Inverse PCR Technique Research Articles

Real Time PCR; A new, fast and accurate technique in diagnosing the severe type of hemophilia A patients

Introduction: Hemophilia A (HA) is one of the most deleterious X-linked bleeding disorders in male patients. Early and rapid detection could provide management strategies for this disease. This study aims to improve the classic method for detection of important intron 22 inversion mutations (INV22). Materials and Methods: Whole blood samples were taken from 21 male children with a history of severe HA, referred to the Iranian comprehensive hemophilia care center. Two groups were involved for detection of INV22 and two methods were examined in a double-blind manner. The first method used a classical method, inverse PCR, and the second method was Real-time inverse PCR. Results: Results showed that both methods could separately detect the INV22 in 11 out of 21 patients with severe HA (52%) in the same accuracy, but with this difference that only one day was needed for detecting of INV22 by Real-time inverse PCR and on other hand 3 days were needed for the classical methods. Concusion: Because of the medical importance of early detection or prenatal/postnatal diagnosis of this disease, this study recommends the Real-time inverse PCR technique for the rapid detection of this mutation in well-equipped genetic laboratories everywhere.

Direct RBS Engineering of the biosynthetic gene cluster for efficient productivity of violaceins in E. coli

BackgroundViolaceins have attracted much attention as potential targets used in medicines, food additives, insecticides, cosmetics and textiles, but low productivity was the key factor to limit their large-scale applications. This work put forward a direct RBS engineering strategy to engineer the violacein biosynthetic gene cluster cloned from Chromobacterium violaceum ATCC 12,472 to efficiently improve the fermentation titers.ResultsThrough four-rounds of engineering of the native RBSs within the violaceins biosynthetic operon vioABCDE, this work apparently broke through the rate-limiting steps of intermediates conversion, resulting in 2.41-fold improvement of violaceins production compared to the titers of the starting strain Escherichia coli BL21(DE3) (Vio12472). Furthermore, by optimizing the batch-fermentation parameters including temperature, concentration of IPTG inducer and fermentation time, the maximum yield of violaceins from (BCDE)m (tnaA−) reached 3269.7 µM at 2 mM tryptophan in the medium. Interestingly, rather than previous reported low temperature (20 ℃), we for the first time found the RBS engineered Escherichia coli strain (BCDE)m worked better at higher temperature (30 ℃ and 37 ℃), leading to a higher-level production of violaceins.ConclusionsTo our knowledge, this is the first time that a direct RBS engineering strategy is used for the biosynthesis of natural products, having the potential for a greater improvement of the product yields within tryptophan hyperproducers and simultaneously avoiding the costly low temperature cultivation for large-scale industrial production of violaciens. This direct RBS engineering strategy could also be easily and helpfully used in engineering the native RBSs of other larger and value-added natural product biosynthetic gene clusters by widely used site-specific mutagenesis methods represented by inverse PCR or CRISPR-Cas9 techniques to increase their fermentation titers in the future.

UPAYA PENINGKATAN KUALITAS TANAMAN KEDELAI (Glycine max L. Merill) MELALUI PEMANFAATAN BIOTEKNOLOGI DALAM MENGATASI KELANGKAAN PANGAN

Population growth the rapidly is currently a major challenges to business world food supply. The development of biotechnology begins with technology genetically engineered has accelerated. Products of biotechnology plants resembling plants provenance, but having the characteristics of certain that causes those plants better. Plant products of biotechnology which has been approved for food as having the nature of : 1) resistance to pests and disease , 2) resistance to herbicide, 3) nutrition changes and 4) content increased save resources. The purpose of drafting this paper is to provide information about improving the quality of the soybean plant ( Glycine max l .Merill ) through the use of biotechnology in addressing scarcity of food.The genetically engineered in plants soybean , includes extraction process dna ( modification ctab ) method of the leaves of plants tangential and soybean R1 R2. Fragments of dna the genome of 0.8 kb involved in tolerant asam-al at b . Japonicum 38 has successfully isolated using inverse PCR technique, Fragments of dna has been successfully diklon into plasmid vector pgemt-easy ( ~ 3kb ) produce recombinant plasmid pgemt-38 a ~ 3.8 family planning. Ttransfer genes pinii in plants soy has successfully done through a . Tumefaciens with one of his event at1 ( tidar ) showing the pcr positive on pinii genes. Protocol best for soybean through the transformation of a . Tumefaciens is using eksplan cotyledon young with the density of bacteria 1 x 108 / cell mls inoculation / long 90 minutes and long kokultivasi 5 days. The soybean plant AT1R1 ( tidar ) transformation through a result. Tumefaciens a little more resistant to pest borer rather than the pods the soybean plant nontransgenik (control)

Detection of DNA Double-Strand Breaks and Chromosome Translocations Using Ligation-Mediated PCR and Inverse PCR.

Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter and gene-specific primers. The rate of appearance and loss of specific PCR products allow detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks and rearrangements introduced into any identifiable genomic location. We have also applied parallel sequencing for the high-throughput analysis of inverse PCR products to facilitate the unbiased recording of all rearrangements located at a specific genomic location.

Characterization of the mating-type locus (MAT) reveals a heterothallic mating system inKnightiella splachnirima

AbstractIn the present study, we characterized the mating-type locus ofKnightiella splachnirima(Icmadophilaceae) using degenerate and inverse PCR techniques. We screened for the presence of both mating-type locus idiomorphs in DNA extracts of minuscule samples of haploid thalline tissue. We found that only one of the two idiomorphs was present in each sample, and the mating-type ratio (MAT1-1:MAT1-2) was very balanced, being 8:10 and 13:14 at local and global scales, respectively. This indicates that the species is probably self-incompatible and requires the presence of compatible mating partners for sexual reproduction (heterothallic mating system). Furthermore, we provide a mating-type screening protocol withK. splachnirimaspecific mating-type locus primers, which could serve as an essential tool for the conservation management of this rare Australasian endemic.

Characterization of endoglucanase from Paenibacillus sp. M33, a novel isolate from a freshwater swamp forest.

The newly isolated Paenibacillus sp. M33 from freshwater swamp forest soil in Thailand demonstrated its potential as a cellulose degrader. One of its endoglucanase genes from Paenibacillus sp., celP, was cloned to study the molecular characteristics of its gene product. The celP gene was recognized firstly by degenerate primer designed from Paenibacillus endoglucanase gene, and subsequently identified flanking region by inverse PCR technique. The celP gene consists of an open reading frame of 1707 bp encoding for 569 amino acids including 33-amino acids signal sequence. CelP is a member of glycoside hydrolase family 5 appended with a family 46 carbohydrate-binding module. CelP from recombinant Escherichia coli was purified by affinity chromatography. SDS-PAGE analysis of purified CelP showed a protein band at about 60 kDa. The purified enzyme gave a specific CMCase activity of 0.03 μmol min-1 mg-1 . It had higher activities on lichenan (0.19 μmol min-1 mg-1 ) and barley β-glucan (0.14 μmol min-1 mg-1 ). Maximum activity on lichenan was obtained at 50 °C, pH 5.0. CelP was stable over a pH range of 3.0-10.0 and retained 80% activity when incubated at 50 °C for 1 h. The properties of its CelP endoglucanase, especially substrate specificity, will make it useful in various biotechnological applications including biomass hydrolysis.

Characterization of a thermostable arginase from Rummeliibacillus pycnus SK31.001

L-arginase from Rummeliibacillus pycnus SK31.001 is newly discovered. A 906bp complete open reading frame, which encodes a 301 amino acid protein, was identified using degenerate PCR and inverse PCR techniques. The arginase was found to have a conserved active site with 6 amino acid residues binding to 2 manganese ions: D123, H125, D228, D230, H100 and D127. Bioinformatics analysis revealed that R. pycnus arginase is a hexamer with a subunit molecular mass of 33kDa and whole molecular mass of 195kDa. R. pycnus arginase is thermostable with an optimal temperature of 80°C and maintains 85% of its initial activity after 24h of incubation at 40 or 50°C. An arginase activity assay showed that R. pycnus arginase has an optimum pH of 9.5 and a preference for Mn2+. Using arginine as the substrate, the Michaelis-Menten constant (Km) and catalytic efficiency (kcat/Km) were measured to be 0.212mM and 2970mM−1s−1, respectively. The biosynthesis yield of L-ornithine by the purified enzyme was 144.4g/L, and the molar yield was 95.2%.

