Occurrence Of Single Nucleotide Polymorphisms Research Articles

The structure of the Cyberlindnera jadinii genome and its relation to Candida utilis analyzed by the occurrence of single nucleotide polymorphisms

The yeast Cyberlindnera jadinii is a close relative of Candida utilis that is being used in the food and feed industries. Here, we present the 12.7Mb genome sequence of C. jadinii strain CBS 1600 generated by next generation sequencing. The deduced draft genome sequence consists of seven large scaffolds analogous to the seven largest chromosomes of C. utilis. An automated annotation of the C. jadinii genome identified 6147 protein-coding sequences. The level of ploidy for both genomes was analyzed by calling single nucleotide polymorphisms (SNPs) and was verified measuring nuclear DNA contents by florescence activated cell sorting (FACS). Both analyses determined the level of ploidy to diploid for C. jadinii and to triploid for C. utilis. However, SNP calling for C. jadinii also identified scaffold regions that seem to be haploid, triploid or tetraploid.

Ocorrência do SNP c.767G>T no gene DNM1responsável pelo colapso induzido pelo exercício em cães da raça Labrador Retriever no Estado de São Paulo

Resumo:O colapso induzido pelo exercício (EIC) é considerado uma síndrome autossômica recessiva que afeta principalmente cães da raça Labrador Retriever. A doença é caracterizada por fraqueza muscular e colapso após exercício intenso. Usualmente, ocorre recuperação clínica após o episódio, mas alguns animais podem vir a óbito. Os sinais clínicos são decorrentes do polimorfismo de base única (SNP) c.767G>T no gene Dynamin 1 (DNM1). O objetivo deste trabalho foi determinar a ocorrência deste SNP em 321 cães da raça Labrador Retriever do Estado de São Paulo. Primers específicos para a amplificação de todo o exon 6 do gene DNM1 foram usados nas PCRs utilizando DNA a partir de amostras de sangue ou swab bucal, a avaliação final foi realizada com sequenciamento direto dos produtos da PCR. Dentre os 321 animais estudados, 3,4 % (11/321) eram homozigotos para o SNP c.767G>T no gene DNM1 e 24,6% (79/321) eram heterozigotos. Somente um dos 11 animais homozigotos apresentavam sinais clínicos compatíveis com a EIC. Este é o primeiro estudo sobre a ocorrência deste SNP no Brasil e considerando que quase 25% dos animais estudados eram heterozigotos, a genotipagem dos animais para este SNP pode ser importante antes dos acasalamentos para cães desta raça. A EIC deve ser considerada nos diagnósticos diferenciais de enfermidades neuromusculares em cães da raça Labrador Retriever.

Polymorphisms in DNA Repair Gene XRCC1 (Arg194Trp) and (Arg399Gln) and their Role in the susceptibility of Bacterial Meningitis

Meningitis is a contagious infectious disease with high rates of mortality. Most pathogenic microbes in humans have the ability to cause bacterial meningitis. However, the most common pathogens are Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae type b (Hib). It was found that the susceptibility to this infectious disease may be related to genetic characteristics of the host, such as the occurrence of single nucleotide polymorphisms (SNPs). In our previous work, association of SNPs in DNA repair genes with bacterial meningitis (BM) was demonstrated. In this study we evaluated two non-synonymous SNPs of the repair gene XRCC1 Arg194Trp (rs 1799782) and Arg399Gln (rs 25487) in patients with BM and health volunteers. The patient genotypes were investigated by PCR-RFLP. DNA damages were quantified using the genomic DNA with formamidopyrimidine DNA-glycosylase (FPG). Cytokines and chemokines were quantified from cerebrospinal fluid samples from BM patients. Concerning the SNP XRCC1 Arg194Trp, none association was found relation to BM. However, a higher frequency of heterozygous genotype for XRCC1 Arg399Gln was observed in the control group compared to the BM group (P=0.043; OR=0.426). DNA damage and cytokine/chemokines levels were not positively correlated with polymorphic genotypes. In conclusion, there is an indication that the SNP XRCC1 Arg399Gln could have a possible protective effect against BM.

