Such a combination of repeat duplication followed by point mutation has been described by Kahl et al. (2005). In another case (patient 12), the observed change could be either Ponatinib duplication or a deletion (t844 and t953, eight and nine repeats; Table 2). Eight samples were collected from this patient at the same time, six of them containing MRSA t844 and two of them MRSA t953. We cannot determine whether t844

or t953 is the ancestor spa type, i.e. if there was a duplication or deletion of a repeat 12. However, at some point a duplication of repeat 12 must have taken place, making it more likely that t844 is the ancestor. The same argument can be applied to t3677 and t4225 (patient 13) with one or two copies of repeat Enzalutamide cost 31. The

spa type t024 was by far the most common spa type found. This spa type was found in 796 isolates from 213 patients. In addition, six patients had only the Variant spa type t430 that was the most common t024 variant. It was first found on a t024-positive resident of a nursing home and subsequently has only been found on residents from that nursing home. In seven patients, the Ancestor and Variant spa types were found simultaneously in the first MRSA-positive sampling (patients 2, 4, 5, 7, 8, 9 and 12; Table 3). No follow-up data were recorded for patients 2, 9 and 12. In patients 4, 5 and 7, only the Ancestor was found in the latest sample, and one patient (patient 8) had both spa types at both sample times. In five cases (patients 1, 3, 10, 11 and 13) the Variant was recovered between 1 and 20 months after the Ancestor. At the latest sampling, the Ancestor alone or both spa types were recovered in one case each (patient 1 and 3) while the Variant alone was found in three patients (patients 10, 11 and 13) (Table 3). In one

case only, Org 27569 the suggested Variant was the first to be found (patient 6). At the second sampling from this patient, both Ancestor and Variant were recovered (patient 6). We found that the spa repeat region was very stable. Of 319 individuals with more than one occasion of MRSA, only 13 (4%) exhibited spa type changes. Of 1536 isolates, only 30 (2%) had changes in the spa repeat region. In a Swiss study, 10% of healthy carriers exhibited spa type mutations within 1 year (Sakwinska et al., 2010). This number is higher than ours but still in the same range of order. This is in contrast to the study by Kahl et al. (2005) of cystic fibrosis patients with persistent S. aureus airway infections. In that study, spa type alterations were found in MSSA isolates from five of 10 patients (50%) and in 14 of 142 isolates (10%). The mutation rates probably reflect differences in selection pressure. The isolates in our study are from infections as well as long-term carriers. The selection pressures under which the S. aureus existed were probably higher for the cystic fibrosis patients (Kahl et al., 2005).

Such a combination of repeat duplication followed by point mutation has been described by Kahl et al. (2005). In another case (patient 12), the observed change could be either this website duplication or a deletion (t844 and t953, eight and nine repeats; Table 2). Eight samples were collected from this patient at the same time, six of them containing MRSA t844 and two of them MRSA t953. We cannot determine whether t844

or t953 is the ancestor spa type, i.e. if there was a duplication or deletion of a repeat 12. However, at some point a duplication of repeat 12 must have taken place, making it more likely that t844 is the ancestor. The same argument can be applied to t3677 and t4225 (patient 13) with one or two copies of repeat CTLA-4 inhibiton 31. The

spa type t024 was by far the most common spa type found. This spa type was found in 796 isolates from 213 patients. In addition, six patients had only the Variant spa type t430 that was the most common t024 variant. It was first found on a t024-positive resident of a nursing home and subsequently has only been found on residents from that nursing home. In seven patients, the Ancestor and Variant spa types were found simultaneously in the first MRSA-positive sampling (patients 2, 4, 5, 7, 8, 9 and 12; Table 3). No follow-up data were recorded for patients 2, 9 and 12. In patients 4, 5 and 7, only the Ancestor was found in the latest sample, and one patient (patient 8) had both spa types at both sample times. In five cases (patients 1, 3, 10, 11 and 13) the Variant was recovered between 1 and 20 months after the Ancestor. At the latest sampling, the Ancestor alone or both spa types were recovered in one case each (patient 1 and 3) while the Variant alone was found in three patients (patients 10, 11 and 13) (Table 3). In one

