Single PCR Product Research Articles

First Report of Southern Blight on Bottle Gourd (Lagenaria siceraria) Caused by Sclerotium rolfsii in South Carolina.

Bottle gourd (Lagenaria siceraria (Mol.) Standl.) is an important rootstock in watermelon production in several countries such as Japan, China, and Israel where 60 to 70% of watermelons are grafted (2). We are evaluating bottle gourds for their ability to improve disease resistance when used as rootstock for watermelon (3). In the summer of 2007, symptoms of wilting and crown necrosis appeared on bottle gourd seedlings 1 month after transplanting in a field in Charleston, SC. Infection was observed on commercial cv. Emphasis and four advanced breeding lines. In October of 2007, 35 of 85 plants examined (41%) had stem rot at the crown area just above the soil line where coarse, white mycelia and abundant sclerotia were observed. The fungus tentatively identified as Sclerotium rolfsii produced sclerotia that were white or light to dark brown and measured 0.6 to 2.5 mm in diameter (mean = 1.1 mm). Diseased tissues with sclerotia from four plants were disinfested for 1 min in 0.5% sodium hypochlorite and plated on acidified potato dextrose agar (APDA). Fungal colonies that produced white mycelia and tan-to-brown sclerotia were isolated from four wilted plants. A single PCR product of approximately 680 bp was amplified from DNA extracted from two isolates using the primers ITS1 and ITS4 (4). One PCR product was cloned into the TOPO TA cloning vector (Invitrogen, Carlsbad, CA) and sequenced (GenBank Accession No. EU338381). BLASTN analysis of the sequence in the NCBI databases revealed 99% similarity to the internal transcribed spacer (ITS) sequences of S. rolfsii and Athelia rolfsii (perfect stage of S. rolfsii), confirming that the pathogen was indeed S. rolfsii. Two S. rolfsii isolates were used to test pathogenicity. Each isolate was used to inoculate five young seedlings and five adult (10-week-old) bottle gourd plants. For inoculation, 10 sclerotia obtained from the APDA plates were placed on the surface of the potting soil 0.5 to 1 cm from the collar region of each bottle gourd plant growing in 10-cm pots. Inoculations were done carefully to ensure that the plants were not injured. After inoculation, the plants were maintained at high humidity and 25°C for 3 days and then transferred to laboratory benches. Four young seedlings and three adult noninoculated plants kept under the same conditions served as controls. The pathogenicity test was repeated once with similar results. All inoculated plants developed symptoms of southern blight. The inoculated plants developed symptoms of wilting 4 to 5 days after inoculation and completely wilted within 7 to 10 days. Symptoms of wilting were soon followed by the appearance of white-to-light brown sclerotia on the collar region. No symptoms were observed on the noninoculated plants. S. rolfsii was reisolated from the inoculated plants on APDA. Although southern blight caused by S. rolfsii has been reported on many crop plants in the southern United States, to our knowledge, this disease has not been reported previously on bottle gourd in North America. However, the disease has been reported on bottle gourd in India (1). Identifying sources of resistance to southern blight in bottle gourds may be necessary to make them suitable as rootstocks in areas where S. rolfsii is present.

Modified Vectors for the Two-Step Directional Cloning of Inverted Repeats for RNA Interference in Drosophila

