Gene-specific Oligonucleotide Primers Research Articles

Next-Generation Sequencing and Bioinformatics-Based Protocol for the Full-Length CYP2E1 Gene Polymorphism Analysis.

Pharmacogenetics studies provide clinically relevant information on the identified associations between genetic variants and individual variability in drug response, which, in turn, offers great promise for guiding personalized drug therapy and clinical trial design. However, there is a lack of information concerning the evidence-based clinical annotations of specific CYP2E1 genetic variants. To design and evaluate the next-generation sequencing-based method for full-length CYP2E1 gene polymorphism analysis. Seven gene-specific oligonucleotide primer pairs targeting overlapping CYP2E1 gene fragments spanning all nine gene exons with interleaving introns, untranslated (UTR) and intergenic regions were designed. Human DNA samples (n = 3) were used as a training set to check the primer performance and to optimize the PCR conditions. The effectiveness of the developed target amplification and sequencing protocol was evaluated using the test set comprising human DNA samples (n = 3) obtained from tuberculosis patients. Sequencing data analysis was performed on the Galaxy online-based platform. The sequencing data quality was sufficient for the detection of genetic variants dispersed throughout the CYP2E1 gene with a high degree of confidence in fully covered regions achieving optimal reading depth of the targeted fragment with high base call accuracy. Developed protocol can be applied in subpopulation-level association studies to determine whether single nucleotide variants (SNVs) or variant combinations from multiple regions of the CYP2E1 gene are of clinical significance.

No Evidence of Influenza A Virus RNA in South African Backyard Swine in the uMgungundlovu District of the KwaZulu-Natal Province

Background: Influenza A virus (IAV) is the most widely reported influenza type in swine populations worldwide. Since no information was available on IAV prevalence in backyard swine populations in South Africa, we, for the first time, performed a molecular surveillance for detecting IAV prevalence in backyard swine in the uMgungundlovu District of the KwaZulu-Natal province of South Africa. Methods: Swine oral secretion (saliva) samples (n=102) were collected from three backyard farms distantly located in the uMgungundlovu District in March 2021. Total RNA was used to detect IAV using a one-step real-time RT-PCR assay with matrix gene-specific oligonucleotide primers and a TaqMan probe. Result: None of the samples amplified the IAV matrix gene suggesting that the swine under investigation were free from IAV active infection; however, the quantified viral RNA of swine saliva samples needs further investigation to determine the presence of other RNA viruses. The present study was conducted during COVID-19 related lockdown restrictions in South Africa, during which a significantly decreased influenza activity was reported in the Southern Hemisphere. Increased alertness and behavioural changes in people, such as maintaining hygiene and using a face mask for day-to-day operations, might have limited the transmission of IAV between humans and swine.

Molecular characterization and polymorphism detection in HSPB6 gene in Sahiwal cattle

The present study was undertaken with the objectives of molecular characterization and detection of genetic polymorphisms in the HSPB6 gene of Sahiwal cattle. A total of 100 Sahiwal cattle maintained at LRC, NDRI, Karnal (Haryana) were included in the study. HSPB6 gene has been mapped on Bos taurus autosome 18 (BTA 18) and spans nearly 2620 bp comprising 3 exons and 2 introns. Four sets of forward and reverse gene-specific oligonucleotide primers of HSPB6 gene were designed using Primer3 software and amplicon size of 529 bp, 460 bp, 670 bp and 502 bp were ascertained. On the basis of comparative sequence analysis, total three polymorphic loci (SNPs) were detected at locus G436A, A2152G and A2417T in HSPB6 gene as compare to Bos taurus (NCBI GenBank AC_000175.1). Out of these three SNPs, two (A2152G & A2417T) were found in 3′-UTR region and third one (G436A) was detected in intronic region 1. However, monomorphic pattern was found in exon 2 of HSPB6 gene in Sahiwal cattle. Further research need be directed to find out the association of allelic variants in major heat shock protein genes to establish markers for the future genetic improvement of livestock through marker assisted selection.

Keeping qRT-PCR rigorous and biologically relevant.

