, 2007). Furthermore, fungi with no known sexual stage still have functional MAT genes (Sharon et al., 1996; Kerényi et al., 2004), indicating that the lack of sexual reproduction in mitotic holomorphic species is caused by adverse mutations at loci other than the MAT locus. The reasons for the occurrence of functional MAT genes in fungi with no known sexual stage are not well understood. One plausible hypothesis is that the MAT transcriptional factors have some functions during the asexual Regorafenib part of the life cycle and may regulate additional genes not involved directly

in sexual events (Hornok et al., 2007). The MAT genes may thus have a selective impact (e.g. through the stimulation of carotenoid production) on asexually reproducing populations. Another explanation is that these fungi have a cryptic sexual stage, but their teleomorphs have not been identified due to the extreme rarity of mating (Leslie & Klein, 1996; Turgeon, 1998). The regulatory mechanism(s) for light-inducible carotenogenesis in Fusarium species are not fully understood. The white collar (WC) proteins are regarded as a universal photoreceptor system regulating

carotenogenesis and other photoregulated processes in fungi (Corrochano & Avalos, 2010). Recent results on WC1-defective mutants in Fusarium oxysporum and F. fujikuroi indicate, however, that carotenogenesis is regulated differentially in members of the genus Fusarium (Avalos & Estrada, 2010). Light-inducible carotenogenesis CAL 101 was retained in WC1 mutants of these Fusarium species, suggesting the existence of WC-independent photoreceptor mechanisms and/or the involvement of unknown factors in light-dependent carotenogenesis. Our present results confirm that F. verticillioides, like F. fujikuroi, has transcriptional control of carotenogenesis in response to light. The induction of car gene expression

and carotenoid biosynthesis are drastically reduced in the absence of a functional MAT1-2-1 gene. Thus, the regulation of light-induced carotenogenesis in F. verticillioides depends at least in part on MAT1-2-1. This gene is absent in the wild strain of enough the opposite sex, FGSC 7600, which, however, exhibits a normal light induction of carotenogenesis. Presumably, the regulatory role played by MAT1-2-1 in FGSC 7603 is played in FGSC 7600 by an equivalent MAT1-1 gene from its MAT locus (Yun et al., 2000). The available information on photoinduction of carotenogenesis in Fusarium suggests that it is a transcriptionally controlled mechanism mediated by a still unknown regulatory system. The attenuation of this photoresponse in the ΔFvMAT1-2-1 mutants of F. verticillioides reveals a novel key regulatory element in the carotenoid pathway whose connection with the light-inducing mechanism remains to be identified.

We are grateful to Dr Ian Toth at the Scottish Crop Research Institute

for supplying the Pa strains, to Rita Monson for providing cDNA and Nick Thomson for help with bioinformatic comparisons. T.J.E. was supported by a Collaborative Award in Science and Engineering from Leatherhead Food Research. This work was funded by the BBSRC. Fig. S1. Multiple sequence alignment of PflA. Table S1. Supplementary strains and primers used in this study. 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 PTC124 cost for the article. “
“Lipopolysaccharide is a major immunogenic structure for the pathogen Yersinia pseudotuberculosis, which contains the O-specific polysaccharide (OPS) that BYL719 manufacturer is presented on the cell surface. The OPS contains many repeats of the oligosaccharide O-unit and

exhibits a preferred modal chain length that has been shown to be crucial for cell protection in Yersinia. It is well established that the Wzz protein determines the preferred chain length of the OPS, and in its absence, the polymerization of O units by the Wzy polymerase is uncontrolled. However, for Y. pseudotuberculosis, a wzz mutation has never been described. In this study, we examine the effect of Wzz loss in Y. pseudotuberculosis serotype O:2a and compare the lipopolysaccharide chain-length profile to that of Escherichia coli serotype O111. In the absence of Wzz, the lipopolysaccharides of the two species showed significant differences in Wzy polymerization. Yersinia pseudotuberculosis O:2a exhibited only OPS with very short chain lengths, which is atypical of wzz-mutant phenotypes that have been observed for other species. We hypothesise that the Wzy