Mapping of deletion breakpoints at the CDKN2A locus in melanoma: detection of MTAP-ANRIL fusion transcripts.

Genomic locus at chromosome 9p21 that contains the CDKN2A and CDKN2B tumor suppressor genes is inactivated through mutations, deletions and promoter methylation in multiple human cancers. Additionally, the locus encodes an anti-sense RNA (ANRIL). Both hemizygous and homozygous deletions at the locus targeting multiple genes are fairly common in different cancers. We in this study investigated breakpoints in five melanoma cell lines, derived from metastasized tumors, with previously identified homozygous deletions using array comparative genomic hybridization (aCGH). For breakpoint mapping, we used primer approximation multiplex PCR (PAMP) and inverse PCR techniques. Our results showed that three cell lines carried complex rearrangements. In two other cell lines, with focal deletions of 141 kb and 181 kb, we identified fusion gene products, involving MTAP and ANRIL. We also confirmed the complex rearrangements and focal deletions in DNA from tumor tissues corresponding to three cell lines. The rapid amplification of 3′cDNA ends (3′RACE) carried out on transcripts resulted in identification of three isoforms of MTAP-ANRIL fusion gene. Screening of cDNA from 64 melanoma cell lines resulted in detection of fusion transcripts in 13 (20%) cell lines that involved exons 4-7 of the MTAP and exon 2 or 5 of the ANRIL genes. We also detected fusion transcripts involving MTAP and ANRIL in two of the seven primary melanoma tumors with focal deletion at the locus. The results from the study, besides identifying complex rearrangements involving CDKN2A locus, show frequent occurrence of fusion transcripts involving MTAP and ANRIL genes.

Internal Amplification Control for a Cryptosporidium Diagnostic PCR: Construction and Clinical Evaluation.

Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (≈375 bp) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects ≈550 bp of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, ≈2 oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.

Evolutionary dynamics of endogenous Jaagsiekte sheep retroviruses proliferation in the domestic sheep, mouflon and Pyrenean chamois

The oncogenic exogenous Jaagsiekte sheep retrovirus (JSRV), responsible for ovine pulmonary adenocarcinoma, has several endogenous counterparts termed enJSRVs. Although many of these elements have been inactivated over time by the accumulation of deleterious mutations or internal recombination leading to solo long terminal repeat (LTR) formation, several members of enJSRVs have been identified as nearly intact and probably represent recent integration events. To determine the level of enJSRV polymorphism in the sheep population and related species, we have undertaken a study by characterizing enJSRVs copies and independent integration sites in six domestic sheep and two wild species of the sheep lineage. enJSRVs copies were detected by amplifying the env-LTR region by PCR, and for the detection of the insertion sites, we used two approaches: (1) an in silico approach based on the recently published Sheep Reference Genome Assembly (OARv3.0) and (2) an experimental approach based on PCR suppression and inverse PCR techniques. In total, 103 enJSRV sequences were generated across 10 individuals and enJSRV integrations were found on 11 of the 28 sheep chromosomes. These findings suggest that there are still uncharacterized enJSRVs, and that some of the integration sites are variable among the different species, breeds of the same species, subspecies and geographic locations.

Raw starch-degrading α-amylase fromBacillus aquimarisMKSC 6.2: isolation and expression of the gene, bioinformatics and biochemical characterization of the recombinant enzyme

The aims were to isolate a raw starch-degrading α-amylase gene baqA from Bacillus aquimaris MKSC 6.2, and to characterize the gene product through in silico study and its expression in Escherichia coli. A 1539 complete open reading frame of a starch-degrading α-amylase gene baqA from B.aquimaris MKSC 6·2 has been determined by employing PCR and inverse PCR techniques. Bioinformatics analysis revealed that B.aquimaris MKSC 6.2 α-amylase (BaqA) has no starch-binding domain, and together with a few putative α-amylases from bacilli may establish a novel GH13 subfamily most closely related to GH13_1. Two consecutive tryptophans (Trp201 and Trp202, BaqA numbering) were identified as a sequence fingerprint of this novel GH13 subfamily. Escherichia coli cells produced the recombinant BaqA protein as inclusion bodies. The refolded recombinant BaqA protein degraded raw cassava and corn starches, but exhibited no activity with soluble starch. A novel raw starch-degrading B.aquimaris MKSC 6.2 α-amylase BaqA is proposed to be a member of new GH13 subfamily. This study has contributed to the overall knowledge and understanding of amylolytic enzymes that are able to bind and digest raw starch directly.