case only, tuclazepam the suggested Variant was the first to be found (patient 6). At the second sampling from this patient, both Ancestor and Variant were recovered (patient 6). We found that the spa repeat region was very stable. Of 319 individuals with more than one occasion of MRSA, only 13 (4%) exhibited spa type changes. Of 1536 isolates, only 30 (2%) had changes in the spa repeat region. In a Swiss study, 10% of healthy carriers exhibited spa type mutations within 1 year (Sakwinska et al., 2010). This number is higher than ours but still in the same range of order. This is in contrast to the study by Kahl et al. (2005) of cystic fibrosis patients with persistent S. aureus airway infections. In that study, spa type alterations were found in MSSA isolates from five of 10 patients (50%) and in 14 of 142 isolates (10%). The mutation rates probably reflect differences in selection pressure. The isolates in our study are from infections as well as long-term carriers. The selection pressures under which the S. aureus existed were probably higher for the cystic fibrosis patients (Kahl et al., 2005).

nidulans argB as a selectable marker. Transformants were streak purified and verified for correct integration into NVP-BEZ235 solubility dmso the IS1 site (Hansen et al., 2011) by two complementing diagnostic PCRs. Strains were inoculated as three point stabs on solid media and incubated for 7 days at 37 °C in the dark. Metabolite extraction was performed according to the micro extraction procedure (Smedsgaard, 1997). Extracts were analyzed by two methods:

(1) Ultra-high performance liquid chromatography-diode array detection (UHPLC-DAD) analyses using a Dionex RSLC Ultimate 3000 (Dionex, Sunnyvale, CA) equipped with a diode-array detector. Separation of 1 μL extract was obtained on a Kinetex C18 column (150 × 2.1 mm, 2.6 μm; Phenomenex, Torrence, CA) at 60 °C using a linear water–acetonitrile gradient starting from Opaganib in vivo 15% CH3CN to 100% (50 ppm trifluoroacetic acid) over 7 min at a flow rate of 0.8 mL min−1. (2) Exact mass, HPLC-DAD-HRMS, was performed on a 5 cm × 3 μm, Luna C18(2) column (Phenomenex) using a water–acetonitrile gradient from 15% CH3CN to 100% over 20 min (20 mM formic acid). LC-DAD-MS analysis was performed on a LCT oaTOF mass spectrometer (Micromass, Manchester, UK) as in Nielsen & Smedsgaard (2003) and Nielsen et al. (2009). 3,5-Dimethylorsellinic acid and dehydroaustinol

were purified from large-scale ethyl acetate extracts prepared from 100 MM agar plates after 4 days’ cultivation in darkness at 37 °C. The compounds were purified using a 10 × 250 mm Phenomenex pentafluorophenyl column (5 μm particles) with a water–acetonitrile gradient from 15% to 100% CH3CN in 20 min using a flow of 5 mL min−1. Arugosin A was isolated from an ethyl acetate extract of the reference strain grown on 200 CYAs agar plates using a Waters 19 × 300 mm C18 Delta Pak column (15 μm particles), gradient from 80% to 90% CH3CN in 10 min, and a flow of 30 mL min−1. The NMR spectra were acquired on a Varian Unity Inova 500 MHz spectrometer using standard ROS1 pulse sequences. Additional details about the compound identification can be found in the supporting information.

The principle of using different media and/or incubation conditions for fungal secondary metabolite production has often been promoted (Oxford et al., 1935; Davis et al., 1966; Pitt et al., 1983; Bode et al., 2002; Scherlach & Hertweck, 2006). Based on our previous experiences (Frisvad, 1981; Frisvad & Filtenborg, 1983; Filtenborg et al., 1990; Frisvad et al., 2007), eight different media, CYA, CYAs, CY20, MM, RT, RTO, YE and YES, were initially selected for the analysis (Fig. 1a). HPLC analyses revealed a large number of different secondary metabolites produced by the A. nidulans reference strain on CYA, CYAs, CY20, RT, RTO and YES (Fig. 1b) and these metabolites served as a source for further investigation. To investigate whether any of the compounds observed in Fig. 1 could be genetically linked to a PKS gene, we decided to take a global approach and individually deleted all 32 (putative and known) PKS genes in the A.

