Flanking Non-coding Regions Research Articles

Determination of genetic predisposition to early breast cancer in women of Kazakh ethnicity.

Breast cancer (BC) is the most common type of cancer among women in Kazakhstan. To date, little data are available on the spectrum of genetic variation in Kazakh women with BC. We aimed to identify population-specific genetic markers associated with the risk of developing early-onset BC and test their association with clinical and prognostic factors. The study included 224 Kazakh women diagnosed with BC (≤40 age). Entire coding regions (>1700 exons) and the flanking noncoding regions of 94 cancer-associated genes were sequenced from blood DNA using MiSeq platform. We identified 38 unique pathogenic variants (PVs) in 13 different cancer-predisposing genes among 57 patients (25.4%), of which 6 variants were novel. In total, 12 of the 38 distinct PVs were detected recurrently, including BRCA1 c.5266dup, c.5278-2del, and c.2T>C, and BRCA2 c.9409dup and c.9253del that may be founder in this population. BRCA1 carriers were significantly more likely to develop triple-negative BC (OR = 6.61, 95% CI 2.44-17.91, p = 0.0002) and have family history of BC (OR = 3.17, 95% CI 1.14-8.76, p = 0.03) compared to non-carriers. This study allowed the identification of PVs specific to early-onset BC, which may be used as a foundation to develop regional expertise and diagnostic tools for early detection of BC in young Kazakh women.

Common calcium-sensing receptor (CASR) gene variants do not modify risk for chronic pancreatitis in a Hungarian cohort

The calcium-sensing receptor (CASR) is expressed in the pancreas where it might regulate calcium concentrations in pancreatic secretions. Two independent studies reported conflicting results claiming that commonly occurring missense variants of the CASR gene are risk factors for chronic pancreatitis (CP). Here, we attempted to replicate the association between CASR variants and CP. We analyzed 337 patients and 840 controls from the Hungarian National Pancreas Registry either by direct sequencing of exon 7 and the flanking noncoding regions or by TaqMan SNP genotyping assays. We identified two common missense variants, c.2956G>T (p.A986S), and c.2968A>G (p.R990G), three low-frequency variants, c.3031C>G (p.Q1011E), c.2610G>A (p.E870=) and c.∗60T>A, and 8 rare variants including the novel variant c.1895G>A (p.G632D). When allelic or genotype distributions were considered, none of the CASR variants associated with CP. Subgroup analysis of nonalcoholic versus alcoholic patients revealed no disease association either. Our results demonstrate that common CASR variants do not modify the risk for CP and should not be considered as genetic risk factors in the clinical setting.

Genomic basis for RNA alterations in cancer

Transcript alterations often result from somatic changes in cancer genomes1. Various forms of RNA alterations have been described in cancer, including overexpression2, altered splicing3 and gene fusions4; however, it is difficult to attribute these to underlying genomic changes owing to heterogeneity among patients and tumour types, and the relatively small cohorts of patients for whom samples have been analysed by both transcriptome and whole-genome sequencing. Here we present, to our knowledge, the most comprehensive catalogue of cancer-associated gene alterations to date, obtained by characterizing tumour transcriptomes from 1,188 donors of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA)5. Using matched whole-genome sequencing data, we associated several categories of RNA alterations with germline and somatic DNA alterations, and identified probable genetic mechanisms. Somatic copy-number alterations were the major drivers of variations in total gene and allele-specific expression. We identified 649 associations of somatic single-nucleotide variants with gene expression in cis, of which 68.4% involved associations with flanking non-coding regions of the gene. We found 1,900 splicing alterations associated with somatic mutations, including the formation of exons within introns in proximity to Alu elements. In addition, 82% of gene fusions were associated with structural variants, including 75 of a new class, termed ‘bridged’ fusions, in which a third genomic location bridges two genes. We observed transcriptomic alteration signatures that differ between cancer types and have associations with variations in DNA mutational signatures. This compendium of RNA alterations in the genomic context provides a rich resource for identifying genes and mechanisms that are functionally implicated in cancer.

Global inference of disease-causing single nucleotide variants from exome sequencing data.