Post-transcriptional gene silencing has become the fastest and most frequently used approach to reduce gene function in cell culture. In an organism without an interferon response, such as Drosophila melanogaster, RNA interference (RNAi) can be evoked using long, double-stranded RNAs (1). In order to achieve a gene knockdown at a stage not amenable to dsRNA injection into early embryos, transgenic approaches have been used in Drosophila (2,3). Commonly, a 500 to 700 bp cDNA product is cloned as an inverted repeat with or without a spacer and expressed under the control of UAS-Gal4 sequences (4). To allow tissue-specific and/or temporally controlled expression of the RNAi transgene, the bimodular UAS-Gal4 system can be used, bypassing any potential toxicity of such transgenes. Inverted repeats tend to be inherently difficult to clone in Escherichia coli due to recombination-mediated DNA repair that occurs during DNA replication (5). Lee and Carthew (6) designed pWIZ, a pUAST-based (4) fly transformation vector in which the two inverted repeats are separated by an intron of the white gene. After transcription, the intron is spliced out and the mRNA forms a hairpin in vivo. The cloning of inverted repeats into pWIZ is very difficult, even when sbcC− E. coli strains reducing recombination repair are used. A smaller shuttle vector in which the repeat/intron cassette is pre-assembled is easier to use, but requires at least 3 or 4 sub-cloning steps (7). Zhu and Stein used a strategy allowing directional cloning of inverted repeats in the germline UAS expression vector pUASp (8,9). pWIZ was also adapted for the GATEWAY in vitro recombineering system (Invitrogen, Carlsbad, CA, USA) (10). This elegant system, however, requires the purchase of expensive, special reagents. In an attempt to simplify the generation of genomic RNAi constructs, we modified pWIZ to improve the cloning efficiency of dsRNA probes. The biggest problem during the cloning of inverted repeats is to obtain the inverted versus the direct orientation of the dsRNA target fragment during the second round of cloning. We therefore constructed pWIZdir by changing the polylinkers of pWIZ to allow the consecutive, directional insertion of a single PCR product using pairs of compatible restriction enzyme sets: SpeI/AvrII and NheI/XbaI adjacent to the white intron, and BglII and BamHI upstream and downstream, respectively (Figure 1). The directionality of the second cloning step greatly increases the frequency of obtaining correctly inverted repeats and obviates the need for the intermediate use of a shuttle plasmid. We have made eight dsRNAi constructs and obtained between 16% and 90% correctly inserted second-round cloning products, a significant improvement over previous attempts (not shown). All eight constructs were successfully used to generate transgenic fly lines. Examples of eye and wing phenotypes obtained in vivo are shown in Figure 2. Figure 1 Schematic representation of pWIZdir and the directional cloning strategy Figure 2 Examples of in vivo phenotypes generated by pWIZdir transgenes

Tandem affinity purification vectors for use in gram positive bacteria

Tandem affinity purification has become a valuable tool for the isolation of protein complexes. Here we describe the construction and use of a series of plasmid vectors for Gram positive bacteria. The vectors utilize the SPA tag as well as variants containing a 3C rather than the TEV protease site as 3C protease has been shown to work efficiently at the low temperatures (4 °C) used to isolate protein complexes. In addition, a further vector incorporates a GST moiety in place of the 3 × FLAG of the SPA tag which provides an additional tagging option for situations where SPA binding may be inefficient. The vectors are all compatible with previously constructed fluorescent protein fusion vectors enabling construction of a suite of affinity and fluorescently tagged genes using a single PCR product.

Role of Nucleosomal Occupancy in the Epigenetic Silencing of the MLH1 CpG Island

Epigenetic silencing of tumor suppressor genes is generally thought to involve DNA cytosine methylation, covalent modifications of histones, and chromatin compaction. Here, we show that silencing of the three transcription start sites in the bidirectional MLH1 promoter CpG island in cancer cells involves distinct changes in nucleosomal occupancy. Three nucleosomes, almost completely absent from the start sites in normal cells, are present on the methylated and silenced promoter, suggesting that epigenetic silencing may be accomplished by the stable placement of nucleosomes into previously vacant positions. Activation of the promoter by demethylation with 5-aza-2'-deoxycytidine involves nucleosome eviction. Epigenetic silencing of tumor suppressor genes may involve heritable changes in nucleosome occupancy enabled by cytosine methylation.

Microfluidic Chips for Detecting the t(4;14) Translocation and Monitoring Disease during Treatment Using Reverse Transcriptase-Polymerase Chain Reaction Analysis of IgH-MMSET Hybrid Transcripts

Diagnosis platforms incorporating low-cost microfluidic chips enable sensitive, rapid, and accurate genetic analysis that could facilitate customized therapies tailored to match the vulnerabilities of any types of cancer. Using ex vivo cancer cells, we have detected the unique molecular signature and a chromosomal translocation in multiple myeloma. Multiple myeloma is characterized by IgH rearrangements and translocations that enable unequivocal identification of malignant cells, detected here with integrated microfluidic chips incorporating genetic amplification via reverse transcriptase-polymerase chain reaction and capillary electrophoresis. On microfluidic chips, we demonstrated accurate and versatile detection of molecular signatures in individual cancer cells, with value for monitoring response to therapy, detecting residual cancer cells that mediate relapse, and evaluating prognosis. Thus, testing for two clinically important molecular biomarkers, the IgH VDJ signature and hybrid transcripts signaling the t(4;14) chro-mosomal translocation, with predictive value in diagnosis, treatment decisions, and monitoring has been efficiently implemented on a miniaturized microfluidic system.

