transcribed with overlapping ,10 promoter regions, and gene fusion studies have shown that they are both activated by Ler. Deletion analysis, using lacZ reporter fusions, of the LEE2 and LEE3 promoters demonstrated that deletions extending closer to the LEE2 transcription start site than ,247 bp lead to loss of activation by Ler, whereas only 70 bp upstream of the LEE3 transcription start site is required for Ler-mediated activation. We have purified Ler as a His-tagged protein and used it to perform DNA-binding assays with LEE2 and LEE3. We observed that Ler bound to a DNA fragment containing the ,300 to +1 region of LEE2; however, it failed to bind to a DNA fragment containing the ,300 to +1 region of LEE3, suggesting that Ler activates both operons by only binding to the regulatory region upstream of LEE2. The Ler-activatable LEE3::lacZ fusions extended to what would be ,246 bp of the LEE2 operon. A lacZ fusion from the ,300 to +1 region of LEE3 failed to be activated by Ler, consistent with our hypothesis that Ler activates the expression of LEE2 and LEE3 by binding to a region located downstream of the LEE3 transcription start site. DNase I footprinting revealed that Ler protected a region of 121 bp upstream of LEE2. Purified Ler mutated in the coiled-coil domain was unable to activate transcription and to bind to the LEE2 regulatory region. These data indicate that Ler may bind as a multimer to LEE2 and activate both divergent operons by a novel mechanism potentially involving changes in the DNA structure. [source] Spontaneously regressed Kaposi's sarcoma and human herpesvirus 8 infection in a human immunodeficiency virus-negative patientPATHOLOGY INTERNATIONAL, Issue 4 2000Yasuko Kondo Kaposi's sarcoma occurring in a 78-year-old woman, with the absence of the human immunodeficiency virus infection, was correctly diagnosed by immunohistochemistry using anti-human herpesvirus 8 (HHV8) antibody (PA1-73N) for the first time. The patient suffered from chronic respiratory failure and was treated with a low dose of steroids for 2.5 years. After her medication dosage was increased for the exacerbation of the respiratory failure, multiple skin tumors in her feet and legs suddenly developed. Histopathologically, skin tumors were suspected as Kaposi's sarcoma at the first biopsy and reactive angiomatosis at the second biopsy. Polymerase chain reaction and immunohistochemistry, however, revealed the presence of HHV8 DNA fragment and positive staining in the majority of spindle cells in the skin tumors. Serological examination confirmed the positivity of anti-HHV8 antibodies. HHV8 infection and steroid-induced immunosuppression, as well as environmental factors played a role in the development of Kaposi's sarcoma in this patient, because she was born in Okinawa, which is a well-known endemic area of Kaposi's sarcoma in Japan. As her general condition improved, the skin lesions regressed without any specific treatment, and disappeared completely 8 months later, in which regression may be associated with evidence of numerous CD8 cell infiltration in the second biopsy tissues. No recurrence was observed during the following 6 month follow up. [source] Mechanisms influencing the evolution of resistance to Qo inhibitor fungicides,,PEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 9 2002Ulrich Gisi Abstract Fungicides inhibiting the mitochondrial respiration of plant pathogens by binding to the cytochrome bc1 enzyme complex (complex III) at the Qo site (Qo inhibitors, QoIs) were first introduced to the market in 1996. After a short time period, isolates resistant to QoIs were detected in field populations of a range of important plant pathogens including Blumeria graminis Speer f sp tritici, Sphaerotheca fuliginea (Schlecht ex Fr) Poll, Plasmopara viticola (Berk & MA Curtis ex de Bary) Berl & de Toni, Pseudoperonospora cubensis (Berk & MA Curtis) Rost, Mycosphaerella fijiensis Morelet and Venturia inaequalis (Cooke) Wint. In most cases, resistance was conferred by a point mutation in the mitochondrial cytochrome b (cyt b) gene leading to an amino-acid change from glycine to alanine at position 143 (G143A), although additional mutations and mechanisms have been claimed in a number of organisms. Transformation of sensitive protoplasts of M fijiensis with a DNA fragment of a resistant M fijiensis isolate