This

research was supported by funding from the Westaim Corporation, selleck inhibitor the Alberta Science and Research Authority (ASRA), the Canadian Institutes for Health Research and the Canadian Cystic Fibrosis Foundation. S.L. holds the Westaim-ASRA Chair in Biofilm Research. R.E.W.H. holds a Canada Research Chair. “
“Yersinia polynucleotide phosphorylase (PNPase), a 3′–5′ exoribonuclease, has been shown to affect growth during several stress responses. In Escherichia coli, PNPase is one of the subunits of a multiprotein complex known as the degradosome, but also has degradosome-independent functions. The carboxy-terminus of E. coli ribonuclease E (RNase E) serves as the scaffold upon which PNPase, enolase (a glycolytic enzyme), and RhlB helicase all have been shown to bind. In the yersiniae, only PNPase has thus far been shown to physically interact with RNase E. We show by bacterial two-hybrid and co-immunoprecipitation assays that RhlB and enolase also interact with RNase E. Interestingly, although PNPase is required for normal growth at cold temperatures, assembly of the yersiniae degradosome was not required. However, degradosome assembly was required for growth in the presence of reactive oxygen species. These data suggest

that while the Yersinia pseudotuberculosis PNPase plays selleckchem a role in the oxidative stress response through a degradosome-dependent mechanism, PNPase’s role during cold stress is degradosome-independent. Like other closely related Gram-negative enteric pathogens, Yersinia pseudotuberculosis employs a type III secretion system (T3SS) to infect host cells, and polynucleotide phosphorylase (PNPase),

a phosphorolytic 3′–5′ exoribonuclease involved in RNA decay, is required for its optimal functioning (Rosenzweig et al., 2005, 2007). Furthermore, we (and others) have observed that PNPase is required for the cold-shock response and/or acclimation for a number of organisms including Yersinia pestis Loperamide and Y. pseudotuberculosis (Rosenzweig et al., 2005, 2007), Escherichia coli (Jones et al., 1987; Mathy et al., 2001; Yamanak & Inouye, 2001; Polissi et al., 2003), and Yersinia enterocolitica (Goverde et al., 1998; Neuhaus et al., 2000; Neuhaus et al., 2003). Intriguingly, PNPase has been shown to physically interact with an essential endoribonuclease, RNase E, in both Escherichia coli (Carpousis et al., 1994; Vanzo et al., 1998; Khemici & Carpousis, 2004) and Y. pseudotuberculosis (Yang et al., 2008) forming a large multiprotein RNA surveillance/quality control complex termed the degradosome. However, the role of the degradosome in various yersiniae stress responses has not been well studied. RNase E, PNPase, RhlB RNA helicase and enolase have all been identified as components of the E. coli degradosome (Carpousis, 2002; Khemici & Carpousis, 2004; Lawal et al., 2010).

This

research was supported by funding from the Westaim Corporation, Neratinib mouse the Alberta Science and Research Authority (ASRA), the Canadian Institutes for Health Research and the Canadian Cystic Fibrosis Foundation. S.L. holds the Westaim-ASRA Chair in Biofilm Research. R.E.W.H. holds a Canada Research Chair. “
“Yersinia polynucleotide phosphorylase (PNPase), a 3′–5′ exoribonuclease, has been shown to affect growth during several stress responses. In Escherichia coli, PNPase is one of the subunits of a multiprotein complex known as the degradosome, but also has degradosome-independent functions. The carboxy-terminus of E. coli ribonuclease E (RNase E) serves as the scaffold upon which PNPase, enolase (a glycolytic enzyme), and RhlB helicase all have been shown to bind. In the yersiniae, only PNPase has thus far been shown to physically interact with RNase E. We show by bacterial two-hybrid and co-immunoprecipitation assays that RhlB and enolase also interact with RNase E. Interestingly, although PNPase is required for normal growth at cold temperatures, assembly of the yersiniae degradosome was not required. However, degradosome assembly was required for growth in the presence of reactive oxygen species. These data suggest

that while the Yersinia pseudotuberculosis PNPase plays Palbociclib solubility dmso a role in the oxidative stress response through a degradosome-dependent mechanism, PNPase’s role during cold stress is degradosome-independent. Like other closely related Gram-negative enteric pathogens, Yersinia pseudotuberculosis employs a type III secretion system (T3SS) to infect host cells, and polynucleotide phosphorylase (PNPase),