Molecular and Structural Characterization of the PezAT Chromosomal Toxin-Antitoxin System of the Human Pathogen Streptococcus pneumoniae

The chromosomal pezT gene of the Gram-positive pathogen Streptococcus pneumoniae encodes a protein that is homologous to the zeta toxin of the Streptococcus pyogenes plasmid pSM19035-encoded epsilon-zeta toxin-antitoxin system. Overexpression of pezT in Escherichia coli led to severe growth inhibition from which the bacteria recovered approximately 3 h after induction of expression. The toxicity of PezT was counteracted by PezA, which is encoded immediately upstream of pezT and shares weak sequence similarities in the C-terminal region with the epsilon antitoxin. The pezAT genes form a bicistronic operon that is co-transcribed from a sigma(70)-like promoter upstream of pezA and is negatively autoregulated with PezA functioning as a transcriptional repressor and PezT as a co-repressor. Both PezA and the non-toxic PezA(2)PezT(2) protein complex bind to a palindrome sequence that overlaps the promoter. This differs from the epsilon-zeta system in which epsilon functions solely as the antitoxin and transcriptional regulation is carried out by another protein designated omega. Results from site-directed mutagenesis experiments demonstrated that the toxicity of PezT is dependent on a highly conserved phosphoryltransferase active site and an ATP/GTP nucleotide binding site. In the PezA(2)PezT(2) complex, PezA neutralizes the toxicity of PezT by blocking the nucleotide binding site through steric hindrance.

Immunolocalization of aquaporin-1 in the mechanoreceptive Ruffini endings in the periodontal ligament

Previous ultrastructural studies have suggested an axon–Schwann cell interaction in the periodontal Ruffini ending, a primary mechanoreceptor. However, no information is available on the transport mechanism between them. The present study examined the immunolocalization of aquaporin-1 (AQP1) and -4 (AQP4), a member of the water-selective channel, in the periodontal Ruffini endings of the rat incisors and trigeminal ganglion. In addition, the expression of mRNA for AQP1 and 4 was detected in the trigeminal ganglion by a RT-PCR technique. A single PCR product of the sizes anticipated for AQP1 and 4 was detectable in a reverse transcripted cDNA sample from the trigeminal ganglion, whose neurons innervate the periodontal Ruffini endings. An AQP1 immunoreaction was recognizable in the axon terminals of the periodontal Ruffini endings as well as their associated terminal Schwann cells, as confirmed with a double staining with AQP1 and either PGP9.5 or S-100 protein. However, no immunoreaction for AQP4 was found in periodontal Ruffini endings. Although the AQP4 immunoreaction was localized in some satellite cells – but never in neurons – of the trigeminal ganglion, 16.1% trigeminal neurons showed the AQP1 immunoreaction. Furthermore, the AQP1 immunoreaction was found in certain satellite cells which surrounded AQP1-positive or -negative neurons. An analysis of a cross-sectional area of these positive neurons demonstrated that approximately 66.9% of the positive neurons were 400–1000 μm 2 (671.4 ± 172.4 μm 2), indicating that they could be categorized as medium-sized neurons which mediate mechanotransduction. These findings suggest that AQP1 controls water transport in the periodontal Ruffini endings.

Unusually short tandem repeats in the chromosome end structure of Rhynchosciara (Diptera: Sciaridae)

The characterisation of chromosome end (terminal and sub-terminal) sequences of Rhynchosciara americana chromosomes was continued with the screening of a plasmid library made of amplified DNA fragments from a microdissected chromosome tip. An insert chosen for analysis hybridised to two chromosome ends and contains two microsatellite arrays in close vicinity to a sequence (named M-47), part of which is significantly similar to minisatellites of Salmonidae that are frequently present in the vicinity of microsatellite arrays. PCR results using a single primer representative of M-47 elements suggest that they are also repetitive in Rhynchosciara genomes. In addition, total single primer PCR products hybridised to the non-telocentric end subset of R. americana chromosomes. Another plasmid microlibrary made of chromosome tips amplified by a single M-47 primer was screened for repeats of Rhynchosciara chromosomes. Selected inserts that hybridised strongly to non-telocentric ends of R. americana and R. hollaenderi have a tandem array of 22 bp repeats (M-22). There is sequence divergence among M-22 repeats but their mean similarity is significantly high in relation to the M-22 consensus sequence derived from the cloned tandem array. M-22 elements lie distal to the 414 bp sub-telomeric satellite array characterised previously as suggested by double labelling for M-22 hybridisation and reverse transcriptase. M-47 elements, formerly identified in Salmonidae, thus contribute to specify unusually short repeats composing the sub-telomeric structure of two Rhynchosciara species.