containing the mutation yielded fully resistant transformants, demonstrating that the G143A substitution may be the most powerful transversion in the cyt b gene conferring resistance. The G143A substitution is claimed not to affect the activity of the enzyme, suggesting that resistant individuals may not suffer from a significant fitness penalty, as was demonstrated in B graminis f sp tritici. It is not known whether this observation applies also for other pathogen species expressing the G143A substitution. Since fungal cells contain a large number of mitochondria, early mitotic events in the evolution of resistance to QoIs have to be considered, such as mutation frequency (claimed to be higher in mitochondrial than nuclear DNA), intracellular proliferation of mitochondria in the heteroplasmatic cell stage, and cell to cell donation of mutated mitochondria. Since the cyt b gene is located in the mitochondrial genome, inheritance of resistance in filamentous fungi is expected to be non-Mendelian and, therefore, in most species uniparental. In the isogamous fungus B graminis f sp tritici, crosses of sensitive and resistant parents yielded cleistothecia containing either sensitive or resistant ascospores and the segregation pattern for resistance in the F1 progeny population was 1:1. In the anisogamous fungus V inaequalis, donation of resistance was maternal and the segregation ratio 1:0. In random mating populations, the sex ratio (mating type distribution) is generally assumed to be 1:1. Therefore, the overall proportion of sensitive and resistant individuals in unselected populations is expected to be 1:1. Evolution of resistance to QoIs will depend mainly on early mitotic events; the selection process for resistant mutants in populations exposed to QoI treatments may follow mechanisms similar to those described for resistance controlled by single nuclear genes in other fungicide classes. It will remain important to understand how the mitochondrial nature of QoI resistance and factors such as mutation, recombination, selection and migration might influence the evolution of QoI resistance in different plant pathogens. © 2002 Society of Chemical Industry [source] Tomato leaf curl geminivirus in Australia: occurrence, detection, sequence diversity and host rangePLANT PATHOLOGY, Issue 3 2003J. Stonor The occurrence of whitefly transmitted geminiviruses in Australia was studied using a mixed DNA probe capable of detecting a range of distinct geminiviruses. The only geminivirus species detected was Tomato leaf curl virus (TLCV), which is spread across a vast geographical region of far-northern coastal Australia, an area inhabited by the Australasian-Oceania biotype of Bemisia tabaci. The newly introduced silverleaf whitefly, B. tabaci biotype B, forms high population densities in the eastern coastal region of Queensland and is currently located approximately 150 km from the nearest known TLCV-infected area. The viral host range appeared to be narrow and of 58 species of crop plants and weeds inoculated using the B biotype, only 11 became infected with the virus, including five that did not show foliar symptoms. A DNA fragment of 694 nt, including the complete C4 open reading frame (ORF), the overlapping N-terminal part of the C1 ORF and the viral iterons involved in replication, was amplified from 11 TLCV field isolates and sequenced. Sequence analysis revealed an overall sequence variation of up to 14% in this region, as well as the presence of distinct viral iterons. [source] A Sarcocystid Misidentified as Hepatozoon didelphydis: Molecular Data from a Parasitic Infection in the Blood of the Southern Mouse Opossum (Thylamys elegans) from ChileTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 6 2008SANTIAGO MERINO ABSTRACT. The blood of 21 adult South American mouse opossums (Thylamys elegans) captured from April through August of 2005 in central Chile was examined for parasites. Light microscopic analysis of blood smears initially suggested that a highly pleomorphic Hepatozoon species typical of American opossums was infecting erythrocytes. Unexpectedly, amplification by PCR and sequencing of a DNA fragment of the small subunit rDNA combined with phylogenetic analyses indicated that the parasite is not a member of the suborder Adeleorina, which includes the Haemogregarina and Hepatozoon species, but