Because of the importance of differential gene expression in the growth, development, and reproduction of organisms, assays of mRNA transcript levels are widely employed in biological research. The most frequently used assay combines reverse transcription (RT) with quantitative polymerase chain reaction (qPCR) and is known as qRT-PCR (or RT-qPCR). In practice, RT generates a single-stranded cDNA copy of each molecule in an mRNA population, after which a qPCR thermocycler is used in conjunction with gene-specific oligonucleotide primers to amplify selected target cDNAs (Bustin et al. 2009). Fluorescent probes allow progress to be monitored through three phases: an initial phase during which the increasing signal cannot be distinguished from background noise, a 2nd phase of observable exponential signal increase, and a third and final phase of slowing signal increase as reagents and other factors become rate limiting. Quantification of each target mRNA requires the determination of the number of cycles (known as Ct, Cq, Cp, or TOP) required to reach a pre-set fluorescence threshold during the second phase. When a transcript is twice as abundant in one sample as it is in another, the Ct will occur about one cycle earlier for the more abundant transcript. The varying transcript levels of target genes must then be normalized against the stable transcript levels of one or more reference genes present in the same mRNA population. Algorithms are available to validate the stability of these reference genes. With use of appropriate controls, standardization, and normalization, measurements of Ct can lead to reliable estimates of the relative amount of a target transcript in the input mRNA population (Bustin et al. 2009). Despite its complexity, qRT-PCR is rapid and cost effective, and, if sufficient care is taken with the design and execution of experiments, it is reliable and sensitive. There are, however, two trends of concern in the publication of qRT-PCR experiments. The first is that many papers do not contain enough information to judge whether the data are reliable. Bustin et al. (2009) addressed this question by defining ‘‘minimum information for publication of quantitative real-time PCR experiments’’ (MIQE). The second trend of concern is the increasing number of manuscripts that claim to have identified reference genes without comparing them to target genes in a well-defined and relevant biological context. We agree with Bustin et al. (2010), who considered it pointless and misleading to publish such ‘reference gene papers’ as stand-alone reports. In response to these trends, we propose that publishable qRT-PCR manuscripts should satisfy two criteria. First, the design, execution, and reporting of qRT-PCR experiments should address the items listed in the MIQE guidelines. Second, the selection and validation of reference genes should be reported in relation to target gene behavior in a well-defined biological context. Here we discuss the background to these criteria and their implementation relative to plant biology studies. Communicated by Neal Stewart.

Folate transporter expression decreases in the human placenta throughout pregnancy and in pre-eclampsia

The transport of folate across the placenta involves a number of different receptors including folate receptor-alpha (FR-α), reduced folate carrier (RFC) and proton coupled folate transporter (PCFT). In addition there are a number of ATP-dependent transporters which have also recently been shown to be involved in folate transport; these include ABCB1, ABCC2 and BCRP (ABCG2). The aim of the current study was to characterise the placental mRNA and protein expression of these folate transporters throughout gestation and also to see if expression is altered in pre-eclampsia. Placental tissue was collected from women undergoing termination of pregnancy (TOP) and from women undergoing elective Caesarean section. To investigate mRNA expression quantitative real time PCR was used with gene specific oligonucleotide primers to FR-α, RFC, PCFT, ABCB1, ABCC2, BCRP and the reference gene YWHAZ. Protein expression was also characterised using immunohistochemistry of paraffin embedded placental tissue. Both protein and mRNA expression of all transporters examined decreased as the gestation progressed. Expression of FR-α and PCFT mRNA and protein were decreased in pre-eclampsia compared with normal term pregnancy. The higher levels of expression of FR-α, RFC, PCFT, ABCB1, ABCC2 and BCRP in early pregnancy indicate that these transporters may have an important role in the establishment and development of the placenta, with expression reducing in preparation for parturition. Reductions in FR-α and PCFT in pre-eclampsia may be a mechanism involved in the pathogenesis of pre-eclampsia by limiting placental folate uptake resulting in reduced levels of angiogenesis, cell proliferation and antioxidant protection.