BackgroundWhole exome sequencing (WES) has recently emerged as an effective approach for identifying genetic variants underlying human diseases. However, considerable time and labour is needed for careful investigation of candidate variants. Although filtration based on population frequencies and functional prediction scores could effectively remove common and neutral variants, hundreds or even thousands of rare deleterious variants still remain. In addition, current WES platforms also provide variant information in flanking noncoding regions, such as promoters, introns and splice sites. Despite of being recognized to harbour causal variants, these regions are usually ignored by current analysis pipelines.ResultsWe present a novel computational method, called Glints, to overcome the above limitations. Glints is capable of identifying disease-causing SNVs in both coding and flanking noncoding regions from exome sequencing data. The principle behind Glints is that disease-causing variants should manifest their effect at both variant and gene levels. Specifically, Glints integrates 14 types of functional scores, including predictions for both coding and noncoding variants, and 9 types of association scores, which help identifying disease relevant genes. We conducted a large-scale simulation studies based on 1000 Genomes Project data and demonstrated the effectiveness of our method in both coding and flanking noncoding regions. We also applied Glints in two real exome sequencing and demonstrated its effectiveness for uncovering disease-causing SNVs. Both standalone software and web server are available at our website http://bioinfo.au.tsinghua.edu.cn/jianglab/glints.ConclusionsGlints is effective for uncovering disease-causing SNVs in coding and flanking noncoding regions, which is supported by both simulation and real case studies. Glints is expected to be a useful tool for human genetics research based on exome sequencing data.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1325-x) contains supplementary material, which is available to authorized users.

Comparative genomic analysis of the compound Brassica napus Rf locus.

BackgroundThe plant trait of cytoplasmically-inherited male sterility (CMS) and its suppression by nuclear restorer-of-fertility (Rf) genes can be viewed as a genetic arms race between the mitochondrial and nuclear genomes. Most nuclear Rf genes have been shown to encode P-type pentatricopeptide repeat proteins (PPRs). Phylogenetic analysis of P-class PPRs from sequenced plants genomes has shown that Rf-proteins cluster in a distinct clade of P-class PPRs, RFL-PPRs, that display hallmarks of positive evolutionary selection. Genes encoding RFL-PPRs (RFLs) within a given plant genome tend to be closely related both in sequence and position, but a detailed understanding of how such species-specific expansion occurs is lacking. In the canola, (oilseed rape) species Brassica napus, previous work has indicated the nuclear restorer genes for the two native forms of CMS, Rfn (for nap CMS) and Rfp (pol CMS), represent alternate haplotypes, or alleles, of a single nuclear locus.ResultsFine genetic mapping indicates that Rfn does indeed localize to the same genomic region as Rfp. We find this region is enriched in RFL genes, three of which, based on their position and expression, represent potential candidates for Rfn; one of these genes, designated PPR4, is a preferred candidate in that it is not expressed in the nap CMS line. Comparison of the corresponding regions of the genomes of B. rapa, B. oleracea, Arabidopsis thaliana and A. lyrata provides insight into the expansion of this group of RFL genes in different lines of evolutionary descent.ConclusionsUnlike other nuclear restorer loci containing multiple RFL genes, the RFL genes in the Rf region of B. napus are not present in tandem arrays but rather are dispersed in genomic location. The genes do not share similar flanking non-coding regions and do not contain introns, indicating that they have duplicated primarily through a retrotransposition-mediated process. In contrast, segmental duplication has been responsible for the distribution of the 10 sequences we annotated as RFL genes in the corresponding region of the A. lyrata genome. Our observations define the Brassica Rf locus and indicate that different mechanisms may be responsible for the proliferation of RFL genes even among closely related genomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3117-0) contains supplementary material, which is available to authorized users.