that it is a clearly distinct member of the suborder Eimeriorina, which includes the cyst-forming family Sarcocystidae. Therefore, a reclassification of this unusual intraerythrocytic apicomplexan will require additional life cycle, microscopic, and molecular analyses. [source] Cattle Pathogen Tritrichomonas foetus (Riedmüller, 1928) and Pig Commensal Tritrichomonas suis (Gruby & Delafond, 1843) Belong to the Same SpeciesTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2 2002JAN TACHEZY ABSTRACT. A number of reports suggest that the sexually transmitted pathogen of cattle, Tritrichomonas foetus, and a gastrointestinal commensal of pigs, Tritrichomonas suis, are very similar and may be co-specific. A conclusive review of the taxonomic and nomenclatural status of these species has not been presented so far. Toward this end, we reexamined and compared porcine and bovine trichomonads with regard to their morphology, pathogenic potential, and DNA polymorphism. Using light and electron microscopy, no distinguishing features between T. foetus and T. suis strains were found in size, general morphology, and karyomastigont structure. Both bovine and porcine trichomonads showed pathogenic potential in the subcutaneous mouse assays and did not separate into distinct groups according to strain virulence. Three DNA fingerprinting methods (i.e. RFLP, RAPD, and PCR-based analysis of variable-length DNA repeats) that produce species-specific DNA fragment patterns did not distinguish between the bovine and porcine strains. Sequencing of a variable 502-bp DNA fragment as well as comparison of 16S rRNA gene sequences did not reveal species-specific differences between the cattle and porcine strains. Therefore, we conclude that T. foetus and T. suis belong to the same species. To prevent confusion that may arise from T. foetus-T. suis synonymy, we propose to suppress the older name suis and maintain its accustomed junior synonym foetus as a nomen protection for both cattle and porcine trichomonads. The case has been submitted to the International Commision on Zoological Nomenclature for ruling under its plenary power. [source] Self-compatibility in Brassica napus is caused by independent mutations in S -locus genesTHE PLANT JOURNAL, Issue 3 2007Shunsuke Okamoto Summary Brassica napus is an amphidiploid species with the A genome from Brassica rapa and the C genome from Brassica oleracea. Although B. rapa, B. oleracea and artificially synthesized amphidiploids with the AC genome are self-incompatible, B. napus is self-compatible. Six S genotypes were identified in B. napus, five of which had class I S haplotypes from one species and a class II S haplotype from the other species, and mutations causing self-compatibility were identified in three of these S genotypes. The most predominant S genotype (BnS-1;BnS-6), which is that of cv. ,Westar', had a class I S haplotype similar to B. rapa S-47 (BrS-47) and a class II S haplotype similar to B. oleracea S-15 (BoS-15). The stigmas of ,Westar' rejected the pollen grains of both BrS-47 and BoS-15, while reciprocal crossings were compatible. Insertion of a DNA fragment of about 3.6 kb was found in the promoter region of the SP11/SCR allele of BnS-1, and transcripts of SP11/SCR were not detected in ,Westar'. The nucleotide sequence of the SP11 genomic DNA of BnS-6 was 100% identical to that of SP11 of BoS-15. Class I SP11 alleles from one species showed dominance over class II SP11 alleles from the other species in artificially synthesized B. napus lines, suggesting that the non-functional dominant SP11 allele suppressed the expression of the recessive SP11 allele in ,Westar'. Two other S genotypes in B. napus also had non-functional class I S haplotypes together with recessive BnS-6. These observations suggest independent origins of self-compatibility in B. napus. [source] Ovine alpha-amylase genes: isolation, linkage mapping and association analysis with milk traitsANIMAL GENETICS, Issue 4 2004J. H. Calvo Summary On the basis of comparisons between cattle and sheep genome mapping information the ovine , - amylase gene was examined as a possible genetic marker for milk traits in sheep. The objective of the present study was to isolate, map and determine whether this gene is a candidate gene for milk traits. DNA fragments (832 and 2360 bp) corresponding to two different AMY