a phosphorolytic 3′–5′ exoribonuclease involved in RNA decay, is required for its optimal functioning (Rosenzweig et al., 2005, 2007). Furthermore, we (and others) have observed that PNPase is required for the cold-shock response and/or acclimation for a number of organisms including Yersinia pestis Galactosylceramidase and Y. pseudotuberculosis (Rosenzweig et al., 2005, 2007), Escherichia coli (Jones et al., 1987; Mathy et al., 2001; Yamanak & Inouye, 2001; Polissi et al., 2003), and Yersinia enterocolitica (Goverde et al., 1998; Neuhaus et al., 2000; Neuhaus et al., 2003). Intriguingly, PNPase has been shown to physically interact with an essential endoribonuclease, RNase E, in both Escherichia coli (Carpousis et al., 1994; Vanzo et al., 1998; Khemici & Carpousis, 2004) and Y. pseudotuberculosis (Yang et al., 2008) forming a large multiprotein RNA surveillance/quality control complex termed the degradosome. However, the role of the degradosome in various yersiniae stress responses has not been well studied. RNase E, PNPase, RhlB RNA helicase and enolase have all been identified as components of the E. coli degradosome (Carpousis, 2002; Khemici & Carpousis, 2004; Lawal et al., 2010).

But this process is limited to systems that can be

placed within the imaging distance of a confocal microscope, to bacteria with a genetic system allowing the use of such expression systems, and to systems with enough oxygen present to allow correct folding of the fluorescent protein with the short half-life. Also, the commonly used glass flow cells are highly artificial surfaces for microbial growth. It took considerable effort to construct a real-time imaging microbial fuel cell, which is currently limited to G. sulfurreducens due to its genetic amiability for fluorescent protein expression. The ability to examine the electricity-producing biofilm nondestructively has allowed Selleckchem Everolimus for the direct measurement of proton accumulation and metabolic activity within the biofilm through the use of fluorescent dyes. A recent development AZD6244 mw that is helping to overcome some of these inherent problems is a technique to spatially extract RNA from a biofilm in sufficient quantity and quality for microarray analysis

from a single biofilm (Franks et al., 2010). Using a single biofilm, a spatial examination of gene expression can be performed, providing valuable information for internal transcriptional differences within the biofilm. Because small differences between biofilms can cause large differences in transcription, these differences can be minimized through the use of a single biofilm. Once again, the experimental design should consider that genes important throughout the biofilm might not be differentially expressed spatially, even though they are fundamental for function. However, gene transcription does not always indicate protein localization. Even though transcription is detected in a biofilm, translation and protein localization may not follow the same pattern, which requires further protein fusion and labelled antibody studies. Microbial biofilms are extremely important, but the examination of gene expression is still fraught with pitfalls

and 5-Fluoracil mouse overgeneralizations. Microarray analysis is a wonderful tool, especially as the costs are continually decreasing, making their use much more routine. However, it is essential to remember that they are only a starting point for biofilm research and not a tool that can be applied without careful consideration of experimental design and follow-up research. Without this caution, researchers may overinterpret results and assign them greater significance than deserved. Although large data sets of significantly up- and downregulated gene expression patterns are created, often only a few genes with real phenotypic importance are identified in biofilm microarray studies. The author is funded by the Office of Science (BER), US Department of Energy, Cooperative Agreement No. DE-FC02-02ER63446 and Office of Naval Research Award No. N00014-07-1-0966.

The SPN has been related to the contingent negative variation (Walter et al., 1964; Tecce, 1972; Hultin et al., 1996; Hamano

et al., 1997), and to pain anticipation (Babiloni et al., 2005b; Brown et al., 2008). The sources of the SPN prior to the onset of a simple finger movement comprise, in addition to primary motor areas, the anterior cingulate cortex and inferior parietal cortex as well as occipital and prefrontal areas (Gómez et al., 2003). Thus, the stronger anticipatory negative drift over the central scalp for needle compared with Q-tip clips in the present study may reflect enhanced preparation for the processing of the subsequently presented electrical stimulus. An aspect that was not addressed by the present study is the effect of viewing a needle prick on the neural responses to electrical stimulation. Selleckchem JAK inhibitor The clips in our study were presented immediately before the onset of the electrical stimuli, triggering anticipatory processes that probably overlap with the responses to the electrical stimulus. Therefore, it is not possible to disentangle whether any poststimulus effects would actually be linked to the processing of the electrical stimuli or are due Z-VAD-FMK in vivo to anticipatory processes that start prior to the electrical stimulation. Future studies may include unimodal visual