Abstract 4983: IRAK4 and breast cancer cell migration in culture and zebrafish xenografts

Abstract Growing evidence indicates that incongruities in the inflammatory and immune response pathways contribute to tumorigenesis. We seek to explore the underlying biological mechanisms that both promote and inhibit breast cancer (BrCa) tumorigenesis via innate immune pathways involving the Toll-like receptors (TLRs) and their associated agonists. Women of African descent (AA) are an understudied group at high risk for developing aggressive BrCa. Therefore, we previously examined the occurrence of single nucleotide polymorphisms (SNPs) in selected genes along TLR pathways among a small cohort of AA women with breast cancer and identified a SNP in the central regulatory kinase IRAK4 that was associated with increased breast cancer risk. In silico predictions suggest that the IRAK4 minor variant rs4251545 codes for an Ala428Thr missense mutation that introduces a potential phosphorylation site in the extreme carboxy terminus (XCT) of the IRAK-4 kinase domain. The current study tests the hypothesis that functional IRAK4 promotes BrCa cell growth and migration using in vitro (cultured human BrCa cell) and in vivo (zebrafish BrCa xenograft) models. Materials and methods: ER-positive MCF7 and triple-negative MDA-MB-231 (wild-type IRAK4) and MDA-MB-468 (IRAK4 w/rs4251545) human BrCa cell lines were compared. Cultured cells were treated for 24 hours with or without IRAK1/4 inhibitor. Cell migration and cell growth among BrCa lines were compared using wound healing and crystal violet assays, respectively. BrCa cell xenografts of zebrafish (ZF) embryos were examined for metastatic potential and the physiologic effects of morpholino inhibition of ZF IRAK4 among embryos with and without BrCa xenograft burdens were compared. In addition, zebrafish/tumor xenograft model was used to address the in vivo metastatic property of BrCa cells and the effect of targeting zebrafish irak4 using morpholino (MO) knockdown approach in the tumor-host environment on the metastatic behavior of MDA-MB-468 cells. Results: Treatment with IRAK1/4 inhibitor delayed BrCa migration and growth in all BrCa cells lines. Relative in vivo migration rates/ (or metastatic property) of MDA-MB-231 and MDA-MB-468 BrCa xenografts in ZF resemble in vitro rates, in which MDA-MB-231s migrate metastasized more rapidly/ (or aggressively). MO inhibition of ZF zebrafish IRAK4 irak4 gene expression in the host environment MDA-MB-468 xenografted fish inhibits MDA-MB-468 metastasis in the zebrafish/tumor xenograft. Conclusion: Inhibition of IRAK4 reduces BrCa growth and migration in vitro and in vivo in zebrafish. The development of drugs that target IRAK4 and other regulators of the innate immune pathway may help modulate BrCa tumor progression. Citation Format: K. Sean Kimbro, Karine Ferri-Lagneau, Jamil Haider, Susan Yeyeodu, Xiaohe Yang, TinChung Leung. IRAK4 and breast cancer cell migration in culture and zebrafish xenografts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4983. doi:10.1158/1538-7445.AM2014-4983

DNA Barcoding for Discriminating the Economically Important Cinnamomum verum from Its Adulterants