polymerase of Y. pseudotuberculosis O:2a has a unique default activity in the absence of the Wzz, revealing the requirement of Wzz to drive O-unit polymerization to greater lengths. “
“Flavodoxin (Fld) is a bacterial electron-transfer protein Temsirolimus solubility dmso that possesses flavin mononucleotide as a prosthetic group. In the genomes of the Pseudomonas species, the mioC gene is the sole gene, annotated Fld, but its function remains unclear. In this study, phenotype microarray analysis was performed using the wild-type and mioC mutant of pathogenic Pseudomonas aeruginosa PAO1. Our results showed that the mioC mutant is very resistant to oxidative stress. Different antibiotics and metals worked differently on the sensitivity of the mutant. Other pleiotropic effects of mutation in the mioC gene, such as biofilm formation, aggregation ability, motility and colony morphology, were observed under iron stress conditions. Most of the phenotypic and physiological changes could be recovered in the wild type by complementation.

More than 50% of the faint type colocalized with NG2 and 91% with oligodendrocyte transcription factor-2, whereas 94% of NG2-immunoreactive and 45% of oligodendrocyte transcription factor-2-immunoreactive cells were faintly CNPase-enhanced green fluorescent protein positive. Based on the complexity of the overall structure, the three types probably represent stages of a maturation process such that one subtype can morph into another. Thus, the least complex ‘smooth’ cell

would represent the youngest oligodendrocyte that matures into the stellar type and eventually progresses to become the most complex ramified oligodendrocyte. Investigation of the distribution pattern revealed that the highest density of oligodendrocytes was IDH mutation found in the stratum lacunosum-moleculare and the hilar region. PD-0332991 clinical trial The distribution analysis of oligodendrocyte subclasses revealed a tendency for different cell types to segregate in large non-overlapping areas. This observation suggests that morphologically, and possible functionally, different oligodendrocytes are topographically segregated. “
“The addictive

properties of morphine limit its clinical use. Learned associations that develop between the abused opiate and the environment in which it is consumed are engendered through Pavlovian conditioning processes. Disruption of the learned associations between the opiate and environmental cues may Dynein be a therapeutic approach to prevent morphine dependence. Although a role for the δ-opioid receptor in the regulation of the rewarding properties of morphine has already been shown, in this study we further characterized the role of the δ-opioid receptor in morphine-induced conditioned responses by examining the effect of a selective δ2-opioid receptor antagonist (naltriben), using a conditioned place preference paradigm in rats. Additionally, we used a subcellular fractionation technique to analyze the synaptic localization of μ-opioid and δ-opioid receptors

in the hippocampus, in order to examine the molecular mechanisms that may underlie this morphine-induced conditioned behavior. Our data show that the administration of 1 mg/kg naltriben (but not 0.1 mg/kg) prior to morphine was able to block morphine-induced conditioned place preference. Interestingly, this naltriben-induced disruption of morphine conditioned place preference was associated with a significant increase in the expression of the δ-opioid receptor dimer at the postsynaptic density. In addition, we also observed that morphine conditioned place preference was associated with an increase in the expression of the μ-opoid receptor in the total homogenate. Overall, these results suggest that modulation of the δ-opioid receptor expression and its synaptic localization may constitute a viable therapeutic approach to disrupt morphine-induced conditioned responses.

This was done more than 1 month before leaving by 47.5% of the responders; 25.1% started preparing 2 weeks to 1 month before departure, 15.7% did so 1 to 2 weeks in advance, and 11.6% did so less than 1 week before leaving. Of those who had not sought health information, the majority stated that they already knew what to do. The most common sources since 2004 for travel health advice to high-risk destinations were the travel clinic or

public health service (66.4%) followed by general practitioner (GP) or family doctor in 21.3% of the respondents. For low-to-intermediate-risk destinations the travel clinic BGJ398 mw or public health service was consulted in 53.2% of the respondents, whereas the GP or family doctor was consulted in 27.8% of the cases. In the 2002 and 2003 questionnaires there was no item concerning source of advice. There were no significant trends over the selleck inhibitor years in the proportion of travelers to high-risk destinations seeking travel