genes were isolated, and one SNP in intron 3 and one GTG deletion in exon 3 of the 2360 bp DNA fragment were found. The 2360 bp ovine AMY DNA fragment was located on chromosome 1 by linkage mapping using the International Mapping Flock. No association was found between estimated breeding values for milk yield, protein and fat contents and AMY genotypes in a daughter design comprising 13 Manchega families with an average of 29 daughters (12,62) per sire. [source] Crystals of ternary protein,DNA complexes composed of DNA-binding domains of c-Myb or v-Myb, C/EBP, or C/EBP, and tom-1A promoter fragmentACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2001Tahir H. Tahirov c-Myb and the C/EBP family are transcriptional regulatory factors that act in concert to regulate the expression of myeloid-specific genes. v-Myb encoded by avian myeloblastosis virus (AMV) is a mutated form of c-Myb that contains point mutations which disrupt the cooperation with C/EBPs. To understand the mechanism of the transcriptional synergy between c-Myb and C/EBPs and the effect of the v-Myb mutations on that synergy, knowledge based on their three-dimensional structures is essential. Crystals of ternary complexes, in which various combinations of the DNA-binding domains of c-Myb or v-Myb and C/EBP, or C/EBP, are bound to a DNA fragment from tom-1A promoter, were obtained by the vapour-diffusion method. Complete diffraction data sets were obtained from each native crystal and two types of iodine-derivative crystals. A three-wavelength MAD data set was also obtained from a bromine-derivative crystal. [source] PCR as a specific, sensitive and simple method suitable for diagnosticsBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 4 2000M. Gonzalo Claros PCR technology is a widespread method that has not reached students laboratory in anything else than a typical amplification reaction. We describe a simple application of PCR in pathogen diagnostics that enables students to identify which ampicillin-resistant organism is present in a cell culture. This experiment has been performed for one year in two "Experimental Biochemistry and Molecular Biology" courses with Biological and Chemical undergraduates. Using specific primers from the Escherichia coli ,-lactamase gene, they have been able to selectively amplify a ,-lactamase DNA fragment in E. coli but not in Staphylococcus aureus and, using different annealing temperatures, test the reaction specificity. By solving the "Study Questions", students understood the specificity and sensitivity of the method, as well as the rationale that should be applied when a molecular weight pattern is used for calculating unknown DNA sizes. © 2000 IUBMB. Published by Elsevier Science Ltd. All rights reserved. [source] Microbial systems engineering: First successes and the way aheadBIOESSAYS, Issue 4 2010Sven Dietz Abstract The first promising results from "streamlined," minimal genomes tend to support the notion that these are a useful tool in biological systems engineering. However, compared with the speed with which genomic microbial sequencing has provided us with a wealth of data to study biological functions, it is a slow process. So far only a few projects have emerged whose synthetic ambition even remotely matches our analytic capabilities. Here, we survey current technologies converging into a future ability to engineer large-scale biological systems. We argue that the underlying synthetic technology, de novo DNA synthesis, is already rather mature , in particular relative to the scope of our current synthetic ambitions. Furthermore, technologies towards rationalizing the design of the newly synthesized DNA fragment are emerging. These include techniques to implement complex regulatory circuits, suites of parts on a DNA and RNA level to fine tune gene expression, and supporting computational tools. As such DNA fragments will, in most cases, be destined for operating in a cellular context, attention has to be paid to the potential interactions of the host with the functions encoded on the engineered DNA fragment. Here, the need of biological systems engineering to deal with a robust and predictable bacterial host coincides with current scientific efforts to theoretically and experimentally explore minimal bacterial genomes. [source] | ||||||||||||||||||||||||||||||||||||||||||||||