Traded forms of spice and spice powders are often subjected to admixing with inferior substances by design or default, affecting public health and national prestige. Cinnamomum verum (true cinnamon), a high-value spice, is often adulterated with its inferior species such as C. cassia and C. malabatrum. The presence and detection of the spurious species in traded barks (whole or powder) of true cinnamon is posing problems. DNA markers are now used to detect such adulteration. Here we report the application of a DNA barcoding method to detect these adulterants in traded market samples of true cinnamon using the barcoding loci rbcL, matK and psbA-trnH. The PCR success rate, sequencing efficiency, inter and intra specific divergence, and occurrence of single nucleotide polymorphisms (SNPs) were utilized to assess the potential of each barcode loci to authenticate C. verum from its related adulterants. The amplification and sequencing success was 100% for rbcL and psbA-trnH while matK failed to amplify in the market samples. The locus of rbcL showed higher interspecific divergence while psbA-trnH exhibited lower interspecific divergence. SNPs specific to C. cassia were detected in rbcL locus in seven out of the ten market samples studied thereby confirming the presence of C. cassia adulteration in commercial samples of true cinnamon. Out of the three loci, rbcL locus proved to be efficient in tracing out adulterants in traded cinnamon. The SNP sites in this locus can be exploited in designing C. cassia specific primers, enabling kit development for easy detection of adulterants at the band level itself thereby bypassing the cost of sequencing.

Higher frequency of C.3435 of the ABCB1 gene in patients with tramadol dependence disorder

Background: Polymorphic variation at the ABCB1 gene has been shown to affect the pharmacodynamics and kinetics of various drugs. Aim: This study aimed to determine the frequency of occurrence of Single Nucleotide Polymorphism (SNP) in position A118G OPRM1 (rs1799971) gene and C.3435 (rs1045642) gene in tramadol users in comparison with normal controls. Methods: This was a cross sectional case-control outpatient study. The study sample consisted of 127 subjects (74 tramadol-dependents and 50 healthy controls). All patients fulfilled the Diagnostic and Statistical Manual IV Criteria for substance dependence (on tramadol). Genotyping of the OPRM1 gene 118 SNP and ABCB1 genes C.3435 SNP was performed by PCR, followed by restriction fragment length polymorphism identification. Results: A significant association was found between the ABCB1 gene T allele at the polymorphic site 3435 and tramadol dependence. No significant association was observed with the A118G OPRM1 gene. Conclusion: The high frequency of ABCB1 gene T allele present at the polymorphic site 3435 could provide a protective mechanism from tramadol dependence disorder. Further study, using a larger sample, would be useful in further evaluating the possible role of ABCB1 gene polymorphisms.

Specialized Dynamical Properties of Promiscuous Residues Revealed by Simulated Conformational Ensembles

The ability to interact with different partners is one of the most important features in proteins. Proteins that bind a large number of partners (hubs) have been often associated with intrinsic disorder. However, many examples exist of hubs with an ordered structure, and evidence of a general mechanism promoting promiscuity in ordered proteins is still elusive. An intriguing hypothesis is that promiscuous binding sites have specific dynamical properties, distinct from the rest of the interface and pre-existing in the protein isolated state. Here, we present the first comprehensive study of the intrinsic dynamics of promiscuous residues in a large protein data set. Different computational methods, from coarse-grained elastic models to geometry-based sampling methods and to full-atom Molecular Dynamics simulations, were used to generate conformational ensembles for the isolated proteins. The flexibility and dynamic correlations of interface residues with a different degree of binding promiscuity were calculated and compared considering side chain and backbone motions, the latter both on a local and on a global scale. The study revealed that (a) promiscuous residues tend to be more flexible than nonpromiscuous ones, (b) this additional flexibility has a higher degree of organization, and (c) evolutionary conservation and binding promiscuity have opposite effects on intrinsic dynamics. Findings on simulated ensembles were also validated on ensembles of experimental structures extracted from the Protein Data Bank (PDB). Additionally, the low occurrence of single nucleotide polymorphisms observed for promiscuous residues indicated a tendency to preserve binding diversity at these positions. A case study on two ubiquitin-like proteins exemplifies how binding promiscuity in evolutionary related proteins can be modulated by the fine-tuning of the interface dynamics. The interplay between promiscuity and flexibility highlighted here can inspire new directions in protein–protein interaction prediction and design methods.

Genetic variations in the beta-tubulin gene and the internal transcribed spacer 2 region of Trichuris species from man and baboons.