health advice (p = 0.315). In contrast, trend analyses in travelers to low-to-intermediate-risk destinations showed a decrease over the years in the proportion of travelers seeking travel health advice (p = 0.0005). The group of older adult travelers comprised 439 respondents. Of them, 365 (83.1%) traveled to a high-risk destination. The group of last-minute travelers comprised 545 respondents; 474 (87.0%) of them traveled to a high-risk destination. Of all respondents, 869 respondents traveled alone and were classified as solo travelers; 650 (74.8%) of them Thiamet G traveled to a high-risk destination. The group of business travelers consisted of 453 individuals of whom 330 (72.8%) traveled to destinations rated as a high risk for hepatitis A. The group of VFRs consisted of 521 respondents; 390 (74.9%) of them traveled to a high-risk destination (Table 1). Older adult travelers to either high-risk (p = 0.076) or low-to-intermediate-risk destinations (p = 0.434) did

not better prepare their vacation than younger-aged travelers to the same risk destination. Older adult travelers visited high-risk destinations more frequently (Table 1). The risk perception and protection rate of older adult travelers to either high-risk or low-to-intermediate-risk destinations was comparable to that of younger travelers (Table 2). Older adult travelers, however, had less intended risk-seeking behavior than younger travelers, irrespective of the hepatitis A risk at the planned destination. As a consequence, as shown in Table 3, the composite risk estimate of KAP of older adult travelers suggested a slight reduction of relative risk for hepatitis A. Solo travelers to either high- (p < 0.001) or low-risk destinations (p < 0.001) had less preparation for their travel than non-solo travelers to the same risk destination. Solo travelers traveled more frequently to low-to-intermediate-risk destinations than to high-risk destinations (Table 1).

Seventy percent of the proteins were assembled into 42 HGs (Supporting Information, Table S1), containing 2–15 members each. The remainder of the proteins form 85 single-member

HGs. The products of wzg, wzz, wzd and wze each fall into a single HG, which is contained in every serotype. These four HGs (Wzg, Wzz, Wzd, and Wze) are the largest groups. The next largest HG consists of nine WcdA CapD-like proteins (HG4), followed by six WchA initial glycosylphosphotransferases (HG5). There are 12 groups of Wzy repeat-unit polymerases and nine groups of Wzx flippases. A pseudogene in serotype 8 cps locus is caused by frame shift. The first four genes, wzg, wzz, wze and wzd (also known as cpsABCD), are conserved with high sequence identity in all 15 serotypes. Wzg and Wzz proteins were predicted to play an important role in the synthesis regulation and the chain Selleckchem Forskolin length determination of CPS in the S. suis serotype 2. Isogenic mutants in wzg

gene cannot produce CPS (Smith et al., 1999a, b, c). The exact function of Wze and Wzd in S. suis is unknown. wze and wzd were also found in other Streptococcus capsule gene clusters (Wessels, 1997). The two proteins are in the MPA1 class of the Paulsen et al. (1997) classification and are thought to be involved in polysaccharide export. It was reported that Wzd is a tyrosine kinase and Wze is a substrate for Wzd kinase in S. pneumoniae (Morona et al., 2003) and the Wzd and Wze proteins may play similar roles in S. suis. The initial glycosylphosphotransferases are responsible Sirolimus cost for linkage of an activated glycosylphosphate to the lipid carrier (Pelosi et al., 2005). The initial glycosylphosphotransferases of all

the 15 serotypes fall into four HGs (WchA, WciI, WcaJ and WcgA). In the group 2 (serotypes 1, 2, 8, 14, 16, 25 and 1/2) cps locus, all the initial transferase genes are wchA, the products of which can add glucose-1-phosphate to undecaprenol phosphate to create Und-PP-Glc (Kolkman, et al., 1997). wchA is absent in the group 1 (serotype 3, 4, 5, 7, 9, 10, 19 and 23) cps locus. The product of the fifth cps gene is a CapD-like protein (WcdA), which can generate amide bonds with peptidoglycan cross-bridges to anchor capsular material within the cell wall envelope (Candela & STK38 Fouet, 2005). In the group 1 locus, the initial transferase genes (wciI, wcaJ and wcgA) are downstream of wcdA. Because the exact composition and structure of most S. suis serotypes CPS is unknown, the transferred sugars of the initial transferases can only be suspected, based on the function of similar proteins of other bacteria. WciI proteins showed a high degree of similarity to that of S. pneumoniae serotype 4 (62% identity). The transferred initial sugar for WciI in S. suis was predicted to be N-acetylgalactosamine pyranose (GalpNAc) or N-acetylglucosamine pyranose (GlcpNAc) (Bentley et al., 2006).