Structural Variation within the Potato Ve Gene Locus and Correlation with Molecular Marker Analysis

Plant breeders rely on molecular markers to guide the selection of traits that are difficult to phenotype or that show wide variation in field trials as they seek to uncover the intricate relationship between an organism's genotype and resulting phenotype. Specificity of molecular markers in wild potato (Solanum spp.) and other less‐adapted germplasm is complicated by the existence of homologous gene clusters. This study examines the consistency and effectiveness of two previously published cleaved amplified polymorphic sequence (CAPS) markers that have been shown to predict Verticillium wilt (VW) resistance. The genome of resistant diploid potato clone C287 was resequenced, and examination of the Ve locus indicated only two homologous regions when compared with the three found in the reference potato genome DM1‐3. The prevalence of each homologous region was examined within a panel of 83 diverse wild potato species. Both structural and genetic sequence variation is predicted to complicate the use of molecular markers developed within specific genetic backgrounds. This study demonstrates that primers designed in the noncoding flanking regions of gene homologs increases specificity and avoids off‐target binding. It is suggested that markers be developed by using more specific primers in these regions coupled with conserved primers within the gene's coding sequence.

First description of small proteins encoded by spRNAs in Methanosarcina mazei strain Gö1.

In Methanosarcina mazei several small RNAs have been identified containing a small putative open reading frame (sORF) and thus classified as spRNAs. Here, we report on the first detection of three small proteins in M.mazei encoded by spRNAs using LC-MS/MS analysis of total protein extracts of cells grown under various stress conditions. Each spRNA shows high conservation in Methanosarcina species with regard to the sORF and the flanking non-coding RNA regions, moreover the predicted RNA structures are as well highly conserved. Characterizing the respective transcript levels in response to several stress conditions by northern blots demonstrated an enormous decrease of spRNA36 and spRNA44 during stationary growth (to less than 5%), and a significant increase of spRNA36 (2.5-fold) in response to nitrogen limitation. spRNA41, however, was only detected by RNA-Seq approaches. Quantification of the small proteins by LC-MS/MS using synthetic stable isotope labeled oligopeptides as standards indicated that the concentration of oligopetide36 and 41 in mid exponential phase is induced under nitrogen limitation, which in case of oligopeptide36 is in accordance with its transcript level. The relative amount of the three oligopeptides did not change upon entering stationary growth phase, even though the transcript levels decreased dramatically. Additional production of the oligopeptides in M.mazei did not result in any evident phenotype under standard or nitrogen limiting growth conditions. However, overall the transcript levels of several genes involved in carbon metabolism or in heat shock response were reduced 2-3 fold due to the overproduction, though no sORF specific change was observed. Based on our findings we hypothesize that oligopeptide36 might have a regulatory function in nitrogen metabolism by modulating the activity of a yet unknown target protein involved in the central nitrogen metabolism.

Association between non-coding polymorphisms of HOPX gene and syncope in hypertrophic cardiomyopathy.

Homeodomain Only Protein X (HOPX) is an unusual homeodomain protein which regulates Serum Response Factor (SRF) dependent gene expression. Due to the regulatory role of HOPX on SRF activity and the regulatory role of SRF on cardiac hypertrophy, we aimed to investigate the relationship between HOPX gene variations and hypertrophic cardiomyopathy (HCM). In this study, designed as a case-control study, we analyzed coding and flanking non-coding regions of the HOPX gene through 67 patients with HCM and 31 healty subjects. Certain regions of the gene were investigated by Single Stranded Conformation Polymorphism (SSCP) and Restriction Fragment Length Polymorphism (RFLP). Statistical analyses of genotypes and their relationship with clinical parameters were performed by chi-square, Kruskal-Wallis and the Fisher's exact test. In 5' Untranslated Region (UTR) and intronic region of the HOPX gene, we found a C>T substitution and an 8-bp insertion/deletion (In/Del) polymorphism, respectively. These two polymorphisms seemed to constitute an haplotype. While the frequency of homozygous genotypes of In/Del and C/T polymorphisms were found significantly lower in the patients with syncope (p=0.014 and p=0.017, respectively), frequency of their heterozygous genotypes were found significantly higher in the patients with syncope (p=0.048 and p=0.030, respectively). CONCLUSİON: Though there was not found any mutation in coding sequence of HOPX gene, two non-coding polymorphisms were found related to syncope in HCM patients. While homozygous status of these polymorphisms was found to be protective against the syncope, their heterozygous status seemed to be a risk factor for syncope in HCM patients. Our results suggest that HOPX may contribute to pathogenesis or manifestation of HCM as a modifier gene.