Molecular identification and further characterization of Arcanobacterium pyogenes isolated from bovine mastitis and from various other origins

The present study was designed to identify phenotypically and genotypically 61 Arcanobacterium pyogenes isolated from bovine mastitis and from various other origins. The A. pyogenes isolates showed the typical cultural and biochemical properties of this species and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the superoxide dismutase A encoding gene sodA of reference strains representing 8 species of genus Arcanobacterium and subsequent design of A. pyogenes sodA gene-specific oligonucleotide primer. The A. pyogenes sodA gene-specific oligonucleotide primer allowed, together with previously described A. pyogenes 16S-23S rDNA intergenic spacer region-specific oligonucleotide primer, a reliable molecular identification of all 61 A. pyogenes of various origins. The additionally performed PCR-mediated amplification of 5 known and putative virulence factor encoding genes revealed that 100, 20, 87, 75, and 98% of the A. pyogenes carried the genes plo, cbpA, nanH, nanP, and fimA, which allowed an individual strain characterization. This might help to elucidate the role the putative virulence factors play in bovine mastitis and in various other infections caused by this bacterial pathogen.

Molecular analysis of antigen receptor variable region repertoires in T lymphocytes infiltrating the intrathyroidal and extrathyroidal manifestations in patients with Graves' disease.

To determine whether T cells infiltrating thyroid, orbital and pretibial tissue of patients with Graves' ophthalmopathy (GO) and pretibial dermopathy (PTD) represent a primary immune response that is directed against certain antigenic determinants shared between these involved tissues, we characterized these T cells at the molecular level. T cell antigen receptor (TcR) variable (V) region gene usage in thyroid, orbital, pretibial tissue and peripheral blood mononuclear cells of patients with GD, GO and PTD was assessed using RT-PCR and 22 V alpha and 23 V beta gene-specific oligonucleotide primers, followed by Southern hybridization analysis using TcR C-region-specific, digoxigenin-labelled oligonucleotide probes. In some instances, CDR3- and junctional regions of TcR V beta genes were sequenced. Marked restriction and similarities of V alpha and V beta gene usage were detected in samples derived from patients with active GO and PTD of recent onset. Moreover, sequence analysis of junctional domains of V beta families revealed oligoclonality of some intrathyroidal, orbital and pretibial T cell populations as well as the presence of conserved junctional motifs shared by T cells derived the thyroid gland and the extrathyroidal sites. These data suggest that similar antigenic determinants may be responsible for the recruitment and oligoclonal expansion of T cells both within the thyroid gland and at the involved extrathyroidal sites in Graves' disease.

Setting up a polymerase chain reaction assay for the detection of toxic cyanobacteria

Cyanobacteria are aquatic and photosynthetic prokaryotes which form harmful algal blooms under certain conditions. Water blooms of cyanobacteria from the genus Microcystis are of increasing concern due to their production of microcystin, a cyclic heptapeptide which is formed nonribosomally by peptide and polyketide synthetases. A number of studies have targeted the PCR amplification of microcystin synthetase gene cluster for the identification of toxic cyanobacteria, both in cultivated strains and environmental samples. In this study, water samples collected from seven sites and the cultures originated from the cyanobacterial bloom of the Lake Beira were analyzed by polymerase chain reaction (PCR) using the cyanobacterial specific oligonucleotide primers for the 16S rRNA gene and genus specific oligonucleotide primers for the mcy E gene. All DNA samples submitted to PCR reactions yielded the unique fragments of about 450 bp for the 16S rRNA gene and 250bp for the mcy E gene. The results of the BLAST EF051239 showed 98% of the nucleotide homology to the Microcystis aeruginosa gene of 16S ribosomal RNA and EF051238, 95% homology to the mcy E gene of M. aeruginosa (PCC 7941).These results confirm the presence of microcystin producing M. aeruginosa in the Beira Lake and the need for rapid identification of toxin producing cyanobacteria from environmental samples. Keywords : Cyanobacteria, mcy gene, microcystin, Microcystis aeruginosa doi:10.4038/jnsfsr.v36i3.159 Journal of the National Science Foundation of Sri Lanka 36 (3) 229-233

A community resource for high-throughput quantitative RT-PCR analysis of transcription factor gene expression in Medicago truncatula.