182 OSTEOPONTIN GENE EXPRESSION IN IMMATURE AND MATURE SWINE CUMULUS CELLS AND OOCYTES

Osteopontin (OPN) is an acidic single-chain phosphorylated glycoprotein found in both the female and the male reproductive tract that is believed to facilitate reproduction. It was recently reported in swine that OPN improves in vitro fertilization (Hao et al. 2006 Biol. Reprod. 75, 726–733). In bovine, OPN improves the efficiency of in vitro embryo production, and it has been detected on the zona pellucida (ZP) of immature and mature oocytes (Monaco et al. 2007 J. Anim. Sci. 85, 529 abst). This study was designed to evaluate the gene expression of OPN (SPP1) in immature and mature swine cumulus cells and oocytes. Ovaries from a local slaughterhouse were collected on three different days. Half of the immature cumulus–oocyte complexes (COCs) from each day were vortexed in 1 mL TCM-199 HEPES containing 10 mg mL–1 hyaluronidase to separate immature cumulus cells, while the other half of the COCs were maturated for 40-42 h and then the cumulus cells were separated. RNA from cumulus cells and oocytes was extracted and genomic DNA was removed by DNase treatment. cDNA was synthesized using 100 ng of RNA and diluted 50% with DNase–RNase-free water. The RT-PCR product from cumulus cells and oocytes was run in a 2% agarose gel stained with ethidium bromide to verify the presence of SPP1. Relative mRNA abundance between immature and mature cumulus cells was assessed by SYBR green real-time RT–PCR run in triplicate using a 6-point twofold dilution standard curve. GAPDH was used as internal control. Primers for SPP1 and GAPDH were designed spanning an exon/exon junction. The presence of a single and specific PCR product was assessed by gel electrophoresis (a single band was expected at 100 and 90 bp, respectively, for SPP1 and GAPDH), dissociation curve, and sequencing. A t-test was used to assess differences between immature (n = 3) and mature (n = 3) cumulus cells. SPP1 was detected in agarose gel in both immature and mature oocytes and cumulus cells. Cumulus cells presented a Ct for SPP1 ranging from 20 (immature) to 29 (mature). Immature cumulus cells showed a 32-fold larger mRNA abundance compared to the mature cumulus cells (P < 0.05) when data were transformed using a standard curve. Results showed expression of SPP1 in porcine oocytes and cumulus cells. However, maturation significantly decreased the expression of SPP1 in cumulus cells. The presence of SPP1 mRNA in oocytes and cumulus cells and the larger mRNA abundance before maturation may suggest a role of this protein prior to maturation of oocytes. Additional studies will be required to determine the specific role of SPP1 in oocyte maturation in the pig.

A real-time PCR assay for the enumeration and detection of Zygosaccharomyces bailii from wine and fruit juices

Zygosaccharomyces bailii is a major food and beverage spoilage organism. Existing methods for its detection involve lengthy enrichment techniques and then the result does not always differentiate between Z. bailii and Saccharomyces cerevisiae. In this work, we developed a quantitative real-time PCR assay for the rapid detection of Z. bailii from fruit juices and wine even in the presence of non-target DNA. Primers were designed to the gene coding for the D1/D2 loop of the 26S ribosomal RNA subunit producing a single PCR product with a melting temperature of 83.5 °C. As few as 2 cells per ml could be detected by the assay in cranberry raspberry and apple juices and 22 cells per ml from grape juice. The assay was equally efficient in wine, detecting 6 cells per ml even in the presence of 10 7 S. cerevisiae cells. The CFU/ml as determined by plating on YM media showed excellent correlation with the CFU/ml established by the QPCR assay for all the beverages examined. Unknown samples of Z. bailii were prepared in the juices and wine and examined by QPCR. The QPCR estimated cell number was in good agreement with the cell counts obtained by plating, the exception being the cranberry raspberry juice sample. It was determined by live/dead cell counts that the Z. bailii cells were less viable in this juice thus leading to an overestimation of CFU/ml by QPCR. However, the correlation was high between QPCR and total cell count as determined by fluorescent microscopy. This assay provides a rapid and accurate method to establish the levels of the total Z. bailii population which consists of both viable and nonviable cells.