BackgroundThe whipworm Trichuris trichiura has been estimated to infect 604 – 795 million people worldwide. The current control strategy against trichuriasis using the benzimidazoles (BZs) albendazole (400 mg) or mebendazole (500 mg) as single-dose treatment is not satisfactory. The occurrence of single nucleotide polymorphisms (SNPs) in codons 167, 198 or 200 of the beta-tubulin gene has been reported to convey BZ-resistance in intestinal nematodes of veterinary importance. It was hypothesised that the low susceptibility of T. trichiura to BZ could be due to a natural occurrence of such SNPs. The aim of this study was to investigate whether these SNPs were present in the beta-tubulin gene of Trichuris spp. from humans and baboons. As a secondary objective, the degree of identity between T. trichiura from humans and Trichuris spp. from baboons was evaluated based on the beta-tubulin gene and the internal transcribed spacer 2 region (ITS2).MethodsNucleotide sequences of the beta-tubulin gene were generated by PCR using degenerate primers, specific primers and DNA from worms and eggs of T. trichiura and worms of Trichuris spp. from baboons. The ITS2 region was amplified using adult Trichuris spp. from baboons. PCR products were sequenced and analysed. The beta-tubulin fragments were studied for SNPs in codons 167, 198 or 200 and the ITS2 amplicons were compared with GenBank records of T. trichiura.ResultsNo SNPs in codons 167, 198 or 200 were identified in any of the analysed Trichuris spp. from humans and baboons. Based on the ITS2 region, the similarity between Trichuris spp. from baboons and GenBank records of T. trichiura was found to be 98 – 99%.ConclusionsSingle nucleotide polymorphisms in codon 167, 198 and 200, known to confer BZ-resistance in other nematodes, were absent in the studied material. This study does not provide data that could explain previous reports of poor BZ treatment efficacy in terms of polymorphism in these codons of beta-tubulin. Based on a fragment of the beta-tubulin gene and the ITS2 region sequenced, it was found that T. trichiura from humans and Trichuris spp. isolated from baboons are closely related and may be the same species.

Medullary thyroid carcinoma (MTC) and RET proto-oncogene: Mutation spectrum in the familial cases and a meta-analysis of studies on the sporadic form

Medullary thyroid carcinoma (MTC) is an uncommon malignant tumor arising from the calcitonin-producing parafollicular cells (C cells) of thyroid. It accounts for 5-10% of all thyroid cancers, and it mostly occurs as a sporadic entity (sMTC), but a familial pattern (fMTC) is also possible. RET proto-oncogene germline mutations are crucial for the onset and the progression of fMTC, and the occurrence of single nucleotide polymorphisms could predispose to the sporadic form. In order to clarify the role of this gene in MTC, we carefully reviewed the PubMed database using appropriate terms. First, we summarized current knowledge of the germline RET mutations, mutation spectrum, and prevalence. We then performed a meta-analysis on the available case-control association studies for sMTC. Finally, we carried out in silico predictions of the best associated variants in the attempt to better define their role in the disease. To date, a total of 39 different RET germline mutations have been identified in fMTC families. The most affected codons are 609, 611, 618, 620 (exon 10) and 634 (exon 11), encoding for the extracellular cysteine-rich domain, and codons 768 (exon 13) and 804 (exon 14) of the intracellular tyrosine kinase domain. Six polymorphisms with at least three studies were included in the meta-analysis (A45A [rs1800858], G691S [rs1799939], L769L [rs1800861], S836S [rs1800862], S904S [rs1800863], and IVS1-126G>T [rs2565206]). The meta-analysis demonstrated a modest association of sMTC susceptibility with S836S and a strong association with the IVS1-126G>T polymorphism. Besides RET polymorphisms, we also investigated the role of a few other low-penetrance alleles of genes involved in the RET pathway or in xenobiotic metabolism, but none of these were confirmed. Thus, despite the well-known molecular basis of fMTC, the genetic variants of the sporadic form are still poorly understood, and functional analyses are needed to better understand the consequence of such RET variants and to improve our knowledge on the disease.