BackgroundMedicago truncatula is a model legume species that is currently the focus of an international genome sequencing effort. Although several different oligonucleotide and cDNA arrays have been produced for genome-wide transcript analysis of this species, intrinsic limitations in the sensitivity of hybridization-based technologies mean that transcripts of genes expressed at low-levels cannot be measured accurately with these tools. Amongst such genes are many encoding transcription factors (TFs), which are arguably the most important class of regulatory proteins. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is the most sensitive method currently available for transcript quantification, and one that can be scaled up to analyze transcripts of thousands of genes in parallel. Thus, qRT-PCR is an ideal method to tackle the problem of TF transcript quantification in Medicago and other plants.ResultsWe established a bioinformatics pipeline to identify putative TF genes in Medicago truncatula and to design gene-specific oligonucleotide primers for qRT-PCR analysis of TF transcripts. We validated the efficacy and gene-specificity of over 1000 TF primer pairs and utilized these to identify sets of organ-enhanced TF genes that may play important roles in organ development or differentiation in this species. This community resource will be developed further as more genome sequence becomes available, with the ultimate goal of producing validated, gene-specific primers for all Medicago TF genes.ConclusionHigh-throughput qRT-PCR using a 384-well plate format enables rapid, flexible, and sensitive quantification of all predicted Medicago transcription factor mRNAs. This resource has been utilized recently by several groups in Europe, Australia, and the USA, and we expect that it will become the 'gold-standard' for TF transcript profiling in Medicago truncatula.

Functional Characterization of Three G Protein-coupled Receptors for Pigment Dispersing Factors in Caenorhabditis elegans

Here, we report the identification, cloning, and functional characterization of three Caenorhabditis elegans G protein-coupled pigment dispersing factor (PDF) receptors, which we designated as Ce_PDFR-1a, -b, and -c. They represent three splice isoforms of the same gene (C13B9.4), which share a high degree of similarity with the Drosophila PDF receptor and are distantly related to the mammalian vasoactive intestinal peptide receptors (VPAC2) and calcitonin receptors. In a reverse pharmacological screen, three bioactive C. elegans neuropeptides, which were recently identified as the Drosophila PDF orthologues, were able to activate these receptors in a dose-dependent manner with nanomolar potency (isoforms a and b). Integrated green fluorescent protein reporter constructs reveal the expression of these PDF receptors in all body wall muscle cells and many head and tail neurons involved in the integration of environmental stimuli and the control of locomotion. Using a custom data analysis system, we demonstrate the involvement of this newly discovered neuropeptide signaling system in the regulation of locomotor behavior. Overexpression of PDF-2 phenocopies the locomotor defects of a PDF-1 null mutant, suggesting that they elicit opposite effects on locomotion through the identified PDF receptors. Our findings strengthen the hypothesis that the PDF signaling system, which imposes the circadian clock rhythm on behavior in Drosophila, has been functionally conserved throughout the protostomian evolutionary lineage.

Genomic location and characterisation of nonclassical MHC class I genes in cattle

The cattle major histocompatibility complex (MHC) region contains a variable number of classical class I genes encoding polymorphic, ubiquitously expressed molecules with a role in antigen presentation. Class I cDNA sequences have previously been reported that are thought to derive from putative nonclassical class I genes. We have located four nonclassical class I genes within the cattle genome; three are close to the MIC genes, and one is close to the classical class I genes. The genomic position relative to anchor genes is very similar to the arrangement reported in the pig MHC region. We have designed gene-specific oligonucleotide primers with which to investigate the presence of these genes in distinct and well-defined MHC haplotypes and to assess transcription in different cell types. Analysis and comparison of all sequences allows an assessment of allelic variation in each case. Partial characterisation gives an indication of the possible role and likely importance of each of these genes.