trials, in which the clips are presented without subsequent electrical stimulation. Neural activity to these stimuli could be subtracted from the activity to bimodal visual-pain stimuli (Busse & Woldorff, 2003; Senkowski et al., 2011). However, the inclusion of unimodal visual stimuli would have substantially changed the stimulation protocol of our original study (Höfle et al., 2012). For this reason, we did not include unimodal visual stimuli in the present study and restricted Montelukast Sodium the analysis of electrophysiological data to the interval prior to electrical stimulation. Our study showed that viewing a needle pricking a hand that is perceived as one’s own enhances the unpleasantness of spatiotemporally aligned painful and nonpainful electrical stimuli. Moreover, our study demonstrated that viewing a needle compared with viewing a Q-tip approaching the body enhances PDRs and reduces anticipatory

alpha-band responses in the PCC and FG. Thus, our study uncovered a spectral signature that was associated with the previously reported effect of viewing a needle prick on the PDR (Höfle et al., 2012). Viewing a needle approaching the body modulates neural activity in the PCC and FG probably to orient the body to the forthcoming stimulation and to prepare adequate defense responses to protect the integrity of one’s body. This study was supported by grants from the German Research Foundation (DFG) (SE 1859/1-2 to D.S.; SFB TRR 58 B04 to A.K.E.) and the European Union (ERC-2010-StG_20091209 to D.S.; ERC-2010-AdG-269716 to A.K.E.). We thank C. Beckmerhagen and R. Zimmermann for help with the preparation of the experimental setup, C. Reißmann and K.

Exclusion criteria included: chronic hepatitis B [hepatitis B virus surface antigen (HBsAg)-positive at screening]; hepatitis C virus infection (RNA positive) that was likely

to require treatment within the next 12 months, or with historical evidence Protease Inhibitor Library of significant fibrosis, cirrhosis and/or hepatic decompensation; a new AIDS-defining condition diagnosed within 35 days prior to the first dose of the study drug; presence of Q151M or 69 insertion mutations in HIV-1 reverse transcriptase at screening; current treatment with zalcitabine; a regimen comprised only of three nucleoside/nucleotide reverse transcriptase inhibitors. Women of childbearing potential were required to have a negative pregnancy test and adequate contraception was required of all patients. Patients were randomly assigned

in a 1:1:1 PARP inhibitor ratio to receive 600 mg ATC twice daily (bid), 800 mg ATC bid or 150 mg 3TC bid, taken orally, plus matching placebos. Double dummy dosing was used in this study. 3TC was given as over-encapsulated 150 mg tablets and patients in the two ATC arms received a placebo capsule matching the 3TC capsule in size, colour and approximate weight. Patients in the 3TC arm received placebo capsules matching the ATC capsules. A centralized randomization scheme was used and randomization was stratified by the number of TAMs present at screening (fewer than three

TAMs or at least three TAMs/K65R), according to the study protocol. Throughout the study, both patients and investigators were blinded to the treatment allocation. The study design is shown in Figure 1. The study was divided into the following treatment periods. On day 0, patients stopped their existing 3TC or FTC treatment and commenced blinded therapy. Abiraterone solubility dmso No other changes to background ART were permitted during this period. On day 21, the background ART could be optimized to contain at least two agents expected to provide activity based on genotype at screening, and blinded therapy continued to week 24. Any approved ART could be used with the exceptions of 3TC, FTC and zalcitabine. After week 24, patients ceased randomized therapy and were offered open-label ATC (800 mg bid) to week 48. After day 21, re-optimization of background ART for lack of response/virological failure was permitted and access to open-label ATC 800 mg bid was provided upon meeting failure criteria (a confirmed<0.5 log10 reduction in HIV RNA from baseline or a confirmed >1 log10 increase in HIV RNA from nadir). The choice of ART for this subsequent regimen was based on genotype at screening or at subsequent evaluations.