The frequency of rash in the week 96 analysis was higher with etravirine than with placebo; however, rash infrequently led to treatment interruption or discontinuation. In addition, the frequency of rash occurring selleck chemicals after 48 weeks was low. Etravirine use does not appear to be associated with an increased risk of neuropsychiatric or hepatic AEs, as the frequency and severity of such events over 96 weeks were similar to those for the placebo group. Similarly, etravirine was not associated with a greater emergence

of lipid abnormalities in treatment-experienced patients. The authors thank the patients and their families, the investigators who recruited patients to the DUET trials, study centre staff, the Data Safety and Monitoring Board and Tibotec study personnel. They also acknowledge

David Anderson, Eric Lefebvre and Frank Tomaka for their important contributions to the manuscript. Medical writing assistance was provided by Karen Pilgram (Medical Writer, Gardiner-Caldwell Communications, Macclesfield, UK); funding for this service was provided by Tibotec Pharmaceuticals Ltd. The DUET trials were sponsored by Tibotec Pharmaceuticals Ltd. Conflicts of interest: The authors disclose the following conflicts. PMG has received support for travel to meetings for the study or other purposes from Abbot, Bristol Cobimetinib supplier Myers Squibb and Gilead Sciences, and renumeration for Board Membership from Abbott, Gilead Sciences and Tibotec/Janssen. before TBC has received support for travel to a scientific meeting for presentation of this study. BG has received research support from Janssen Pharmaceutic Inc., Merck and Co Inc. and Schering Plough Corporation and has served as a consultant for ARDEA Biosciences. JH and AR have received support for travel to meetings from Tibotec/Janssen. SN and JW are full-time employees of Tibotec. JW is a J&J stockholder. “
“Darunavir was designed for activity against HIV resistant to other protease inhibitors (PIs). We assessed the efficacy, tolerability and risk factors for virological failure of darunavir for treatment-experienced patients seen in

clinical practice. We included all patients in the Swiss HIV Cohort Study starting darunavir after recording a viral load above 1000 HIV-1 RNA copies/mL given prior exposure to both PIs and nonnucleoside reverse transcriptase inhibitors. We followed these patients for up to 72 weeks, assessed virological failure using different loss of virological response algorithms and evaluated risk factors for virological failure using a Bayesian method to fit discrete Cox proportional hazard models. Among 130 treatment-experienced patients starting darunavir, the median age was 47 years, the median duration of HIV infection was 16 years, and 82% received mono or dual antiretroviral therapy before starting highly active antiretroviral therapy.

About 0.5 g of the surface-disinfected reed roots were frozen with liquid nitrogen and ground to a fine powder in a sterilized and precooled mortar. Then, the hot cetyltrimethylammonium bromide (CTAB) procedure (Xie et al., 1999) was used to extract the total DNA. The DNA was then resuspended in 25 μL of sterile Milli-Q water. The pair of primers 799f (5′-AACAGGATTAGATACCCTG-3′) and 1492r (5′-GGTTACCTTGTTACGACTT-3′) (Chelius & Triplett, 2001) was selected to amplify the DNA of reed endophytic bacteria. The 50-μL PCR mixture contained 100 ng of DNA extract, 5 μL 10 × Taq reaction buffer (including 1.5 mM MgCl2), 10 pmol of each primer, check details 200 μM each dNTP, and 1.5 U of Taq DNA polymerase (Takara