Detection and quantitation of Solenopsis invicta virus in fire ants by real-time PCR

A quantitative real-time PCR (QPCR) method was developed to detect and quantify the amount of Solenopsis invicta virus (SINV) infecting individual ants of S. invicta. The two-step method utilized a gene-specific oligonucleotide primer targeting the SINV RNA-dependent RNA polymerase (RdRp) for cDNA synthesis. Dithiothreitol used in the cDNA synthesis step was found to significantly decrease the detection sensitivity for SINV RdRp and was therefore omitted. SINV RdRp cDNA was then quantified by QPCR using SYBR Green dye and a standard curve generated from SINV RdRp plasmid clones. A standard curve was successfully constructed from clones of the SINV RdRp region. A strong linear relationship [ r 2 = 0.998; y = (−3.63 ± 0.37) x + (39.19 ± 1.33)] between C T and starting SINV RdRp copy number was observed within a dynamic range of 5–5 × 10 6 copies. SINV RdRp copy number was determined with the optimized QPCR method in individual S. invicta ants taken from an infected field colony. Worker ants exhibited the highest RdRp copy number (2.1 × 10 9 copies/worker ant) and pupae exhibited the lowest (4.2 × 10 2 copies/pupa). Mean RdRp copy number was lowest in early larvae and pupae. Overall, SINV RdRp copy number increased through larval development, sharply declined during pupation, then sharply increased in adults.

Diversity of Ca 2+-activated K + channel transcripts in inner ear hair cells

Hair cells express a complement of ion channels, representing shared and distinct channels that confer distinct electrophysiological signatures for each cell. This diversity is generated by the use of alternative splicing in the α subunit, formation of heterotetrameric channels, and combinatorial association with β subunits. These channels are thought to play a role in the tonotopic gradient observed in the mammalian cochlea. Mouse Kcnma1 transcripts, 5′ and 3′ ESTs, and genomic sequences were examined for the utilization of alternative splicing in the mouse transcriptome. Comparative genomic analyses investigated the conservation of KCNMA1 splice sites. Genomes of mouse, rat, human, opossum, chicken, frog and zebrafish established that the exon–intron structure and mechanism of KCNMA1 alternative splicing were highly conserved with 6–7 splice sites being utilized. The murine Kcnma1 utilized 6 out of 7 potential splice sites. RT-PCR experiments using murine gene-specific oligonucleotide primers analyzed the scope and variety of Kcnma1 and Kcnmb1–4 expression profiles in the cochlea and inner ear hair cells. In the cochlea splice variants were present representing sites 3, 4, 6, and 7, while site 1 was insertionless and site 2 utilized only exon 10. However, site 5 was not present. Detection of KCNMA1 transcripts and protein exhibited a quantitative longitudinal gradient with a reciprocal gradient found between inner and outer hair cells. Differential expression was also observed in the usage of the long form of the carboxy-terminus tail. These results suggest that a diversity of splice variants exist in rodent cochlear hair cells and this diversity is similar to that observed for non-mammalian vertebrate hair cells, such as chicken and turtle.

Patterns of B-type cyclin gene expression during adventitious rooting of micropropagated cork oak

The aim of our study is the identification of the critical points in the rooting process at the molecular level for possible improvement of the propagation of cork oak. The rhizogenic potential of woody plants depends on both genotype and developmental stage. To investigate the role of cyclin transcription in the regulation of cell division during adventitious root induction, we have analysed the temporal expression of a member of the cyclin gene family. The histological modifications that occur during the root induction and formation processes as a consequence of auxin treatments can be detected by the technique of tissue printing for hydrogen peroxide (H2O2). Thus a H2O2 printing assay was performed in parallel with cyclin expression studies. We report the cloning and sequencing of a B-type cyclin genomic fragment from micropropagated cork oak using gene-specific and genotype-independent oligonucleotide primers to conserved regions of the gene. After 48 h of auxin induction, the expression of this B-type cyclin increased, possibly in association with the first cell divisions. The expression observed in control shoots may reflect unorganised cell divisions associated with callus formation.