EFL GTPase in Cryptomonads and the Distribution of EFL and EF-1α in Chromalveolates

EFL (EF-like protein) is a member of the GTPase superfamily that includes several translation factors. Because it has only been found in a few eukaryotic lineages and its presence correlates with the absence of the related core translation factor EF-1alpha, its distribution is hypothesized to be the result of lateral gene transfer and replacement of EF-1alpha. In one supergroup of eukaryotes, the chromalveolates, two major lineages were found to contain EFL (dinoflagellates and haptophytes), while the others encode EF-1alpha (apicomplexans, ciliates, heterokonts and cryptomonads). For each of these groups, this distribution was deduced from whole genome sequence or expressed sequence tag (EST) data from several species, with the exception of cryptomonads from which only a single EF-1alpha PCR product from one species was known. By sequencing ESTs from two cryptomonads, Guillardia theta and Rhodomonas salina, and searching for all GTPase translation factors, we revealed that EFL is present in both species, but, contrary to expectations, we found EF-1alpha in neither. On balance, we suggest the previously reported EF-1alpha from Rhodomonas salina is likely an artefact of contamination. We also identified EFL in EST data from two members of the dinoflagellate lineage, Karlodinium micrum and Oxyrrhis marina, and from an ongoing genomic sequence project from a third, Perkinsus marinus. Karlodinium micrum is a symbiotic pairing of two lineages that would have both had EFL (a dinoflagellate and a haptophyte), but only the dinoflagellate gene remains. Oxyrrhis marina and Perkinsus marinus are early diverging sister-groups to dinoflagellates, and together show that EFL originated early in this lineage. Phylogenetic analysis confirmed that these genes are all EFL homologues, and showed that cryptomonad genes are not detectably related to EFL from other chromalveolates, which collectively form several distinct groups. The known distribution of EFL now includes a third group of chromalveolates, cryptomonads. Of the six major subgroups of chromalveolates, EFL is found in half and EF-1alpha in the other half, and none as yet unambiguously possess both genes. Phylogenetic analysis indicates EFL likely arose early within each subgroup where it is found, but suggests it may have originated multiple times within chromalveolates as a whole.

Isotachophoresis preconcentration integrated microfluidic chip for highly sensitive genotyping of the hepatitis B virus

The genotyping of hepatitis B virus (HBV) has become recently a valuable tool not only for epidemiological reasons but also for the clinical practice. Conventional methods for HBV genotyping typically include amplification of the target DNA sequences with a two-round nested PCR followed by separation of the amplified fragments by gel electrophoresis. A microfluidic chip that couples isotachophoresis (ITP) preconcentration and zone electrophoresis (ZE) separation may provide great advantages for sensitive, rapid and cost-effective clinical analysis. In this study, an HBV genotyping method with only one amplification round was developed by the application of the ITP-ZE chip. All the analysis steps of the ITP-ZE separation including sample injection, stacking and separation were performed continuously, controlled by sequential high-voltage switching. A 2.1 cm sample plug was preconcentrated between discontinuous buffers in ITP process, followed by ZE separation. Sensitivity enhancement was obtained through the increase of sample loading volume. The average LOD value of the ITP-ZE microfluidic chip was determined to be 0.0021 pg/μL. In a large-scale HBV genotyping test, single round PCR products were analyzed by ITP-ZE microfluidic chip, and the results were compared with that of the conventional method. Among the 200 cases studied, the classification rate obtained with microfluidic chip was 93%, which was 6% higher than that obtained with the conventional method. Method with ITP-ZE chip analysis provides HBV genotyping information in reduced PCR amplification time with higher detection rate when compared with conventional method. This method holds great potential for extrapolation to the abundance of similar molecular biology-based techniques in clinical diagnosis.