Other studies suggest continued immunological and clinical benefits if the HIV RNA level is maintained <10 000–20 000 copies/mL [66]. Continuing or commencing NRTIs, even in the presence of known resistance may contribute partial ARV activity [54, 55]. Hence, if the CD4 cell count is well maintained (>200 cells/μL), it may be better to continue the failing regimen and not change treatment until investigational agents are available that can be put together with drugs, which may have only partial activity

at best, to increase the likelihood of constructing virologically suppressive and durable regimen options. In general, selleck screening library adding a single, fully active ARV to a failing regimen is not recommended because of the risk of rapid development of resistance. However, in patients with a high likelihood of clinical progression (e.g. CD4 cell count <100 cells/mL) and limited drug options, adding a single drug may reduce the risk of immediate clinical

progression, because even transient decreases in HIV RNA and/or transient increases in CD4 cell counts have been associated with clinical benefits [67]. Potential benefits must be balanced with the ongoing risk of accumulating additional resistance mutations and patients should maintain that regimen for the shortest period possible [68, 69]. Where feasible, patients should be given the opportunity to enrol in research studies or expanded access programmes evaluating investigational new drugs. Some drugs are likely to be available in the near future that might be find more sequenced in the same class (e.g. dolutegravir) although others with novel sites of action (e.g. maturation Ribonucleotide reductase inhibitors, CD4 receptor antagonists, etc.) are still in earlier phases of development and some years off randomized trials. Drugs developed

for, and used in, other settings such as pegylated interferon that have been incidentally demonstrated to decrease VL should not be used without discussion with an experienced HIV physician as data are either too limited or contradictory. Several studies and an early meta-analysis suggested that CCR5 receptor antagonists were associated with significant gains in CD4 cell counts even in the presence of C-X-C chemokine receptor type 4 tropic virus. However, a more recent meta-analysis refuted this finding (P=0.22) when comparing with other new drugs [53]. A priority question that the Writing Group addressed was whether 3TC/FTC should be used in maintaining an RT mutation at codon 184 in patients with limited or no therapeutic options. Although the M184V mutation is associated with resistance to 3TC/FTC, the mutation has a broad influence on the RT enzyme. In vitro studies have shown that M184V-possessing enzymes have lower processivity and higher fidelity and replicate more slowly than WT enzymes [70].

3f ). As indicated above, IAL does not inhibit the growth of S. aureus; therefore, it can be concluded that IAL did not decrease S. aureus CFUs, which then led to a decrease in A549 cell injury. The in vitro results show that low concentrations of IAL inhibit the production of α-toxin by S. aureus and attenuate α-toxin-mediated injury of human lung cells, which indicates that IAL has potential therapeutic relevance. To investigate the in vivo protective effects of IAL on mouse S. aureus-related pneumonia, we first assessed its pharmacokinetic characteristics in mice. Time–concentration

profiles of plasma for three single subcutaneous IAL doses are presented in Fig. 4. The maximum concentrations of IAL in plasma (Cmax) were 6.16, 15.67, and 32.66 μg mL−1 for doses of 10, 25, and 50 mg kg−1, respectively. The area under

each of the concentration–time selleckchem curves (AUC) for plasma was calculated from 0.25 to 24 h and was 29.73, 82.69, and 206.31, for doses of 10, 25, and 50 mg kg−1, respectively. Mice were infected via the intranasal route with 4 × 108 CFUs of S. aureus 8325-4. Following treatment with IAL as described in the ‘Materials and methods’, mortality was monitored over 72 h. As a control, the mortality following infection with an hla−S. aureus strain DU 1090 was also determined. As shown in Fig. 5a, MAPK Inhibitor Library mice that received 50 mg kg−1 of IAL were significantly protected from S. aureus pneumonia (P < 0.05); however, the mortality was much higher than that in mice infected with S. aureus DU 1090. The protective effect was less evident in mice that received 25 mg kg−1

of IAL, and little protective effect was observed in mice that were given 10 mg kg−1 Sirolimus of IAL. To evaluate the impact of IAL treatment on pathological manifestations of lung injury, we performed histopathologic analysis of lungs from S. aureus-infected mice that received 50 mg kg−1 of IAL or PBS as a control. Gross inspection indicated that the lung tissue of infected mice was crimson and had a tight texture. Following treatment with IAL, the lung tissue of infected mice was light pink and fungous (Fig. 5b). As shown in the Fig. 5c, there were significant accumulations of inflammatory cells (dark blue or purple) in alveolar space in the group infected with S. aureus 8325-4. Notably, treatment with IAL resulted in a marked alleviation of pulmonary inflammation; treated mice had less accumulation of cellular infiltrates in the alveolar space. The increase in resistance of S. aureus to β-lactam antibiotics as well as the decreased clinical performance of vancomycin and linezolid (Mandell et al., 2007; Nguyen & Graber, 2010), combined with a decrease in the discovery of new antibiotics (Liu et al., 2008), warrants the search for new therapeutic targets to combat infections caused by S. aureus.