Detection ofVibrio parahaemolyticusin Shellfish by Use of Multiplexed Real-Time PCR with TaqMan Fluorescent Probes

We developed a multiplexed real-time PCR assay using four sets of gene-specific oligonucleotide primers and four TaqMan probes labeled with four different fluorophores in a single reaction for detection of total and pathogenic Vibrio parahaemolyticus, including the pandemic O3:K6 serotype in oysters. V. parahaemolyticus has been associated with outbreaks of food-borne gastroenteritis caused by the consumption of raw or undercooked seafood and therefore is a concern to the seafood industry and consumers. We selected specific primers and probes targeting the thermostable direct hemolysin gene (tdh) and tdh-related hemolysin gene (trh) that have been reported to be associated with pathogenesis in this organism. In addition, we targeted open reading frame 8 of phage f237 (ORF8), which is associated with a newly emerged virulent pandemic serotype of V. parahameolyticus O3:K6. Total V. parahaemolyticus was targeted using the thermolabile hemolysin gene (tlh). The sensitivity of the combined four-locus multiplexed TaqMan PCR was found to be 200 pg of purified genomic DNA and 10(4) CFU per ml for pure cultures. Detection of an initial inoculum of 1 CFU V. parahaemolyticus per g of oyster tissue homogenate was possible after overnight enrichment, which resulted in a concentration of 3.3x10(9) CFU per ml. Use of this method with natural oysters resulted in 17/33 samples that were positive for tlh and 4/33 samples that were positive for tdh. This assay specifically and sensitively detected total and pathogenic V. parahaemolyticus and is expected to provide a rapid and reliable alternative to conventional detection methods by reducing the analysis time and obviating the need for multiple assays.

Crystal structure of dihydrodipicolinate synthase (BA3935) from Bacillus anthracis at 1.94 Å resolution

Introduction. As part of a structural genomics program, we are determining structures of proteins from the causative agent of anthrax, Bacillus anthracis, a Grampositive spore-forming bacterium. Among our initial candidates for crystallographic analysis are the products of essential genes based on knock-out studies in Bacillus subtilis. The BA3935 gene of B. anthracis (www.tigr.org), annotated as DapA2, encodes a putative protein consisting of 292 amino acid residues with a subunit molecular weight of 31,233 Da. The predicted protein has 60% identity with dihydrodipicolinate synthase (DHDPS or DapA) from B. subtilis and 40% amino acid sequence identity to its orthologue in Escherichia coli. dapA is one of only 271 out of the total of 4118 genes in B. subtilis that are indispensable for growth of the organism on standard laboratory media. 1 DHDPS catalyses the condensation of aspartate semialdehyde and pyruvate and is the first committed step on the pathway to diaminopimelate and L-lysine in prokaryotes, some fungi, and higher plants (Scheme 1). The product released from the E. coli enzyme has been shown to be 4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid (HTDPA) rather than L-2,3-dihydrodipicolinic acid (DHDPA); as the name of the enzyme suggests, 2 DHDPA can be formed spontaneously from HTDPA by elimination of water. The steps of aspartate semialdehyde synthesis from aspartate are shared with the biosynthetic pathways leading methionine and threonine. The diaminopimelate/ lysine pathway is thought to be of particular importance in Gram-positive bacteria because diaminopimelate makes up a higher proportion of the dry cell weight than it does in Gram-negative bacteria, a consequence of the thicker cell wall in the former. In Bacilli, the product of the DHDPS reaction is further reduced by dipicolinate synthase to dipicolinate, which makes up 10% of the dry weight of spores. Crystal structures have been determined of DHDPS from E. coli, 3 Nicotiana sylvestris, 4 and most recently from Thermotoga maritima. 5 In this article, we report the crystal structure of DHDPS from B. anthracis (Ba DHDPS), the first structure of a dihydrodipicolinate synthase from a Gram-positive bacterium. The structure of Ba DHDPS was determined by molecular replacement using the coordinate set for the E. coli orthologue (PDB code 1DHP) as the search model. 3 Two structures have been determined to 1.9 and 2.2 A resolution in different orthorhombic crystal forms. Data collection, refinement, and model-building statistics are summarized in Table I. The structures in the two crystal forms are very similar and unless otherwise stated, the commentary here will refer to the structure refined to higher resolution

Crystal structure of PurE (BA0288) from Bacillus anthracis at 1.8 Å resolution

Crystal structure of PurE (BA0288) from <i>Bacillus anthracis</i> at 1.8 Å resolution

N-acetyl- d-galactosamine/ N-acetyl- d-glucosamine – recognizing lectin from the snail Cepaea hortensis: purification, chemical characterization, cloning and expression in E. coli