Mitochondrial SCAR and SSR Markers for distinguishing cytoplasmic male sterile lines from their isogenic maintainer lines in cotton

With 8 figures and 3 tablesAbstractCytoplasmic male sterility (CMS) line P30A of Upland cotton has been used in commercial production of F1 seeds. However, cytoplasm‐specific molecular markers in this ‘three‐line’ (CMS line P30A, maintainer line P30B and restorer line Y18R) system are mostly unknown. Twenty mitochondrial gene probes were used to identify the differences between P30B fertile (N‐) and P30A sterile (S‐) cytoplasm. Among six genes that revealed restriction fragment length polymorphisms (RFLPs), the gene for α‐subunit of F1ATPase (atpA) revealed significant differences between N‐ and S‐cytoplasm. All of the EcoRI restriction bands of atpA were amplified by inverse PCR (iPCR) technique, indicating that both N‐ and S‐cytoplasm contained an intact and a 3′ truncated atpA copy, but the truncating site (breakpoint) was different. According to the chimerical sequences following the breakpoints, three cytoplasm‐specific sequence characterized amplified region (SCAR) markers were developed. In addition, a simple sequence repeat (SSR) locus in the 3′ flanking sequences of the intact atpA gene was found between N‐ and S‐cytoplasm. The four PCR markers were used to distinguish CMS lines from maintainer lines.

Isolation, Chromosome Assignment, and Expression Assay of Nitrite Reductase Gene and Regulatory Sequence in Wheat

Nitrite reductase is wildly involved in N metabolism in plants and has shown excellent relevance to regeneration poten-tial in the tissue culture of rice(Oryza sativa L.).In this study,in silico and inverse PCR techniques were employed to isolate nitrite reductase encoded gene and its regulatory sequences from wheat(Triticum aestivum L.).This gene was predicted to contain four exons and three introns.The gDNA and cDNA sequences were 2 881 bp and 1 830 bp in length,respectively,and they were both submitted to GenBank under the accession numbers FJ555239 and FJ527909.Its deduced encoding protein product was ap-proximately 65.7 kD,sharing high(more than 60%) identity with other nitrite reductase genes deposited in the NCBI database,especially with those from other monocot cereal crops(more than 80%).The 5′ flanking region was isolated and extended to -2 924 bp(counting from the start code: ATG) through inverse PCR.After induced by 1 mmol L-1 IPTG,a protein of ca.70 kD was obtained in prokaryotic expression vector pET-28a,including a histidine tag of ca.3.8 kD.The highest expression of gene in wheat seedlings was induced by 30 mmol L-1 KNO3 for 1 h.Measurement on nitrite reductase activity showed the enzyme acti-vity increased following the treatment time extension at 5-d intervals under the above same condition.According to the result of AS-PCR analysis,at least one copy of the gene existed on chromosome 6A and 6B each in common wheat.The study provides a basis for functional determination of wheat nitrite reductase gene in subsequent research.

Porcine insulin receptor substrate 4 (IRS4) gene: cloning, polymorphism and association study

Using PCR and inverse PCR techniques we obtained a 4,498 bp nucleotide sequence FN424076 encompassing the complete coding sequence of the porcine insulin receptor substrate 4 (IRS4) gene and its proximal promoter. The 1,269 amino acid porcine protein deduced from the nucleotide sequence shares 92% identity with the human IRS4 and possesses the same domains and the same number of tyrosine phosphorylation motifs as the human protein. We detected substitution FN424076:g.96C<G in the promoter region that segregates in Meishan and a synonymous substitution FN424076:g.1829T<C in the coding sequence with allele C present only in Meishan. Linkage mapping placed the IRS4 gene at position 82 cM on the current USDA-USMARC linkage map of porcine chromosome X. Association analyses were performed on 555 animals of 12th-15th generation of the Meishan × Large White cross and showed that both SNPs were highly significantly associated with backfat depth (P=0.0005) and that the SNP FN424076:g1829T<C was also associated with loin depth (P=0.017). The Meishan alleles increased back fat depth and decreased loin depth. IRS4 can be considered a positional candidate gene for at least some of the QTL located at the centromeric region of porcine chromosome X.