Functional Characterization of a Novel Aquaporin from Dictyostelium discoideum Amoebae Implies a Unique Gating Mechanism

The social amoeba Dictyostelium discoideum is a widely used model organism for studying basic functions of protozoan and metazoan cells, such as osmoregulation and cell motility. There is evidence from other species that cellular water channels, aquaporins (AQP), are central to both processes. Yet, data on D. discoideum AQPs is almost absent. Despite cloning of two putative D. discoideum AQPs, WacA, and AqpA, water permeability has not been shown. Further, WacA and AqpA are expressed at the late multicellular stage and in spores but not in amoebae. We cloned a novel AQP, AqpB, from amoeboidal D. discoideum cells. Wild-type AqpB was impermeable to water, glycerol, and urea when expressed in Xenopus laevis oocytes. Neither stepwise truncation of the N terminus nor selected point mutations activated the water channel. However, mutational truncation by 12 amino acids of an extraordinary long intracellular loop induced water permeability of AqpB, hinting at a novel gating mechanism. This AqpB mutant was inhibited by mercuric chloride, confirming the presence of a cysteine residue in the selectivity filter as predicted by our structure model. We detected AqpB by Western blot analysis in a glycosylated and a non-glycosylated form throughout all developmental stages. When expressed in D. discoideum amoebae, AqpB-GFP fusion constructs localized to vacuolar structures, to the plasma membrane, and to lamellipodia-like membrane protrusions. We conclude that the localization pattern in conjunction with channel gating may be indicative of AqpB functions in osmoregulation as well as cell motility of D. discoideum.

Translational Database Selection and Multiplexed Sequence Capture for Up Front Filtering of Reliable Breast Cancer Biomarker Candidates

Biomarker identification is of utmost importance for the development of novel diagnostics and therapeutics. Here we make use of a translational database selection strategy, utilizing data from the Human Protein Atlas (HPA) on differentially expressed protein patterns in healthy and breast cancer tissues as a means to filter out potential biomarkers for underlying genetic causatives of the disease. DNA was isolated from ten breast cancer biopsies, and the protein coding and flanking non-coding genomic regions corresponding to the selected proteins were extracted in a multiplexed format from the samples using a single DNA sequence capture array. Deep sequencing revealed an even enrichment of the multiplexed samples and a great variation of genetic alterations in the tumors of the sampled individuals. Benefiting from the upstream filtering method, the final set of biomarker candidates could be completely verified through bidirectional Sanger sequencing, revealing a 40 percent false positive rate despite high read coverage. Of the variants encountered in translated regions, nine novel non-synonymous variations were identified and verified, two of which were present in more than one of the ten tumor samples.

TET2 SNP rs2454206 (I1762V) Correlates with Improved Survival In Pediatric Acute Myelogenous Leukemia, a Report From the Children's Oncology Group