Tumor Necrosis Factor α Induces Spermidine/Spermine N1-Acetyltransferase through Nuclear Factor κBin Non-small Cell Lung Cancer Cells

Tumor necrosis factor alpha (TNFalpha) is a potent pleiotropic cytokine produced by many cells in response to inflammatory stress. The molecular mechanisms responsible for the multiple biological activities of TNFalpha are due to its ability to activate multiple signal transduction pathways, including nuclear factor kappaB (NFkappaB), which plays critical roles in cell proliferation and survival. TNFalpha displays both apoptotic and antiapoptotic properties, depending on the nature of the stimulus and the activation status of certain signaling pathways. Here we show that TNFalpha can lead to the induction of NFkappaB signaling with a concomitant increase in spermidine/spermine N(1)-acetyltransferase (SSAT) expression in A549 and H157 non-small cell lung cancer cells. Induction of SSAT, a stress-inducible gene that encodes a rate-limiting polyamine catabolic enzyme, leads to lower intracellular polyamine contents and has been associated with decreased cell growth and increased apoptosis. Stable overexpression of a mutant, dominant negative IkappaBalpha protein led to the suppression of SSAT induction by TNFalpha in these cells, thereby substantiating a role of NFkappaB in the induction of SSAT by TNFalpha. SSAT promoter deletion constructs led to the identification of three potential NFkappaB response elements in the SSAT gene. Electromobility shift assays, chromatin immunoprecipitation experiments and mutational studies confirmed that two of the three NFkappaB response elements play an important role in the regulation of SSAT in response to TNFalpha. The results of these studies indicate that a common mediator of inflammation can lead to the induction of SSAT expression by activating the NFkappaB signaling pathway in non-small cell lung cancer cells.

Typing of Human Adenoviruses in Specimens from Immunosuppressed Patients by PCR-Fragment Length Analysis and Real-Time Quantitative PCR

Currently, 51 human adenovirus (AdV) serotypes, which are divided into six species (A to F), are known. AdV infections are a major cause of morbidity and mortality in immunosuppressed individuals, particularly in allogeneic stem cell transplant (SCT) recipients. Any AdV species may cause life-threatening disease, but little information is available on the clinical relevance of individual serotypes. The use of serological testing for serotype identification is limited due to the impaired immune response during the posttransplant period. A new molecular approach to serotype identification is presented here that exploits variable regions within the hexon gene. All serotypes belonging to the species A, B, C, E, and F can be determined by fragment length analysis of a single PCR product. For species C, which is the most prevalent in many geographic regions, an alternative technique based on serotype-specific real-time quantitative PCR was established. Of 135 consecutive pediatric patients screened for AdV infections after allogeneic SCT, 40 tested positive. Detailed analysis revealed the presence of 10 different serotypes; serotypes 1 and 2 from species C (C01 and C02) showed the highest prevalence, accounting for 77% of the AdV-positive cases. Representatives of other species were observed less commonly: serotype A12 in 6.5%; serotype A31 in 4.5%; and B03, B16, C05, C06, D19, and F41 in 2%. The approach to rapid molecular serotype analysis presented here provides a basis for detailed studies on adenovirus epidemiology and on the transmission of nosocomial infections. Moreover, in view of the increasing importance of tailored therapy approaches, serotype identification may in the future have implications for the selection of the most appropriate antiviral treatment.

The use of the Dhcr7 knockout mouse to accurately determine the origin of fetal sterols

Mice with a targeted mutation of 3beta-hydroxysterol Delta(7)-reductase (Dhcr7) that cannot convert 7-dehydrocholesterol to cholesterol were used to identify the origin of fetal sterols. Because their heterozygous mothers synthesize cholesterol normally, virtually all sterols found in a Dhcr7 knockout fetus having a Delta(7) or a Delta(8) double bond must have been synthesized by the fetus itself but any cholesterol had to have come from the mother. Early in gestation, most fetal sterols were of maternal origin, but at approximately E13-14, in situ synthesis became increasingly important, and by birth, 55-60% of liver and lung sterols had been made by the fetus. In contrast, at E10-11, upon formation of the blood-brain barrier, the brain rapidly became the source of almost all of its own sterols (90% at birth). New, rapid, de novo sterol synthesis in brain was confirmed by the observation that concentrations of C24,25-unsaturated sterols were low in the brains of all very young fetuses but increased rapidly beginning at approximately E11-12. Reduced activity of sterol C24,25-reductase (Dhcr24) in brain, suggested by the abundance of C24,25-unsaturated compounds, seems to be the result of suppressed Dhcr24 expression. The early fetal brain also appears to conserve cholesterol by keeping cholesterol 24-hydroxylase expression low until approximately E18.