Retroviral Insertional Mutagenesis Reveals Genes That Cooperate with FLT3-ITD in Leukemogenesis.

Abstract 1960Poster Board I-983FLT3 is a receptor tyrosine kinase that plays an important role in hematopoiesis and is one of the most mutated genes in acute myeloid leukemia (AML). The internal tandem duplication (ITD) mutation, which causes autophosphorylation and constitutive activation of the receptor, has been found in up to 35% of AML and 3% of ALL patients. Our lab has created and described a FLT3ITD/wt knock-in mouse with the mutation expressed under the endogenous promoter. These mice develop myeloproliferative disorder (MPD) with a median time to disease of 52 weeks, but do not progress to acute leukemia, indicating that an additional genetic event is required for leukemogenesis. We have used the MOL4070LTR retrovirus in a retroviral insertional mutagenesis (RIM) screen to identify genes that can cooperate with the FLT3/ITD to cause leukemia. FLT3ITD/wt mice were injected with the retrovirus at birth and 78% developed leukemia with a median time to disease of 18 weeks (50% B-ALL/lymphoma, 18% AML, 10% biphenotypic leukemia). By comparison, retrovirus-injected FLT3wt/wt mice developed leukemias with a much longer median time to disease of 69 weeks. The decreased latency in the retrovirally-infected FLT3ITD/wt mice implies that the viral integrations have created mutations that collaborate with FLT3/ITD signaling to generate leukemia. To identify the genes responsible, we cloned the insertion sites using an inverse PCR technique. 343 integration sites were cloned from 25 FLT3ITD/wt mice that developed leukemia, 29 of which were found in two or more mice. These common insertion sites (CIS) are considered putative cooperating genes. The most common insertion, found in 5 mice, involved the Evi1 gene, with the virus inserting approximately 17kb upstream of the gene in three of the mice or approximately 100kb upstream of the gene in the other two mice. All of the mice showed overexpression of Evi1 by quantitative RT-PCR. This is an interesting collaborating gene because its overexpression has been described in AML and predicts a poor prognosis in patients, but mouse models have shown that, like FLT3/ITD, it is not leukemogenic on its own. Though singularly insufficient, together these two mutations may cooperate to cause leukemia. Another putative cooperating gene found in the screen is Ring1a. Insertions upstream of the gene (22kb, 96kb and 209kb) were found in 3 mice displaying myeloid-associated disease, and in all cases caused overexpression of Ring1a, as seen by quantitative RT-PCR. Strong expression of Ring1a, part of the PRC1 polycomb repressor complex, has been observed in several cancer types but not in myeloid leukemia; however, another member of PRC1, Bmi-1, seems to play an extensive role in myeloid leukemia. Integrations near Ring1a have not previously been described in the Retrovirus Tagged Cancer Gene Database (RTCGD mm8), indicating a specific cooperation with FLT3/ITD and a role in the disease. With these studies we have shown that additional mutations cooperate with the FLT3/ITD to cause leukemia and we have identified several possible collaborating genes, including Evi1 and Ring1a. Disclosures:No relevant conflicts of interest to declare.

Cloning of glyceraldehyde‐3‐phosphate dehydrogenase from an Antarctic psychrophilic bacterium by inverse and splinkerette PCR

Psychrophiles are organisms that thrive in cold environments. One of the strategies for their cold adaptation is the ability to synthesize cold-adapted enzymes. These enzymes usually display higher catalytic efficiency and thermolability at lower temperatures compared to their mesophilic and thermophilic counterparts. In this work, a psychrophilic bacterium codenamed pi9 was selected for the cloning of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme in the glycolytic pathway. Here, the cloning of an 1,113 bp fragment of GAPDH gene which covers the 1,002 bp open reading frame by using multiple PCR steps is described. The partial sequence of this gene was PCR amplified by using degenerate primers followed by the cloning of the flanking sequences by inverse and splinkerette PCR techniques. The success in cloning the GAPDH gene by PCR has bypassed the more time consuming genomic library construction and screening method. The full length GAPDH protein was subsequently expressed in E. coli, purified as His-tag protein and confirmed to be catalytically active. This work demonstrated the use of multiple PCR techniques to clone a gene based solely on sequence comparison. It also laid the foundation for further biochemical and structural characterizations of GAPDH from a psychrophilic bacterium by providing a highly purified recombinant protein sample.