Haploinsufficiency of Tumor Suppressor Genes is Driven by the Cumulative Effect of microRNAs, microRNA Binding Site Polymorphisms and microRNA Polymorphisms: An in silico Approach

Haploinsufficiency of tumor suppressor genes, wherein the reduced production and activity of proteins results in the inability of the cell to maintain normal cellular function, is one among the various causes of cancer. However the precise molecular mechanisms underlying this condition remain unclear. Here we hypothesize that single nucleotide polymorphisms (SNPs) in the 3′untranslated region (UTR) of mRNAs and microRNA seed sequence (miR-SNPs) may cause haploinsufficiency at the level of proteins through altered binding specificity of microRNAs (miRNAs). Bioinformatics analysis of haploinsufficient genes for variations in their 3′UTR showed that the occurrence of SNPs result in the creation of new binding sites for miRNAs, thereby bringing the respective mRNA variant under the control of more miRNAs. In addition, 19 miR-SNPs were found to result in non-specific binding of microRNAs to tumor suppressors. Networking analysis suggests that the haploinsufficient tumor suppressor genes strongly interact with one another, and any subtle alterations in this network will contribute to tumorigenesis.

Evaluation of the occurrence possibility of SNP in Brassica napus with sliding window features by using RBF networks

We extract some physical and chemical features related to the occurrence of single nucleotide polymorphism (SNP) from three groups of sliding windows around SNP site, and then make the predictions about accuracy by using radial basis function (RBF) networks. The result of the forward sliding windows suggests that the accuracies and Matthews correlation coefficient (MCC values) ascend with the increasing of length of sliding windows. The accuracies range from 73.27 % to 80.69 %, and MCC values range from 0.465 to 0.614. The backward sliding windows and the sliding windows with fixed length three are designed to find the crucial sites related to SNP. The results imply that the occurrence possibility of SNP relies heavily on the above physical and chemical features of sites which are at a distance around 20 bases from the SNP site. Compared with the support vector machine (SVM), our RBF network approach has achieved more satisfactory results.

Single Nucleotide Polymorphisms in the Human Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) Gene

Species' variation(s) in gene homologues can result in differences among species in their quantitative and qualitative susceptibility and responsiveness to environmental contaminants. In the case of dioxin-like compounds (DLCs), it has been hypothesized that single nucleotide polymorphisms (SNPs) in genes associated with aryl hydrocarbon receptor (AHR)-regulated pathways may result in greater susceptibility to DLC toxicity. A key step in the activation of AHR involves heterodimerization with the AHR nuclear translocator (ARNT) protein before binding to its DNA response element. The objective of this study was to identify SNPs in the human ARNT gene that could potentially affect the sensitivity of AHR-dependent gene transcription. Results from DNA sequencing of 101 human samples demonstrated the presence of five unique SNPs at the ARNT locus, including three non-synonymous SNPs, of which two were novel: V304M and T462A. The genetic frequencies of the non-synonymous SNPs were very low (≤0.02), and the novel SNPs occurred in the Per-ARNT-Sim (PAS) functional domain. In silico analysis indicated that V304M was the only SNP identified in the current population with the potential to significantly alter ARNT protein function. Our findings indicated a very limited occurrence of SNPs with predicted functional consequence in key domains of human ARNT.