Since the first report of ESBLs in 2002 (Chanawong

et al., 2002), blaCTX-M has been predominant in mainland (Yu et al., 2007; Liu et al., 2009). In this multicentre study, the prevalence of ESBL production in K. pneumoniae has been demonstrated to be about 40%. Of 158 ESBL-producers, the isolates harboring ESBL genes and blaCTX-M-14 were 94.3% and 49.4%, respectively, and were shown to increase 10% and 9% to those in another large-scale study (Yu et al., 2007), respectively. The proportion of blaCTX-M increased 12% compared to the percentage (72.3%) described in a report of southern China three years ago (Liu et al., 2009) and doubled the percentage reported nine years ago (Li et al., 2003). Because the usage of plasmid-based amplification method in this study and the potential Angiogenesis inhibitor false-negative products

owing to the unbinding on some novel bla, the detection of β-lactamase genes LDE225 chemical structure may have been underestimated. Although there are some differences in the source of the isolates in our study as compared to the studies mentioned above, our results clearly suggest the increasing prevalence of blaCTX-M in K. pneumoniae in China. CTX-M-type ESBLs exhibit powerful activity against cefotaxime and ceftriaxone but generally not against ceftazidime, and several variants with enhanced ceftazidimase activity have been reported (Poirel et al., 2002; Bonnet et al., 2003; Amisulpride Rossolini et al., 2008). In this study, it was observed that the isolates harboring CTX-M-15 or CTX-M-27 alone exhibited higher resistance rates to ceftazidime and aztreonam than that in subgroup CTX-M-14 without other ESBLs

(Table 2). Further, a high percentage of isolates harboring blaCTX-M-27 demonstrated the MDR phenotype. To our knowledge, this is the first study about the high prevalence of CTX-M-27 in Enterobacteriaceae in China. This warrants for an active surveillance to monitor these resistant bacteria. The overall resistance rates to the tested β-lactam antimicrobial agents were over 30% except for cefepime, piperacillin/tazobactam, and cefotetan in this study. As shown in Table 2, only 9.3% isolates harboring CTX-M-14 alone showed resistance to cefepime, but 50% isolates harboring CTX-M-15 exhibited resistance (P < 0.01), and a 100% resistance rate when CTX-M-15 coexisted with other ESBLs. Nevertheless, piperacillin/tazobactam show only 10.1% resistance rate in vitro, although the proportion increased to 26.7% when the isolates contained two types of ESBLs(blaCTX-M + blaSHV)(Table 1). Several clinical intervention studies also supported that piperacillin/tazobactam may contribute to preventing the ESBL-producing K. pneumoniae outbreaks (Lee et al., 2007; Tangden et al., 2011). These properties highlight the value of piperacillin/tazobactam as empirical therapy for infections by suspected organisms possessing a single ESBL (especially the blaCTX-M).

That is, sPBP 656 (PBP 6 containing the MMD of PBP 5) exhibited a dd-CPase activity toward both substrates that was more like PBP 5 than PBP 6, but sPBP 565 PF-02341066 research buy (PBP 5 containing the MMD of PBP 6) was not active on either of two substrates. In addition to its decreased dd-CPase activity, PBP 565 also bound and hydrolyzed the β-lactam BOCILLIN FL much less well than any of the other proteins. These behavioral changes

of sPBP 565 may not have been due to the improper folding of the molecule because CD spectral analyses predict that there is no gross alteration in the composition of the overall secondary structure of sPBP 565 compared with sPBP 5, except that there is 3% less β-sheet

structure in the former. Although the reason for the altered behavior of sPBP 565 is not clear, a gross change in the microarchitecture of the active site cannot be ruled out. Because β-sheet structures are not usual components of active sites, the lower percentage of the β-sheet structure in sPBP 565 is not likely the key feature for its altered behavior. Nevertheless, the possible changes include altering the size and volume of the substrate-binding pocket that may change the affinity or the activity of the chimeric protein toward specific substrates. In any case, the ability of each PBP to act as a dd-CPase correlates exactly with its ability to complement cell shape changes in vivo, 3-deazaneplanocin A clinical trial strongly suggesting that this activity is responsible for the shape maintenance phenotype. We thank Robert A. Nicholas for providing the plasmid pT7-cPBP5 and for suggestions regarding the construction of sPBPs. We also acknowledge Rakesh Sikder for initiating the computational work. This work was supported by a grant from the Department of Science and Technology, the Government of India, to A.S.G., and K.D.Y. was supported ID-8 by a grant R01-GM061019 from the US National Institutes of Health and by the Arkansas Biosciences Institute, the