Cloning, Characterization and Functional Analysis of a 1-FEH cDNA from Vernonia herbacea (Vell.) Rusby

Variations in the inulin contents have been detected in rhizophores of Vernonia herbacea during the phenological cycle. These variations indicate the occurrence of active inulin synthesis and depolymerization throughout the cycle and a role for this carbohydrate as a reserve compound. 1-Fructan exohydrolase (1-FEH) is the enzyme responsible for inulin depolymerization, and its activity has been detected in rhizophores of sprouting plants. Defoliation and low temperature are enhancer conditions of this 1-FEH activity. The aim of the present work was the cloning of this enzyme. Rhizophores were collected from plants induced to sprout, followed by storage at 5 degrees C. A full length 1-FEH cDNA sequence was obtained by PCR and inverse PCR techniques, and expressed in Pichia pastoris. Cold storage enhances FEH gene expression. Vh1-FEH was shown to be a functional 1-FEH, hydrolyzing predominantly beta-2,1 linkages, sharing high identity with chicory FEH sequences, and its activity was inhibited by 81% in the presence of 10 mM sucrose. In V. herbacea, low temperature and sucrose play a role in the control of fructan degradation. This is the first study concerning the cloning and functional analysis of a 1-FEH cDNA of a native species from the Brazilian Cerrado. Results will contribute to understanding the role of fructans in the establishment of a very successful fructan flora of the Brazilian Cerrado, subjected to water limitation and low temperature during winter.

P.2.c.014 Mecamylamine as an augmentation treatment for depressed patients who were non-responders to citalopram

Using a combination of reverse transcription polymerase chain reaction (RT-PCR) and inverse-PCR techniques, we amplified, cloned and sequenced a full-length porcine 5-hydroxytryptamine 1F (5-ht1F) receptor complementary DNA (cDNA) derived from porcine trigeminal ganglion. Sequence analysis revealed 1101 base pairs (bp) encoding an open reading frame of 366 amino acids showing a high similarity (>90%) with the 5-ht1F receptor sequences from other species, including human. The recombinant porcine 5-ht1F receptor was expressed in African green monkey kidney cell lines (COS-7 cells) and its ligand binding profile was determined using [3H]5-HT. The affinities of several agonists (LY334370 (5-(4-fluorobenzoyl)amino-3-(1-methylpiperidin-4-yl)-1H-indole fumarate)>CP122638 (N-methyl-3 [pyrrolidin 2(R)-yl methyl]-1H-indol-5-ylmethyl sulphonamide)=naratriptan=5-HT>eletriptan>sumatriptan>frovatriptan=avitriptan>dihydroergotamine>zolmitriptan>5-carboxamidotryptamine>rizatriptan>alniditan=donitriptan>L694247 (2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indole-3-yl] ethylamine) and putative antagonists (methiothepin>GR127935 (N-[4-methoxy-3-(4-methyl-1-piperazinyl) phenyl]-2′-methyl 4′-(5-methyl-1,2,4-oxadiazol-3-yl) [1,1-biphenyl]-4-carboxamide hydrochloride)>ritanserin>SB224289 (2,3,6,7-tetrahydro-1′-methyl-5-[2′-methyl-4′(5-methyl-1,2,4-oxadiazol-3-yl) biphenyl-4-carbonyl] furo [2,3-f] indole-3-spiro-4′-piperidine hydrochloride)>BRL155572 ([1-(3-chlorophenyl)-4-[3,3-diphenyl (2-(S,R) hydroxypropanyl)piperazine] hydrochloride)>ketanserin=pindolol) correlated highly with those described for the recombinant human 5-ht1F receptor (Spearman correlation coefficient; rs=0.942). Nevertheless, as compared to the human homologue, some triptans (i.e. sumatriptan, zolmitriptan and rizatriptan) displayed a 10- to 15-fold lower affinity for the porcine 5-ht1F receptor. Using RT-PCR technique, the expression of porcine 5-ht1F receptor mRNA was observed in cerebral cortex, trigeminal ganglion and several blood vessels, but not in skeletal muscles. In conclusion, we have cloned and established the amino acid sequence and ligand binding profile of the porcine 5-ht1F receptor as well as the distribution of its mRNA. This information may be helpful in exploring the role of 5-ht1F receptor in physiological processes and diseases, such as migraine.