Rolling circle amplification-RACE: a method for simultaneous isolation of 5′ and 3′ cDNA ends from amplified cDNA templates

Isolation of full-length gene transcripts is important to determine the protein coding region and study gene structure. However, isolation of novel gene sequences is often limited to expressed sequence tags (ESTs) (i.e., short cDNA fragments that predominantly represent the 3′ end of the transcript). Rapid amplification of cDNA ends (RACE) is today by far the most popular approach for obtaining full-length cDNAs when only part of the transcript’s sequence is known.Since its original description (1,2) numerous modifications and improve-ments of the method have been developed and consist of a collection of PCR-based cloning procedures that extend a known cDNA fragment toward the 3′ (3′ RACE) or the 5′ (5′ RACE) cDNA end. The original method is based on attachment of an anchor sequence to one end of the cDNA that can be used as a primer binding template in PCR with a second gene-specific primer from the known part of the gene. Although this procedure seems in theory fast and simple, it is technically difficult and usually requires substantial optimi-zation and several repetitions before satisfactory results can be obtained (3). This is particularly due to the use of a universal primer corresponding to the anchor sequence present in all cDNAs, which may result in a high background of nonspecific products even after a nested PCR with a gene-specific primer internal to the first gene-specific primer is performed. Another drawback of the method is the difficulty of obtaining the full-length 5′ end of the transcript due to the presence of many truncated transcripts in the messenger RNA (mRNA) pool. Several strategies aimed at eliminating these problems have been developed (4–9) and have proven to be very useful in certain applica-tions. One improvement is based on the utilization of a pair of gene-specific primers in inverse PCR on circularized cDNA templates, which would avoid the use of a universal primer and the problems it may generate (4,6,8–10). This strategy also allows the simulta-neous isolation of both cDNA ends in a single reaction (9,10). Some of these procedures require the generation of double-stranded cDNA, including the use of template-switching reverse transcription (9) or a post-reverse transcription adaptor ligation step (10). Methods that are performed directly on first-strand cDNA are complicated by the low efficiency of RNA ligase for the circularization reaction (6) or the need for bridging oligonucleotides for this step (8). Furthermore, existing inverse-RACE methods typically require nested PCR to amplify the transcript of interest, and only a limited number of transcripts can be isolated from a single reverse transcription reaction, making it difficult to analyze rare transcripts from scarce tissue.We describe here an improved inverse-RACE method, which uses CircLigase™ (Epicentre Biotechnologies, Madison, WI, USA) for cDNA circularization, followed by rolling circle amplification (RCA) of the circular cDNA with ϕ29 DNA polymerase (New England Biolabs, Ipswich, MA, USA). In this way, a large amount of the PCR template is produced, allowing the simultaneous isolation of the 3′ and 5′ unknown ends of a virtually unlimited number of transcripts after a single reverse transcription reaction. Figure 1 illus-trates this method, named RCA-RACE. The process takes advantage of the properties of CircLigase to circularize

Free Fatty Acids Induce JNK-dependent Hepatocyte Lipoapoptosis

Elevated serum free fatty acids (FFAs) and hepatocyte lipoapoptosis are features of non-alcoholic fatty liver disease. However, the mechanism by which FFAs mediate lipoapoptosis is unclear. Because JNK activation is pivotal in both the metabolic syndrome accompanying non-alcoholic fatty liver disease and cellular apoptosis, we examined the role of JNK activation in FFA-induced lipoapoptosis. Multiple hepatocyte cell lines and primary mouse hepatocytes were treated in culture with monounsaturated fatty acids and saturated fatty acids. Despite equal cellular steatosis, apoptosis and JNK activation were greater during exposure to saturated versus monounsaturated FFAs. Inhibition of JNK, pharmacologically as well as genetically, reduced saturated FFA-mediated hepatocyte lipoapoptosis. Cell death was caspase-dependent and associated with mitochondrial membrane depolarization and cytochrome c release indicating activation of the mitochondrial pathway of apoptosis. JNK-dependent lipoapoptosis was associated with activation of Bax, a known mediator of mitochondrial dysfunction. As JNK can activate Bim, a BH3 domain-only protein capable of binding to and activating Bax, its role in lipoapoptosis was also examined. Small interfering RNA-targeted knock-down of Bim attenuated both Bax activation and cell death. Collectively the data indicate that saturated FFAs induce JNK-dependent hepatocyte lipoapoptosis by activating the proapoptotic Bcl-2 proteins Bim and Bax, which trigger the mitochondrial apoptotic pathway.