Polymorphic variation within the VWF gene contributes to the failure to detect mutations in patients historically diagnosed with type 1 von Willebrand disease from the MCMDM-1VWD cohort

Polymorphic variation within the VWF gene contributes to the failure to detect mutations in patients historically diagnosed with type 1 von Willebrand disease from the MCMDM-1VWD cohort

Analysis of single nucleotide polymorphisms in three chromosomes of European sea bass Dicentrarchus labrax

Single nucleotide polymorphisms (SNPs) are believed to contain relevant information and have been therefore extensively used as genetic markers in population and conservation genetics, and molecular ecology studies. This study reports on the identification of potential SNPs in a diploid European sea bass Dicentrarchus labrax genome by using reference sequences from three assembled chromosomes and mapping all WGS datasets onto them (3× Sanger, 3× 454 and 20× SOLEXA). A total of 20,779 SNPs were identified over the 1469 gene loci and intergenic space analysed. Within chromosomes the occurrence of SNPs was the lowest in exons and higher in introns and intergenic regions, which may be explained by the fact, that coding regions are under strong selective pressure to maintain their biological function. The ratio of nonsynonymous to synonymous mutations was smaller than one for all the chromosomes, suggesting that most of deleterious nonsynonymous mutations were eliminated by negative selection. SNPs were not uniformly distributed over the chromosomes. Two chromosomes exhibited large regions with extremely low SNP density, which might represent homozygous regions in the diploid genome. The results of this study show how SNP detection can take profit from sequencing a single diploid individual, but also uncover the limits of such an approach. SNPs that have been identified will support marker development for genetic linkage mapping, population genetics and aquaculture related questions in general.

The first set of EST resource for gene discovery and marker development in pigeonpea (Cajanus cajan L.).

BackgroundPigeonpea (Cajanus cajan (L.) Millsp) is one of the major grain legume crops of the tropics and subtropics, but biotic stresses [Fusarium wilt (FW), sterility mosaic disease (SMD), etc.] are serious challenges for sustainable crop production. Modern genomic tools such as molecular markers and candidate genes associated with resistance to these stresses offer the possibility of facilitating pigeonpea breeding for improving biotic stress resistance. Availability of limited genomic resources, however, is a serious bottleneck to undertake molecular breeding in pigeonpea to develop superior genotypes with enhanced resistance to above mentioned biotic stresses. With an objective of enhancing genomic resources in pigeonpea, this study reports generation and analysis of comprehensive resource of FW- and SMD- responsive expressed sequence tags (ESTs).ResultsA total of 16 cDNA libraries were constructed from four pigeonpea genotypes that are resistant and susceptible to FW ('ICPL 20102' and 'ICP 2376') and SMD ('ICP 7035' and 'TTB 7') and a total of 9,888 (9,468 high quality) ESTs were generated and deposited in dbEST of GenBank under accession numbers GR463974 to GR473857 and GR958228 to GR958231. Clustering and assembly analyses of these ESTs resulted into 4,557 unique sequences (unigenes) including 697 contigs and 3,860 singletons. BLASTN analysis of 4,557 unigenes showed a significant identity with ESTs of different legumes (23.2-60.3%), rice (28.3%), Arabidopsis (33.7%) and poplar (35.4%). As expected, pigeonpea ESTs are more closely related to soybean (60.3%) and cowpea ESTs (43.6%) than other plant ESTs. Similarly, BLASTX similarity results showed that only 1,603 (35.1%) out of 4,557 total unigenes correspond to known proteins in the UniProt database (≤ 1E-08). Functional categorization of the annotated unigenes sequences showed that 153 (3.3%) genes were assigned to cellular component category, 132 (2.8%) to biological process, and 132 (2.8%) in molecular function. Further, 19 genes were identified differentially expressed between FW- responsive genotypes and 20 between SMD- responsive genotypes. Generated ESTs were compiled together with 908 ESTs available in public domain, at the time of analysis, and a set of 5,085 unigenes were defined that were used for identification of molecular markers in pigeonpea. For instance, 3,583 simple sequence repeat (SSR) motifs were identified in 1,365 unigenes and 383 primer pairs were designed. Assessment of a set of 84 primer pairs on 40 elite pigeonpea lines showed polymorphism with 15 (28.8%) markers with an average of four alleles per marker and an average polymorphic information content (PIC) value of 0.40. Similarly, in silico mining of 133 contigs with ≥ 5 sequences detected 102 single nucleotide polymorphisms (SNPs) in 37 contigs. As an example, a set of 10 contigs were used for confirming in silico predicted SNPs in a set of four genotypes using wet lab experiments. Occurrence of SNPs were confirmed for all the 6 contigs for which scorable and sequenceable amplicons were generated. PCR amplicons were not obtained in case of 4 contigs. Recognition sites for restriction enzymes were identified for 102 SNPs in 37 contigs that indicates possibility of assaying SNPs in 37 genes using cleaved amplified polymorphic sequences (CAPS) assay.ConclusionThe pigeonpea EST dataset generated here provides a transcriptomic resource for gene discovery and development of functional markers associated with biotic stress resistance. Sequence analyses of this dataset have showed conservation of a considerable number of pigeonpea transcripts across legume and model plant species analysed as well as some putative pigeonpea specific genes. Validation of identified biotic stress responsive genes should provide candidate genes for allele mining as well as candidate markers for molecular breeding.