Cloning and Sequence Analysis of the Gene Encoding NiFe-hydrogenase from Klebsiella pneumoniae

Degenerate PCR primers were designed by multiple alignment of the protein sequences of known structural genes encoding the catalytic subunits of NiFe-hydrogenases obtained from Swiss-Prot Protein Sequence Database through CLUSTAL-W software and compared for conserved sequence motifs. A 1 kb amplified PCR product was obtained from the genomic DNA of Klebsiella pneumoniae using a set of degenerate primers, and then the inverse PCR technique was used to obtain the full hydrogenase coding region. A predicted secondary structure and a 3D structural model were constructed by homology modeling and docking. On the basis of these results, it was inferred that NiFe-hydrogenase from Klebsiella pneumoniae belongs to the membrane-bound H 2 evolving hydrogenase group (Ech hydrogenase group).

The Evolution of Techniques Used for the Detection of the F8 IVS 22 Inversion Mutation.

Almost half of patients with severe hemophilia A have a genomic rearrangement of the F8 gene, resulting in the separation of exons 1–22 from exons 23–26. This disruption is due to a recombination hotspot involving a 9.5kb region within intron 22 (int22h-1) and one of at least two extragenic copies (int22h-2 and inth22-3) located telomeric to the F8 gene. The 3 copies of int22h are estimated to be 99.9% homologous, with consistent differences between them confined to only 3 nucleotides. The more distal Int22h-3 lies in opposite orientation to the intragenic copy, and recombination between these two results in an inversion mutation, and a total lack of F8 protein. It was recently shown that int22h-2 is oriented in the same direction as int22h-1, and recombination events between these will result in either deletion or duplication rather than inversion.Three techniques have been published for the detection of the IVS22 recombination mutations: Southern blot, Long-PCR and Inverse PCR. We present data on a significant number of patients tested by more than one technique, and highlight difficulties in the detection/positive identification of mutations by each. Southern blotting (the only available method for many years) requires a large amount of DNA, not readily available from immuno-compromised patients, relies on radioisotope activity, and takes several days to obtain a result. All forms of recombination can be distinguished, but polymorphic variants can be misinterpreted as rare inversions resulting in patients being incorrectly assigned as inversion positive. In 1998, a long range PCR method was published for the analysis of IVS22 inversions. This technique, more rapid and less hazardous than Southern blotting, has proved to be problematic in many laboratories. The primers degrade rapidly, do not consistently permit multiplexing, and amplification is highly dependant on freshly extracted, high purity DNA. In 2005, a technique based on genomic digestion, followed by self ligation and PCR was published. This inverse PCR methodology has proved to be robust and can be used for fresh or archived samples, generating reproducible results within 36 hours. Proximal and distal recombinations are indistinguishable, and non-causative polymorphisms are not detected. During evaluation of the emerging technologies this laboratory has parallel tested a significant number of samples. 36 patients, including 6 carrier females, gave concordant results with Southern blot and inverse PCR. 12 patients with polymorphic, non-haemophilia associated, banding patterns gave a normal result by inverse PCR. 3/12 had been incorrectly assigned as mutation positive by southern blot; with 1 discovered only by the subsequent detection of non-linkage in the family. 25 patients gave identical results by long PCR and inverse PCR, including 4 carrier females. 2 patients were wrongly defined as positive by long PCR due to poor quality DNA. A result was unobtainable for 23 patients by southern blot, and for 6 patients by long PCR, due to insufficient or poor quality DNA. Of these, 8/23 and 6/6 were subsequently resolved by inverse PCR. Of the remaining 15/23 failures, the causative mutation has since been identified elsewhere in the F8 gene in 8 patients, while 7 require repeat bleeding.In conclusion, the inverse PCR technique provides a fast, reliable, reproducible and safe method for the detection of IVS 22 recombination mutations, and is recommended as the method of choice for diagnostic laboratories.