From the albumin gland of the snail Cepaea hortensis we isolated and characterized a new N-acetyl- d-galactosamine/ N-acetyl- d-glucosamine (GalNAc/GlcNAc) specific lectin (CHA-II) which was purified by a combination of affinity chromatography on GalNAc-agarose and gel filtration. The purified native lectin was found to be a multimeric protein, as revealed by SDS–PAGE and MALDI-TOF analysis. In SDS–PAGE the denatured and reduced lectin showed two bands of molecular masses with 17 and 15.5 kDa which reacted equally with anti-CHA-II rabbit antiserum. The lectin was O- and N-glycosylated with [(Gal) 2-Man] 2-Man-GlcNAc-GlcNAc-Asn as a probable structure for the oligosaccharide. Isoelectric focusing revealed a heterogeneous protein of at least four bands around pH 8.7. Tryptic peptides of CHA-II were N-terminally sequenced and highly degenerated gene specific oligonucleotide primers (GSPs) had been constructed. Using total RNA isolated from albumin glands, cDNAs were produced by the running race technique. Specific PCR fragments were obtained by PCR using GSPs, the universal primer and 5′- or 3′-RACE-cDNAs. The amplified fragments were cloned into the vector pDrive and were sequenced. The resulting total cDNA sequence consisted of 496 base pairs including an open reading frame of 360 base pairs which encoded a protein of 120 amino acids. The protein carried a putative signal peptide. The mature protein was predicted to comprise 99 amino acid residues with a calculated molecular weight of 11,239 Da. The PCR fragment encoding the mature protein was cloned into the vector pQE30 and expressed in E. coli. Recombinant CHA-II lectin was produced as inclusion bodies and extracted by 6 M guanidine hydrochloride. After refolding, the recombinant CHA-II agglutinated specifically human red blood cells of groups A and AB. In immunodiffusion experiments using rabbit antiserum raised against the native lectin, the protein showed a precipitation line of identity with the native lectin.

Cloning and expression of a sialic acid-binding lectin from the snail Cepaea hortensis

Highly degenerated gene-specific oligonucleotide primers (GSPs) were constructed from the amino acid sequence of tryptic fragments produced from the purified sialic acid-specific lectin of the garden snail Cepaea hortensis. From the albumin glands, the total RNA or the mRNA was prepared. Combination of a universal primer with the GSPs delivered gene-specific fragments of about 650, 620 and 280 bp by polymerase chain reaction (PCR). These fragments were cloned into the vector pDrive (Qiagen) and sequenced. The resulting cDNA sequence consisted of 744 bp, including an open reading frame of 480 bp. The encoded protein consists of 159 amino acids, including the putative signal sequence peptide. The mature protein should comprise 141 amino acid residues with a calculated molecular mass of 15 529 Da. The expression of the recombinant lectin in Escherichia coli resulted in a soluble protein reacting specifically with rabbit antiserum raised against the native lectin.

The use of the S haplotype-specific F-box protein gene, SFB, as a molecular marker for S-haplotypes and self-compatibility in Japanese apricot (Prunus mume).

Japanese apricot ( Prunus mume) exhibits the S-RNase-based gametophytic self-incompatibility system as do other self-incompatible Prunus species. This report identifies the S haplotype-specific F-box protein gene ( SFB), a candidate gene for pollen- S, of Japanese apricot, which leads to the development of a molecular typing system for S-haplotype in this fruit species. Both 5'- and 3'-RACE (rapid amplification of cDNA ends) were performed with SFB gene-specific oligonucleotide primers to clone Pm-SFB(1) and Pm-SFB(7) of 'Nanko ( S(1) S(7))'. As in the case of SFB of other Prunus species, Pm-SFB(1) and Pm-SFB(7) showed a high level of S-haplotype-specific sequence polymorphism and their expression was specific to pollen. Genomic DNA-blot analyses of 11 Japanese apricot cultivars with the Pm-SFB probes under low stringency conditions yielded RFLP bands specific to the S(1)- to S(8)-haplotypes as well as a self-compatible S(f)-haplotype. A practical usage of SFB as a molecular marker for S-haplotypes and self-compatibility in Japanese apricot is discussed.