Co.). After initial denaturation at 94 °C for 5 min, each thermal cycling was as follows: denaturation at 94 °C for 1 min, annealing at 53 °C for 1 min, and elongation at 72 °C for 1 min. At the end of 30 cycles, the final extension step was at 72 °C for 15 min. Products of four parallel PCRs were combined and separated electrophoretically. A band approximately 700 bp in size in the electrophoresis pattern was excised from a 1% agarose gel and purified using the Gel Extraction Kit (Omega Co.) as described by the manufacturer. The purified PCR products were ligated into the pMD18-T vector (Takara Co.). Escherichia coli Top10 competent cells (Tiangen

Co.) were transformed with the ligation LY294002 mouse products and spread onto LB agar plates with ampicillin (100 mg L−1) for standard blue and white screening (Sambrook et al., 1989). Randomly selected colonies were screened directly for inserts by performing colony PCR with of primers RV-M (5′-GAGCGGATAACAATTTCACACAGG-3′) and M13-47 (5′-CGCCAGGGTTTTCCCAGTCACGAC-3′) for the vector (Takara Co.). A total of 180 clones containing inserts of the correct size were sequenced

using an ABI PRISM 3730 automatic sequencer (Shanghai Sangon Co. Ltd). After being trimmed by removing the vector sequences using the editseq program in the dnastar package (Burland, 2000), clones with >97% sequence identity were grouped into one operational taxonomic unit (OTU) by sequencher 4.8 (Gene Codes, Ann Arbor, MI). All the nucleotide sequences, approximately 700 bases, were compared with the NCBI database using blastn or aligned by the identify analysis of EzTaxon server 2.1 (Chun et al., 2007). Sequences with >97% similarity were assigned to the same species and those with >95% similarity were assigned to the same genus. The sequences were aligned using clustal w (Thompson et al., 1994), and tree constructions were performed with the mega 3 program package (Kumar et al., 2004) using the neighbor-joining method. Bootstrap analysis was performed using data resampled 1000 times. The trees were constructed by calculating Kimura distances (Kimura, 1980).

Owing to this study, the University of Utah now requires that new nurses observe a minimum of five pre-travel consults, participate in five mock travel consults, and are observed for five complete pre-travel consults. The model presented here is reliable, reproducible, and can be tailored according to the local needs and legal requirements regarding the scope of nursing practice. It could be used in large urban areas where physicians and subspecialists are in high demand with many factors competing for their time. It also has application for training and maintaining qualified personnel in rural and other remote

areas where it is difficult to regularly serve the critical number of travelers to receive adequate experience. There is a growing need for standardized travel clinics throughout the world. The University of Utah

has learn more created a model where multiple LDK378 nmr pre-travel clinics throughout the state can be staffed by nurses, who are effectively trained, consistently supervised and who maintain a high level of expertise. Further work is needed to gather data to objectively demonstrate the effectiveness of these clinics in safely providing for the travel needs of the population and in preventing disease in the international traveler. The authors would like to thank Charles Langelier, MD, PhD candidate, for his help with editing and proof reading the manuscript. The authors state that they have no conflicts of interest to declare. “
“Background. Health-care professionals can help travelers by providing accurate pre-travel counseling for mosquito-transmitted diseases such as malaria, yellow fever, and dengue fever. Governments and international organizations will benefit from knowledge survey among health professionals in this field to promote the development of travel health profession. This study investigates

physicians’ and nurses’ knowledge regarding malaria, yellow fever, and dengue fever. Methods. A cross-sectional questionnaire survey was distributed to physicians and nurses in Taiwan interested in travel medicine between April and September of 2008. The self-administered, single-choice questionnaire evaluated knowledge regarding epidemiology, prophylactic medication for Ribose-5-phosphate isomerase malaria, yellow fever, and dengue fever, and vaccinations for yellow fever as well as background information of participants. Results. Complete information was collected from 82 physicians and 203 nurses. (Out of 289, effective response rate = 99.9%). The mean percentage of accurate responses was similar for all three diseases: malaria 67.3% (range, 16.8%–90.5%); yellow fever 65.4% (39.6%–79.3%); and dengue fever 74.4% (14.4%–96.5%). The items with the lowest accuracy were (1) behavior of the dengue fever vector Aedes aegypti mosquito (14.4%) and (2) incubation period of malaria (16.8%). There were 60.4% participants who did not know the current revaccination interval for the yellow fever vaccine.