NOD2/Card Polymorphism And The Development Of Idiopathic Pulmonary Syndrome

In hematopoietic stem cell transplantation (HSCT) the occurrence of single nucleotide polymorphisms (SNPs) within the NOD2/CARD15 gene are associated with higher susceptibility for graft-versus-host disease (GvHD). Idiopathic pulmonary syndrome (IPS) is a non-infectious early onset lung disease (<100days) after HSCT and associated with high mortality rates. Exact pathogenesis is unknown, but IPS could be considered as an allo-reactive disease. We retrospectively studied the clinical impact of NOD2-variants in pediatric HSCT patients with IPS.

Identification of single nucleotide polymorphisms in different Populus species

Partial sequences of six genes in 54 trees belonging to five different Populus species were analyzed for occurrence of single nucleotide polymorphisms (SNPs). Genes selected are involved in wood formation and quality (CAD), defence reactions (PPO), hormone biosynthesis (GA20ox), or transcription factors (CBF1, TB1, LFY). The number of polymorphisms identified for each gene fragment varied between different genes, and also between exon and intron regions. The six genes resolved the phylogenetic relationships between the five species to different degrees. Only PPO is resolving all five Populus species as monophyletic in all three phylogenetic approaches. CAD resolves all species with the exception of Populus tremula. For CBF1 and TB1, a monophyletic group consisting of P. tremula and P. tremuloides is resolved by some of the reconstruction approaches. Indels in three out of the six genes analyzed were detected in “consensus”-sequence comparisons between the Populus species. In the CAD-like and LFY genes, these were found only in introns but in the case of TB1 gene these were also found in coding regions. Sizes of the indels range from 1 up to 8 nucleotides. This study confirms the main split between section Leuce and a group-combining sections Aigeiros and Tacamahaca that is perfectly supported with 100% bootstrap supports and 1.0 posterior probability. The SNP markers developed as well as the indels identified can be used for differentiation of Populus species and characterization of hybrids.

Search for Polymorphism in Exon 2 of the Equine Leptin Gene

The aim of the current research was to investigate the possible occurrence of a single nucleotide polymorphism (SNP) in exon 2 of the equine leptin gene in obese mares determined to be hyperleptinemic. Three experiments were conducted: one to determine the prevalence of hyperleptinemic horses in the resident herd; another to complete the sequencing of exon 2 and flanking introns of the equine leptin gene, which had been partially sequenced by others; and a third to compare the exon 2 sequences of obese, hyperleptinemic mares with those of obese mares not displaying hyperleptinemia. In experiment 1, jugular blood was collected from 31 mares, and they were categorized by age, body condition score (BCS), and average plasma leptin concentrations. Mean BCS was correlated ( P < .001) with leptin concentrations; age was not. Five obese, hyperleptinemic and five obese, nonhyperleptinemic mares were selected to study the possibility of polymorphism in exon 2. First, in experiment 2, forward and reverse primers were designed from the Bos taurus leptin gene (GenBank Accession # U50365) to identify and subsequently clone the equine leptin gene to provide material for sequencing. The multiple copies of genomic DNA were then used for sequencing of exon 2 and the flanking introns. Comparative genomics was used to further identify and characterize regulatory elements in the 5' and 3' flanking regions of the leptin gene. In experiment 3, DNA from the five mares of each type (previously selected in experiment 1) was extracted, and exon 2 was sequenced and analyzed for possible SNP. The sequences of exon 2 for the 10 mares were identical; thus, no polymorphism was present. It was concluded that approximately one third of obese, pasture-maintained mares and geldings in the resident herd display hyperleptinemia relative to other horses of similar body condition, but this condition was not associated with the occurrence of SNP in exon 2 of the leptin gene.