AbstractAbstract 949Deletions and rearrangements involving chromosome 4q24 in patients with myelodysplastic syndrome led to the discovery that the TET2 gene found in this genomic region may contribute to myeloid leukemogenesis. Mutations in this gene were identified in AML that correlated with clinical outcome. We screened genomic DNA extracted from diagnostic specimens of a cohort of 169 pediatric de novo AML patients treated on the Children's Cancer Group study CCG-2961 for genomic alterations in the TET2 gene. Twenty one primer pairs were used to amplify and sequence the entire coding sequence (11 exons) as well as flanking promoter and non-coding regions of TET2. We identified 10 different previously annotated single nucleotide polymorphisms (SNPs) with prevalence greater than 4% in addition to 12 disease associated mutations. Presence of each SNP as well as the mutations as a group were correlated with disease characteristics and clinical outcome.Of the 10 SNPs evaluated, one SNP correlated significantly with outcome. The minor allele of SNP rs2454206 (I1762V) was found in 54% of samples tested. There was no significant difference in the age or gender of patients with or without SNP rs2454206. The prevalence of SNP rs2454206 varied in different ethnic groups. Of the 92 patients with SNP rs2454206, 85% were Caucasian and 3% were African American compared to 56% and 15% of those without the SNP, respectively (p<0.001 and p=0.018). Conversely, 65% of all Caucasian patients and 21% of all African American patients with AML had SNP rs2454206, similar to the reported SNP frequency in general Caucasian and African American populations. There was no significant difference in presenting white blood cell count, blast percentage or FAB group in patients with and without SNP rs2454206. The prevalence of specific cytogenetic groups and mutations of FLT3/ITD, CEBPA and WT1 did not differ between the groups. There was a lower prevalence of NPM mutation in the SNP rs2454206 group compared to the SNP negative group (2.5% vs. 12.7%, p= 0.035). Patients with SNP rs2454206 were more likely to achieve a complete remission than those without the SNP (80% vs. 63%, p=0.028). Actuarial overall survival at 5 years from diagnosis for patients with SNP rs2454206 was 61±11% vs. 36±12% for those without the SNP (p=0.007). Corresponding event-free survival was 41±12% vs. 27±11% for those with and without the SNP (p=0.051). Of those who achieved a complete remission, overall survival from CR for those with and without SNP rs2454206 was 66% and 43%, respectively (p=0.022). Improved outcome may in part be due to lower treatment related mortality (TRM) where those with SNP rs2454206 had a lower rate of TRM 6±5% vs. 17±9% (p=0.043).We further evaluated the clinical implications of TET2 mutations. Disease associated mutations of the TET2 gene were identified in 11 patients with 9 heterozygous missense mutations (K423R, N767D, R814H, E1010D, S1039L, A1443V, V1718L, H1817N and E1973K), 2 heterozygous nonsense mutations (Q958X and E1323X) and one heterozygous single base insertion (ins1870-1871) which caused a frame shift beginning with T624N and causing an early termination codon (E637X). Each mutation was found in a single patient sample except for V1718L which was found in two samples. Also, one patient sample had two missense mutations (E1010D and E1973K) and another patient sample contained both nonsense mutations. Therefore a total of 11 out of 169 samples (6.5%) had a TET2 mutation. Of the TET2 mutant patients with available cytogenetics, 50% had t(8;21) compared to 14% of those without a mutation (p=0.026), and there was not an association of TET2 mutation with high risk cytogenetic features. Of the 11 patients with TET2 mutations, 6 patients failed to achieve a complete remission with a CR rate of 45% compared to that of 74% for those without a mutation (p=0.074). Patients with a TET2 mutation had a disease-free survival of 26±31% vs. 42±10% for those without the mutation (p=0.247).This study demonstrates the potential significance of genomic alterations of the TET2 gene in childhood AML. Further evaluation of TET2 mutations and the SNP rs2454206 in a larger cohort of pediatric AML patients is in process in order to determine if these will be useful markers for risk based stratification and allocation of therapy. The TET2 SNP rs2454206 may represent a new marker of favorable disease in pediatric AML as it does not overlap with other favorable prognostic markers. Disclosures:No relevant conflicts of interest to declare.

Tools for neuroanatomy and neurogenetics in Drosophila

We demonstrate the feasibility of generating thousands of transgenic Drosophila melanogaster lines in which the expression of an exogenous gene is reproducibly directed to distinct small subsets of cells in the adult brain. We expect the expression patterns produced by the collection of 5,000 lines that we are currently generating to encompass all neurons in the brain in a variety of intersecting patterns. Overlapping 3-kb DNA fragments from the flanking noncoding and intronic regions of genes thought to have patterned expression in the adult brain were inserted into a defined genomic location by site-specific recombination. These fragments were then assayed for their ability to function as transcriptional enhancers in conjunction with a synthetic core promoter designed to work with a wide variety of enhancer types. An analysis of 44 fragments from four genes found that >80% drive expression patterns in the brain; the observed patterns were, on average, comprised of <100 cells. Our results suggest that the D. melanogaster genome contains >50,000 enhancers and that multiple enhancers drive distinct subsets of expression of a gene in each tissue and developmental stage. We expect that these lines will be valuable tools for neuroanatomy as well as for the elucidation of neuronal circuits and information flow in the fly brain.