The MICs of H2O2 and t-BHP were 100 μM and 1 mM, respectively, for IK-1 and 10 and 100 μM, respectively, for IK-1Δ8 (Fig. 1a). IK-1 was more resistant to the two ROS tested than was IK-1Δ8. The same tendency was observed when cells of IK-1 and IK-1Δ8 were treated with various kinds of water-soluble antibiotics including ampicillin sodium, kanamycin sulphate, streptomycin sulphate, and tetracycline hydrochloride. The results are summarized in Table 1. The proton ionophore, CCCP, and the ATP synthase inhibitor, DCCD, are water-insoluble

and ethanol-soluble compounds. CCCP and DCCD were dissolved in absolute ethanol. The final concentration of ethanol in the culture medium was 1% (v/v), and this concentration Hydroxychloroquine solubility dmso of ethanol had no effect on the growth of IK-1 or IK-1Δ8. The MICs of CCCP and DCCD were 1 μM and 1 mM, respectively, for IK-1 and 10 μM and >10 mM, respectively, for IK-1Δ8 (Fig. 1b and Table 1). Although the growth of IK-1Δ8 at 1 and 10 mM DCCD appeared to be lower than that at ≤0.1 mM DCCD Fulvestrant after 4 days at

20 °C (Fig. 1b), prolonged incubation of all IK-1Δ8 cultures at a DCCD concentration of ≤10 mM produced almost the same turbidity. In contrast, the growth of IK-1 was never observed at a concentration of DCCD of ≥1 mM. The cell surface hydrophobicity is expressed as the percent adhesion of bacterial cells to water measured using the BATH method (Rosenberg et al., 1980). In cells grown at 20 °C, the values were 94±1% and 99±1% for IK-1 and IK-1Δ8, respectively: the surface hydrophobicity was greater

in IK-1 cells, in which EPA comprised 8% of the total fatty acids, than in IK-1Δ8 cells. IK-1 with EPA was more resistant than IK-1Δ8 with no EPA to H2O2 and to t-BHP, an analogue of H2O2 (Fig. 1a and Table 1), suggesting that catalases or other H2O2-decomposing enzymes are not involved in the resistance of IK-1. The finding that IK-1 was slightly more resistant to all the water-soluble antibiotics tested than was IK-1Δ8 (Table 1) suggests that hydrophilic compounds other than ROS may be hindered from entering the cell through the cell membrane by the membrane-shielding effect more efficiently in IK-1 Digestive enzyme than in IK-1Δ8 cells, as was the case for hydrophilic ROS. However, in Gram-negative bacteria, hydrophilic antibiotics with a molecular weight less than about 600 pass nonspecifically through porin channels on the outer membrane and not by diffusion (Nikaido & Vaara, 1985) and the compounds that enter the cells can be pumped out from the cells (Walsh, 2000; Martinez et al., 2009). Therefore, the membrane-shielding effects of EPA are not necessarily involved directly in the higher resistance to these antibiotics in IK-1 cells. However, because the entry of streptomycin sulphate, whose molecular weight (1457.

The MICs of H2O2 and t-BHP were 100 μM and 1 mM, respectively, for IK-1 and 10 and 100 μM, respectively, for IK-1Δ8 (Fig. 1a). IK-1 was more resistant to the two ROS tested than was IK-1Δ8. The same tendency was observed when cells of IK-1 and IK-1Δ8 were treated with various kinds of water-soluble antibiotics including ampicillin sodium, kanamycin sulphate, streptomycin sulphate, and tetracycline hydrochloride. The results are summarized in Table 1. The proton ionophore, CCCP, and the ATP synthase inhibitor, DCCD, are water-insoluble

and ethanol-soluble compounds. CCCP and DCCD were dissolved in absolute ethanol. The final concentration of ethanol in the culture medium was 1% (v/v), and this concentration Selisistat cost of ethanol had no effect on the growth of IK-1 or IK-1Δ8. The MICs of CCCP and DCCD were 1 μM and 1 mM, respectively, for IK-1 and 10 μM and >10 mM, respectively, for IK-1Δ8 (Fig. 1b and Table 1). Although the growth of IK-1Δ8 at 1 and 10 mM DCCD appeared to be lower than that at ≤0.1 mM DCCD selleck inhibitor after 4 days at