Single Nucleotide Variants of the TGACTCA Motif Modulate Energetics and Orientation of Binding of the Jun-Fos Heterodimeric Transcription Factor

The Jun-Fos heterodimeric transcription factor is the terminal link between the transfer of extracellular information in the form of growth factors and cytokines to the site of DNA transcription within the nucleus in a wide variety of cellular processes central to health and disease. Here, using isothermal titration calorimetry, we report detailed thermodynamics of the binding of bZIP domains of Jun-Fos heterodimer to synthetic dsDNA oligos containing the TGACTCA cis element and all possible single nucleotide variants thereof encountered widely within the promoters of a diverse array of genes. Our data show that Jun-Fos heterodimer tolerates single nucleotide substitutions and binds to TGACTCA variants with affinities in the physiologically relevant micromolar to submicromolar range. The energetics of binding are richly favored by enthalpic forces and opposed by entropic changes across the entire spectrum of TGACTCA variants in agreement with the notion that protein-DNA interactions are largely driven by electrostatic interactions and intermolecular hydrogen bonding. Of particular interest is the observation that the Jun-Fos heterodimer binds to specific TGACTCA variants in a preferred orientation. Our 3D atomic models reveal that such orientational preference results from asymmetric binding and may in part be attributable to chemically distinct but structurally equivalent residues R263 and K148 located within the basic regions of Jun and Fos, respectively. Taken together, our data suggest that the single nucleotide variants of the TGACTCA motif modulate energetics and orientation of binding of the Jun-Fos heterodimer and that such behavior may be a critical determinant of differential regulation of specific genes under the control of this transcription factor. Our study also bears important consequences for the occurrence of single nucleotide polymorphisms within the TGACTCA cis element at specific gene promoters between different individuals.

Application of degenerate oligonucleotide primed PCR (DOP-PCR) for SNP discovery in soybean

As the majority of methods for single nucleotide polymorphism (SNP) identification are highly cost-prohibitive, it is necessary to develop new strategies that are more suitable for small and medium-scale laboratories. In this paper, we investigate the potential of degenerate oligonucleotide primed PCR (DOP-PCR) for SNP discovery in soybean. PCR fragments were amplified from two soybean cultivars, ‘Pureunkong’ and ‘Jinpumkong 2,’ shotgun cloned and sequenced. The sequences of both cultivars were then assembled and examined for occurrence of SNPs. The effectiveness of SNP discovery was much lower than expected. Over 1,300 analyzed sequences were grouped in 144 contigs, but only 51 putative SNP sites were found in 18 of these contigs. About 50% of the contigs contained identical sequences and in more than one-third (35.4%), putative paralogous fragments were assembled. Subsequent validation of SNPs allowed the confirmation of only eight SNP sites. The failure to validate the remaining SNPs was mostly due to amplification of duplicated or multiplicated genomic regions. A relatively high proportion of chloroplast and mitochondrial DNA sequences was another limitation of effective SNP detection. Although the DOP-PCR technique was not efficient enough for SNP discovery because of the degree of soybean genome complication, the relatively large number of paralogous sequences in our data collection can be used for further detailed analysis of the genome structure of this species.