Circulating Angiogenic Precursors in Idiopathic Pulmonary Arterial Hypertension

Vascular remodeling in idiopathic pulmonary arterial hypertension (IPAH) involves hyperproliferative and apoptosis-resistant pulmonary artery endothelial cells. In this study, we evaluated the relative contribution of bone marrow-derived proangiogenic precursors and tissue-resident endothelial progenitors to vascular remodeling in IPAH. Levels of circulating CD34+ CD133+ bone marrow-derived proangiogenic precursors were higher in peripheral blood from IPAH patients than in healthy controls and correlated with pulmonary artery pressure, whereas levels of resident endothelial progenitors in IPAH pulmonary arteries were comparable to those of healthy controls. Colony-forming units of endothelial-like cells (CFU-ECs) derived from CD34+ CD133+ bone marrow precursors of IPAH patients secreted high levels of matrix metalloproteinase-2, had greater affinity for angiogenic tubes, and spontaneously formed disorganized cell clusters that increased in size in the presence of transforming growth factor-beta or bone morphogenetic protein-2. Subcutaneous injection of NOD SCID mice with IPAH CFU-ECs within Matrigel plugs, but not with control CFU-ECs, produced cell clusters in the Matrigel and proliferative lesions in surrounding murine tissues. Thus, mobilization of high levels of proliferative bone marrow-derived proangiogenic precursors is a characteristic of IPAH and may participate in the pulmonary vascular remodeling process.

Vibrio cholerae CTX?? Phage Repressor Gene is Borne on an Insertion Sequence-Like Element and Encodes a Putative Transposase

Bioinformatics analyses show the presence of a novel insertion sequence-like element in the CTXPhi phage of Vibrio cholerae. The solitary open reading frame encompassed by the element is known to encode a repressor (RstR) of phage DNA replication and is responsible for phage heteroimmunity. Analysis of the nucleotide and protein sequence of the repressor and its flanking non-coding regions indicates that it resembles distinctly simple bacterial insertion elements in numerous aspects. Based on the results of extensive sequence analysis, we propose that the rstR gene is borne on an insertion sequence-like element and that the RstR protein encodes a putative transposase. We put forward the possibility that a bi-directional promoter could regulate the divergent transcription of the neighbouring genes of rstR and rstA. The possibility of overlapping reading frames yielding distinct repressor and transposase proteins is also expanded based on the analyses.

Pharmacogenetics of β&lt;sub&gt;2&lt;/sub&gt;-Agonist Drugs in Asthma

Genetic variation in the structure of the beta2-adrenoceptor (ADRB2) is a potentially important source of variability in the response to beta2-agonist drugs. The relevant polymorphisms are common, with allele frequencies as high as 50% in some cases. "Nonsynonymous" variants result in amino acid substitutions in the receptor structure, of which substitution of Gly for Arg at position 16 and Glu for Gln at position 27 are the most clinically relevant. Other polymorphisms occur in the noncoding flanking regions of the gene and may be important in regulating ADRB2 expression, thus affecting individual drug responses. Linkage disequilibrium involving polymorphisms in both the coding and noncoding regions may explain the diversity of outcomes regarding functional in vitro studies. To date, analysis by haplotype, rather than genotype, has tended to confuse the issues. Functional in vitro studies provide the basis by which these polymorphisms are believed to be relevant in vivo. However, follow-up investigations have not substantiated the conventional wisdom that Gly-16 confers increased propensity to downregulation, whereas Glu-27 confers resistance. The in vivo picture is further complicated by the dynamic status of ADRB2s. Paradoxically, downregulation of receptors with exogenous beta2-agonist drugs may occur more readily in cells that are relatively resistant to downregulation with exposure to endogenous catecholamine. Numerous studies have highlighted that the Arg-16 variant is associated with an enhanced bronchodilator response in vivo, although the picture is not a consistent one. Outcomes during chronic treatment of asthma with beta2-agonists have been more consistent. In patients who are homozygous for Arg-16, reduced lung function and increased frequency of exacerbations occur during chronic treatment with short-acting beta2-agonists. With long-acting beta2-agonists, the overall clinical benefits may be worse for Arg-16 than for the Gly-16 genotype. Therefore, ADRB2 polymorphisms are an important consideration in assessing individual patients with difficult asthma that may be drug-induced for reasons of ADRB2 genotype. They are also relevant in the design of clinical trials involving beta2-agonist therapy.