20 °C (Fig. 1b), prolonged incubation of all IK-1Δ8 cultures at a DCCD concentration of ≤10 mM produced almost the same turbidity. In contrast, the growth of IK-1 was never observed at a concentration of DCCD of ≥1 mM. The cell surface hydrophobicity is expressed as the percent adhesion of bacterial cells to water measured using the BATH method (Rosenberg et al., 1980). In cells grown at 20 °C, the values were 94±1% and 99±1% for IK-1 and IK-1Δ8, respectively: the surface hydrophobicity was greater

in IK-1 cells, in which EPA comprised 8% of the total fatty acids, than in IK-1Δ8 cells. IK-1 with EPA was more resistant than IK-1Δ8 with no EPA to H2O2 and to t-BHP, an analogue of H2O2 (Fig. 1a and Table 1), suggesting that catalases or other H2O2-decomposing enzymes are not involved in the resistance of IK-1. The finding that IK-1 was slightly more resistant to all the water-soluble antibiotics tested than was IK-1Δ8 (Table 1) suggests that hydrophilic compounds other than ROS may be hindered from entering the cell through the cell membrane by the membrane-shielding effect more efficiently in IK-1 Resminostat than in IK-1Δ8 cells, as was the case for hydrophilic ROS. However, in Gram-negative bacteria, hydrophilic antibiotics with a molecular weight less than about 600 pass nonspecifically through porin channels on the outer membrane and not by diffusion (Nikaido & Vaara, 1985) and the compounds that enter the cells can be pumped out from the cells (Walsh, 2000; Martinez et al., 2009). Therefore, the membrane-shielding effects of EPA are not necessarily involved directly in the higher resistance to these antibiotics in IK-1 cells. However, because the entry of streptomycin sulphate, whose molecular weight (1457.

(2010) on cell growth and metabolite concentration profiles. Izumi et al. (1994) reported that R. erythopolis D-1 desulfurized DBT to 2-hydroxybiphenyl (HBP) successfully. They used 500 mL of PXD101 a glucose-based biosynthetic medium with 0.125 mM DBT as the sole sulfur source at 30 °C to examine the desulfurization activity of growing cells.

They measured pH, cell growth, DBT concentration, and HBP concentration at various times during their experiment. In another study, Davoodi-Dehaghani et al. (2010) isolated R. erythropolis SHT87. They used growing cells at 30 °C in a 50 mL solution of glycerol containing a synthetic medium with 0.25 mM of DBT as the sole sulfur source. They also measured cell growth, DBT concentration, and HBP concentration at different times over 120 h. The experimental data from the above two independent studies provided a sound basis for validating our proposed model. We used their cell growth data and DBT/HBP concentration profiles from the exponential Daporinad concentration phase to compute specific cell growth rates (1 h−1) and DBT (HBP) uptake (secretion) rates (mmol g−1 dcw h−1). Our reconstructed model consists of 87 intracellular metabolic reactions, 66 transport reactions, and 196 metabolites related to either sulfur or

central metabolism. The sulfur metabolism includes the 4S pathway; the CoA biosynthetic pathway; metabolism of inorganic sulfur, cysteine, and methionine; and biosynthesis of cysteine, methionine, mycothiol, biotin, and thiamine. The central metabolism includes gluconeogenesis, citric acid cycle, pentose phosphate pathway, and Embden Meyerhoff next Paranas pathway for glycolysis. Figure 1 shows a complete picture of the pathways and reactions in our model, with full details in the Supporting information. We simulated the experiments