A family of E. coliexpression vectors for laboratory scale and high throughput soluble protein production

BackgroundIn the past few years, both automated and manual high-throughput protein expression and purification has become an accessible means to rapidly screen and produce soluble proteins for structural and functional studies. However, many of the commercial vectors encoding different solubility tags require different cloning and purification steps for each vector, considerably slowing down expression screening. We have developed a set of E. coli expression vectors with different solubility tags that allow for parallel cloning from a single PCR product and can be purified using the same protocol.ResultsThe set of E. coli expression vectors, encode for either a hexa-histidine tag or the three most commonly used solubility tags (GST, MBP, NusA) and all with an N-terminal hexa-histidine sequence. The result is two-fold: the His-tag facilitates purification by immobilised metal affinity chromatography, whilst the fusion domains act primarily as solubility aids during expression, in addition to providing an optional purification step. We have also incorporated a TEV recognition sequence following the solubility tag domain, which allows for highly specific cleavage (using TEV protease) of the fusion protein to yield native protein. These vectors are also designed for ligation-independent cloning and they possess a high-level expressing T7 promoter, which is suitable for auto-induction. To validate our vector system, we have cloned four different genes and also one gene into all four vectors and used small-scale expression and purification techniques. We demonstrate that the vectors are capable of high levels of expression and that efficient screening of new proteins can be readily achieved at the laboratory level.ConclusionThe result is a set of four rationally designed vectors, which can be used for streamlined cloning, expression and purification of target proteins in the laboratory and have the potential for being adaptable to a high-throughput screening.

Simplified screening for the detection of soluble fusion constructs expressed in E. coli using a modular set of vectors

BackgroundThe solubility of recombinant proteins expressed in bacteria is often disappointingly low. Several strategies have been developed to improve the yield and one of the most common strategies is the fusion of the target protein with a suitable partner. Despite several reports on the successful use of each of these carriers to increase the solubility of some recombinant proteins, none of them was always successful and a combinatorial approach seems more efficient to identify the optimal combination for a specific protein. Therefore, the efficiency of an expression system critically depends on the speed in the identification of the optimal combination for the suitable fusion candidate in a screening process. This paper describes a set of expression vectors (pETM) designed for rapid subcloning, expression and subsequent purification using immobilized metal affinity chromatography (IMAC).ResultsA single PCR product of two Yellow Fluorescent Proteins (EYFPs) was cloned into 18 vectors comprising identical restriction sites and varying fusion partners as well as differing protease recognition sites. After a small-scale expression, the yields of the different constructs were compared using a Coomassie stained SDS-polyacrylamide gel and the results of this preliminary screening were then confirmed by large-scale purification. The yields were calculated and the stability of the different constructs determined using three independent conditions. The results indicated a significant correlation between the length and composition of non-native amino acid tails and stability. Furthermore, the buffer specificity of TEV and 3C proteases was tested using fusion proteins differing only in their protease recognition sequence, and a His-GST-EYFP construct was employed to compare the efficiency of the two alternative affinity purification methods.ConclusionThe experiments showed that the set of pETM vectors could be used for the rapid production of a large array of different constructs with specific yield, stability, and cleavage features. Their comparison allowed the identification of the optimal constructs to use for the large-scale expression. We expect that the approach outlined in this paper, i.e. the possibility to obtain in parallel fusion products of the target protein with different partners for a preliminary evaluation, would be highly beneficial for all them who are interested in the rapid identification of the optimal conditions for protein expression.

The β-tubulin genes of two Strongyloides species

The World Health Organization is sponsoring major treatment programs with the aim of controlling helminth infection throughout the tropical world. Prominent among the anthelmintics recommended for use in these programs are drugs in the benzimidazole (BZ) class. Resistance to these drugs has been associated with polymorphisms in the β-tubulin gene. We have cloned and sequenced the β-tubulin genes of Strongyloides stercoralis and Strongyloides ratti and have proceeded to develop a protocol for genotyping single worms for polymorphisms in β-tubulin. Our findings indicate that S. ratti has a single β-tubulin gene, making DNA sequence analysis of a single larva PCR product a feasible means of studying BZ resistance in these species. Our genotyping test allows the identification of polymorphisms at codons 167, 198, and 200 in the Strongyloides β-tubulin gene, thus enabling survey for BZ resistant genotypes.