Long-term potentiation and long-term depression, long held as the principal means of producing lasting change in cerebral circuits, are easily induced in the hippocampus (Bliss & Lomo, 1973; Dudek & Bear, 1992) but are more difficult to produce in the cortex (Trepel & Racine, 1998). Induction of synaptic plasticity in the cortex requires multiple sessions of tetanising trains to be Selleck E7080 effective and reflects the relative stability of neocortical circuits. While the mechanisms underlying the ability of tDCS to produce lasting neural changes in these circuits have not yet been fully established (see Stagg & Nitsche, 2011; Márquez-Ruiz et al.,

2012), the number of sessions required for recovery is probably due to tDCS overcoming cortical resistance to synaptic plasticity, a delay period in the accumulation of critical Gefitinib chemical structure neuromodulators or growth factors (e.g., brain-derived neurotrophic factor; Fritsch et al., 2010),

or both. Recovery was only observed to more peripherally located visual targets, and this finding may reflect a limited capacity of the tDCS to penetrate into the depths of the cortex. The targeted cortex is retinotopically organised: the representation of the contralateral peripheral visual field is located near the skull on the crest of each gyrus, and the neurons in the fundus of the sulcus represent central and pericentral locations (Palmer et al., 1978). The behavioral results, therefore, may reflect a selective reduction in activity or in the firing probability of the neurons that represent peripheral targets Thymidylate synthase and that are located closer to the skull. The results also may reflect selective activation of neurons in this cortex whose somatodendritic or axonal axes is optimally oriented to the electric field (e.g., Bikson et al., 2004; Radman et al., 2009; Kabakov et al., 2012). The behavioral results

also indicate that the resting membrane potential of neurons near the depth of the sulcus, which correspond to central visual field locations (Palmer et al., 1978), may not be sufficiently modulated by tDCS to produce a behavioral change. In as much as functional alterations in these neurons are the basis for the recovery, this result runs counter to predictions of modeling studies that show a preferential effect of tDCS on neurons at the bases of sulci (Miranda et al., 2013). Moreover, the present results suggest that the tDCS-mediated reduction in activity also does not feed down to neurons in the depth of the sulcus through substantial intra-areal circuits demonstrated to fill this region (Norita et al., 1996). Further modeling of tDCS currents and biological study is required to provide a definitive answer to the mechanisms and the precise neuronal elements underlying the present results. It is notable that one animal did not respond to tDCS treatment. Examination of the lesion showed no identifiable differences in terms of size or extent of lesion.

The DDBs were characterized by Gram staining and the 16S rRNA genes were analysed as described by Ikunaga et al. (2011). This yielded approximately 1200 bp of useful 16S rRNA gene sequence. Sequences similar to the 16S rRNA gene of isolated strains were identified using blast searches (National Center AZD2281 for Biotechnology Information, http://www.ncbi.nlm.nih.gov). The

16S rRNA gene sequences determined in this study have been deposited in the DNA Data Bank of Japan (DDBJ) under accession numbers AB627753 to AB627765. Culture samples were filtered through 0.45-μm membranes (Advantec, Tokyo, Japan) and 10 μL was directly injected on to an HPLC system. The HPLC system (Waters, Milford, MA) consisted of a 600E pump, a 2487 dual absorbance detector, a Waters Symmetry C18 column (3.9 mm ID × 150 mm; Waters) and empower 2 software. The mobile phase contained methanol and water (15:85, v/v) at a flow rate of 1.0 mL min−1. A wavelength of 220 nm was used. The column was heated to 40 °C. Authentic DON and 3-epi-DON were detected at retention

times of 6.5 and 4.5 min, respectively. The DDBs were cultured on the agar plates at 28 °C for 5 days. These bacteria (OD600 nm of 0.2) were then preincubated in DON mineral medium (DMM, MM containing 20 μg mL−1 DON), 1/3R2A and 1/3LB liquid media at 28 °C for 24 h. After incubation, bacterial cells were recovered by centrifugation at 6000 g for 10 min, washed twice with 50 mM potassium phosphate buffer (pH 7.0), and resuspended