major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000. Fig. S1. Structures of the peptide substrates. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. “
“3-Methoxy-2-methyl-carbazole-1,4-quinone (1) together with carbazomycins D (2) and F (3) were isolated from the crude extract of Streptomyces CMU-JT005, an actinomycete with nematicidal activity. 3-Methoxy-2-methyl-carbazole-1,4-quinone is reported here for the first time from nature. In this paper, we describe the isolation and structure elucidation of the compounds together with the characterization of the Streptomyces strain CMU-JT005.

, 2001a, b). Mutator bacteria do not constitute a large fraction of natural bacterial isolates because they accumulate adaptive and neutral mutations in the current environment that can be deleterious in a secondary environment, thus imparting long-term disadvantage (Giraud et al., 2001a, b). The sediment in Lake Oneida from which S. oneidensis MR-1 was isolated is a highly eutrophic environment, prone to frequent wind mixing events and the establishment of temporary redox gradients in the sediments and water (Dean et al., 1981; Mitchell et al., 1996; Ausubel, 2008; Domack, 2008). These conditions result in the creation of temporary microenvironments in sediments (Greeson,

1971; Ausubel, 2008; Domack, 2008). Such an environment would select for mutator bacteria phylotypes capable of survival through the development of environmental adaptations including the ability to use glucose as the only carbon source with high frequency. The ability www.selleckchem.com/products/jq1.html of S. oneidensis MR-1 to use glucose HIF inhibitor as a sole carbon source via a mutator population or a GASP mutation (although these are not mutually exclusive) suggests interesting ecological implications. Members of the Shewanella genus have great flexibility in terms of growth strategy and metabolisms (Tang et al., 2009),

allowing them to proliferate in diverse and changing environments. The ability to maintain a mutator population within Shewanella species and/or gain GASP mutations indicates that the genus and specifically S. oneidensis MR-1 have other understudied mechanisms to assist them with establishing populations in highly variable environments. We thank Preston A. Fulmer for laboratory assistance. We also thank Russell Kirk Pirlo, Lisa A. Fitzgerald, Justin C. Biffinger, and anonymous reviewers for helpful comments. This work was funded by the Office of Naval Research through NRL Program Element Number 62123N and NRL Program Element Number 17-DMAG (Alvespimycin) HCl 61153N. This

work was carried out while E.C.H. held a National Research Council Post-Doctoral Associateship. “
“The hetero-oligomeric FlhD/FlhC complex is a global regulator of transcription in Escherichia coli. FlhD alone, independent of FlhC, has also been reported to control when E. coli cells stop dividing and enter the stationary phase. This work is frequently cited as evidence that FlhD regulates cell division; however, our data indicate that this is not the case. The results presented here show that the previously observed phenotype is not due to the flhD locus, but is instead due to differences in the thyA alleles present in the flhD+ and flhD− strains used in the original studies. We find that when the strains being compared have the same thyA allele (wild type or mutant), flhD mutations have no effect on growth. The hetero-oligomeric FlhD/FlhC complex is a global regulator of gene expression in Escherichia coli.

For visualization of EGFP expression, spores of pHxk1-EGFP transformed strains were inoculated in MM and grown for 10 h at 28 °C. These cultures were used directly for microscopic observation. Fluorescence and light microscopy were performed using a Nikon Eclipse 80i fluorescence microscope and images were captured under control of nis-elements ar software. Previous work showed that H. jecorina hexokinase-negative strains were not able to grow on MM containing d-fructose as the sole carbon source (Hartl & Seiboth 2005).