of Izumi et al. (1994) and Davoodi-Dehaghani et al. (2010) and compared our predicted cell growth rates with their measured data. As the 4S pathway is aerobic, we assumed unlimited oxygen flux in all of our validation studies and analyses. Sulfur was a limiting substrate in the experiments of Izumi et al. (1994) and Davoodi-Dehaghani et al. (2010). We inferred this from the fact that the stationary phase in their experiments was triggered, when DBT concentration went to zero and HBP concentration reached its maximum. Therefore, we allowed unlimited glucose flux for simulating the experiment of Izumi et al. (1994) and unlimited glycerol flux for Davoodi-Dehaghani et al. (2010). Then, we fixed the DBT uptake and HBP production rates (mmol g−1 dcw h−1) to be at some values computed from their data, and predicted specific cell growth rates at those values. Figure 2 shows that our growth predictions are in close agreement with the two experimental data. The accuracy of our predictions is confirmed by the argument that the limiting sulfur solely determines the growth.

For each strain, one cosmid carrying hiC6 was analyzed by physical mapping and sequencing. For construction of physical maps,

cosmids were digested by single or double restriction enzymes, and the sizes of restricted fragments FXR agonist were calculated based on their migration distances in agarose gel electrophoresis. hiC6 genes were localized to restriction fragments by PCR. For sequencing of the hiC6 region in the NJ-7 cosmid, a library of 2–4 kb Sau3AI DNA fragments (partial digestion) was constructed by insertion into the BamHI site of pUC19. hiC6-containing subclones were selected by PCR screening and sequenced. The sequence of the NJ-7 hiC6 region was assembled from overlapping subclone sequences. With the reference of the NJ-7 sequence, this website PCR fragments were generated for the hiC6 region of UTEX259 and sequenced. In addition, restriction fragments of this region in the UTEX259 cosmid were cloned and sequenced. The whole sequence of the UTEX259 hiC6 region was assembled from those of PCR and restriction fragments. In each case, the sequence was confirmed by

a series of PCRs using genomic DNA as the template. DNA sequences were deposited in the NCBI GenBank under accession numbers JF333588 (NJ-7 hiC6 genes) and JF333589 (UTEX259 hiC6 genes). Genomic DNA of C. vulgaris was extracted using the cetyltrimethylammonium bromide (CTAB) method (Murray & Thompson, 1980). A 10-μg aliquot of DNA was digested completely with one or two restriction enzymes. Separation of digested DNA with 0.7% agarose electrophoresis and capillary transfer of the separated DNA fragments onto Immobilon-Ny+ membrane (Millipore) were performed as standard methods (Sambrook et al., 1989). The digoxigenin (DIG)-labeled hiC6 probe for hybridization was prepared by PCR using hiC6-5 and hiC6-6 as primers and genomic DNA of NJ-7 as the template. Labeling,

hybridization and detection were performed with DIG High Prime DNA Labeling and Detection Starter Terminal deoxynucleotidyl transferase kit I (Roche) according to the manufacturer’s recommendations. Total RNA was extracted using Trizol reagent (Invitrogen) from C. vulgaris strains according to manufacturer’s instructions, separated by agarose/formaldehyde gel electrophoresis and blotted onto Immobilon-Ny+ membranes by capillary transfer. The hiC6 transcripts were probed by a PCR-generated 322-bp fragment overlapping the 3′-end of hiC6-3/4 cDNA (nt.380-701) of NJ-7. NJ-7 and UTEX259 were grown at 20 °C for 7 days and exposed to 4 °C for 24 h. Total RNA extracted from the algal cells with or without exposure to 4 °C was treated with RNase-free DNase I to remove residual DNA until no DNA could be detected by PCR, and then converted into cDNA using M-MLV reverse transcriptase (Promega). The transcription of each hiC6 gene was shown with RT-PCR with gene-specific primers listed in Supporting Information, Table S1.