Cyclopamine cost in the same buffer containing 100 μg mL−1 DON to achieve an OD600 nm of 0.8 (equivalent to 1.3 mg dry weight mL−1). Samples were incubated at 25 °C, collected at various time points, filtered and subjected to HPLC. Initial DON degradation rates were measured within the period of linear DON degradation. Three buffers, noninoculated cells containing DON, inoculated cells without DON and inoculated autoclaved cells (121 °C, 20 min) containing DON, were also analysed as controls. Cells were inoculated into MM with or without 100 μg mL−1 DON and incubated at 120 r.p.m. and 28 °C after precultivating on the agar plates at 28 °C for 5 days. Culture media were RG7420 datasheet collected every other day, appropriately diluted and spread onto 1/3LB agar plates for strains SS5 and RS1, and onto 1/3R2A agar plates for the other strains. These agar plates were incubated at 28 °C for 7 days, and the numbers of colonies were counted. Statistical analysis of the data was carried out using jmp software (version 5.01J; SAS Institute Japan, Tokyo, Japan). Significant differences between the means were determined by using a t-test and Tukey test variance analysis (P < 0.05). A total of 169 environmental samples (61 soil, 78 wheat leaf and 30 wheat spikelet samples) were used for enrichment culture.

The ‘core’ of the S. coelicolor linear chromosome from c. 1.5 to 6.4 Mb contains genes unconditionally essential for growth and propagation, while the two ‘arms’ (c. 1.5 Mb for the left and 2.3 Mb for the right), carrying conditionally adaptive genes, are presumptively deletable (Bentley et al., 2002; Hopwood, 2006). Deletions of these large segments near telomeres will make a compact S. coelicolor genome for studying the functions of the linear chromosome.

Here, we report experimental determination of extent of the two deletable arm regions and sequential Dabrafenib cell line deletion of all the PKS and NRPS biosynthetic genes, together with a 900-kb subtelomeric sequence. Actinorhodin production was enhanced when the act gene cluster was reintroduced into some of the deleted

strains. Strains and plasmids used in this work are listed in Table 1 and all oligonucleotides in Supporting information, Table S1. Plasmid isolation, transformation of Escherichia coli DH5α, and PCR amplification GSK2126458 followed Sambrook et al. (1989). Escherichia coli DH10B was used as the host for propagating cosmids. Escherichia coli ET12567 (pUZ8002) was used as a nonmethylating strain for conjugation with Streptomyces strains. Escherichia coli BW25113 was used to propagate plasmid pIJ790. Streptomyces cultures and isolation of Streptomyces genomic DNA followed Kieser et al. (2000). For observation of sporulation, Streptomyces strains were grown on MS medium (mannitol, 20 g; soya flour, 20 g; agar, 20 g; H2O,

1 L) covered with cellophane disks. The cells were fixed with 2% glutaraldehyde (pH 7.2) and 1% osmium tetroxide. After dehydration, ethanol was replaced AZD9291 ic50 by amyl acetate. The samples were then dried by the supercritical drying method in HCP-2 (Hitachi Inc.), coated with gold by Fine Coater JFC-1600, and examined with a JSM-6360LV scanning electron microscopy (Jeol Inc.). Genomic DNA of S. coelicolor M145 was partially digested with Sau3AI, and fragments were sized by sucrose gradient centrifugation (Kieser et al., 2000). The 35–45 kb fractions were dephosphorylated with calf-intestine alkaline phosphatase (CIAP) and then ligated with pHAQ31 (BamHI). The ligation mixture was packaged in vitro using the Giga-pack® III XL Gold Packaging Extract kit (Stratagene Inc.). Approximately 2000 cosmids were isolated, and the inserted sequences were determined by PCR sequencing with two primers from the flanking sequences of the pHAQ31-BamHI site. The insertion sequences were blasted on the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST). By comparison with the complete nucleotide sequence of the S. coelicolor chromosome (Bentley et al., 2002), we obtained an ordered cosmid library.