To test the utility of the two polyols d-mannitol and d-sorbitol as osmotic stabilizer and as a selective carbon source we tested the growth of H. jecorina TU-6H and the parent strain on MM agar plates with different carbon sources. Growth tests showed that TU-6H strains were Opaganib price not able to grow on MM containing 10 g L−1d-mannitol and d-sorbitol, whereas the parent strain was able to use both polyols as sole carbon source. This indicates that, in H. jecorina, the two polyols d-mannitol and d-sorbitol are catabolized via d-fructose as intermediate (Elorza & Arst, 1971; Solomon et al., 2007). To demonstrate the utility of this transformation system, H. jecorina TU-6H was transformed

with the reporter plasmid pHxk1-EGFP. For determination of the optimal selective carbon source and osmotic stabilizer, we compared the effect of 1 M d-sorbitol and 1 M d-mannitol in the regeneration medium on transformation efficiency. Results from three independent parallel transformation experiments showed that d-mannitol leads to higher transformation efficiencies than d-sorbitol. Using d-mannitol, approximately 500–1000 colonies μg−1

Selumetinib plasmid DNA were obtained, whereas using d-sorbitol, only 100–200 colonies were found. Branched chain aminotransferase In control transformation assays with the EGFP expression, plasmid pIG1783 alone or without plasmid DNA, no colonies were found on the selective plates. As shown in Fig. 1a, transformed colonies of variable sizes were seen on selective plates. After purification of the transformants, their growth on different carbon sources was compared with growth on the parental strains (Fig. 1b). The growth of the transformants was fully restored on MM with d-mannitol or d-glucose as carbon source. The presence of the reporter plasmid pHxk1-EGFP in d-mannitol-utilizing transformants was confirmed by the presence of both hxk1 and egfp in the isolated gDNA of different transformants as detected by PCR (Fig. 2a). Three randomly picked transformants were further analyzed by Southern blot to determine the pattern of integration. Figure 2b shows that pHxk1-EGFP integrated into the genome of all analyzed transformants at the hxk1 locus; in all cases, the plasmid integrated next to the promoter region of hxk1. Some of the transformants showed additional hybridizing bands, most likely due to tandem integration or insertion of the plasmid by ectopic integration as well as targeted integration.

For visualization of EGFP expression, spores of pHxk1-EGFP transformed strains were inoculated in MM and grown for 10 h at 28 °C. These cultures were used directly for microscopic observation. Fluorescence and light microscopy were performed using a Nikon Eclipse 80i fluorescence microscope and images were captured under control of nis-elements ar software. Previous work showed that H. jecorina hexokinase-negative strains were not able to grow on MM containing d-fructose as the sole carbon source (Hartl & Seiboth 2005).

To test the utility of the two polyols d-mannitol and d-sorbitol as osmotic stabilizer and as a selective carbon source we tested the growth of H. jecorina TU-6H and the parent strain on MM agar plates with different carbon sources. Growth tests showed that TU-6H strains were find more not able to grow on MM containing 10 g L−1d-mannitol and d-sorbitol, whereas the parent strain was able to use both polyols as sole carbon source. This indicates that, in H. jecorina, the two polyols d-mannitol and d-sorbitol are catabolized via d-fructose as intermediate (Elorza & Arst, 1971; Solomon et al., 2007). To demonstrate the utility of this transformation system, H. jecorina TU-6H was transformed

with the reporter plasmid pHxk1-EGFP. For determination of the optimal selective carbon source and osmotic stabilizer, we compared the effect of 1 M d-sorbitol and 1 M d-mannitol in the regeneration medium on transformation efficiency. Results from three independent parallel transformation experiments showed that d-mannitol leads to higher transformation efficiencies than d-sorbitol. Using d-mannitol, approximately 500–1000 colonies μg−1

Selleck PI3K Inhibitor Library plasmid DNA were obtained, whereas using d-sorbitol, only 100–200 colonies were found. Adenosine triphosphate In control transformation assays with the EGFP expression, plasmid pIG1783 alone or without plasmid DNA, no colonies were found on the selective plates. As shown in Fig. 1a, transformed colonies of variable sizes were seen on selective plates. After purification of the transformants, their growth on different carbon sources was compared with growth on the parental strains (Fig. 1b). The growth of the transformants was fully restored on MM with d-mannitol or d-glucose as carbon source. The presence of the reporter plasmid pHxk1-EGFP in d-mannitol-utilizing transformants was confirmed by the presence of both hxk1 and egfp in the isolated gDNA of different transformants as detected by PCR (Fig. 2a). Three randomly picked transformants were further analyzed by Southern blot to determine the pattern of integration. Figure 2b shows that pHxk1-EGFP integrated into the genome of all analyzed transformants at the hxk1 locus; in all cases, the plasmid integrated next to the promoter region of hxk1. Some of the transformants showed additional hybridizing bands, most likely due to tandem integration or insertion of the plasmid by ectopic integration as well as targeted integration.