Regulation and Evolution of Genes in Ascidians

Understanding how the interaction between transcription factors and cis -regulatory systems has evolved and controls gene expression is a key to uncover mechanisms of evolution and development of organisms. As a model system to study gene regulation in development, we have been using a group of primitive chordates, ascidians. Ascidians are also favorable for evolutionary research because of their phylogenetic position near the vertebrates and of the existence of closely-related species with different modes of development. Our recent research focuses on development and function of nervous systems in ascidians. The ascidian larva has a simple central nervous system (CNS) derived from the dorsal neural tube. Here we present two topics in our studies on development and evolution of chordate features in ascidians. Computational and experimental analyses of cis -regulatory DNAs Genes regulated by the same transcription factors are expected to share cis -regulatory elements in their flanking and/or intronic noncoding regions. DNA motifs over-represented in 5’ flanking regions of potentially co-regulated genes were identified by computational analyses of 5’ flanking regions of tissue-specific genes of Ciona intestinalis and Ciona savignyi . Comparative genomics analysis between C. intestinalis and C. savignyi can efficiently predict cis -regulatory sequences important for tissue-specific gene expression. In vivo functional analysis demonstrated the validity of computational prediction of cis -regulatory elements. The sequences and distribution patterns of potential cis -regulatory DNAs can be incorporated into a model promoter structure, and the model can then be used for genome-wide prediction of co-regulated promoters.

Sequential gene deletions in Hypocrea jecorina using a single blaster cassette

In Hypocrea jecorina (anamorph: Trichoderma reesei) multiple gene deletions are limited by the number of readily available selection markers. We have therefore constructed a blaster cassette which enables successive gene knock-outs in H. jecorina. This 3.5 kb pyr4 blaster cassette contains the H. jecorina pyr4 marker gene encoding orotidine-5'-monophosphate (OMP) decarboxylase flanked by two direct repeats of the Streptoalloteichus hindustanus bleomycin gene (Sh ble), which facilitate the excision of the blaster cassette by homologous recombination after each round of deletion. Functionality of this pyr4 blaster cassette was demonstrated by deletion of the glk1 encoding glucokinase and hxk1 encoding hexokinase. 1.4-1.8 kb of the non-coding flanking regions of both target genes were cloned into the respective blaster cassettes and transformation of a pyr4 negative H. jecorina strain with the two cassettes resulted in 10-13% of the transformants in the deletion of one of the two kinase genes. For excision of the pyr4 blaster cassettes, Deltaglk1 strains were selected for growth in the presence of 5-fluoroorotic acid. Recombination between the two Sh ble elements resulted in uridine auxotrophic strains which retained their respective glucokinase negative phenotype. Subsequent transformation of one of these auxotrophic Deltaglk1 strains with the hexokinase blaster cassette resulted in pyr4 prototrophic strains deleted in both glk1 and hxk1. Deltaglk1 strains showed reduced growth on d-glucose and d-fructose whereas Deltahxkl strains showed reduced compact growth on d-glucose but were unable to grow on d-fructose as carbon source. The double Deltaglk1Deltahxk1 deletion strain was completely unable to grow on either d-glucose or d-fructose.

Functional analysis of the noncoding regions of the Uukuniemi virus (Bunyaviridae) RNA segments.

The role of the variable portion of the noncoding regions (NCRs) of the three Bunyaviridae RNA segments (L, M, S) in transcription, replication, and packaging was studied using the recently developed plasmid-driven RNA polymerase I minigenome system for Uukuniemi (UUK) virus, genus Phlebovirus (11), as a model. Comparison of the different segments showed that all NCRs were sufficient to mediate transcription/replication of a minigenome but demonstrated decreased promoter strength in the order M > L > S. Chimeric minigenomes with flanking NCRs from different genome segments revealed that the number of total base pairs within the inverted, partially complementary ends was important for transcription and replication. Point mutations increasing the base-pairing potential produced increased reporter expression, indicating that complementarity between the 5' and 3' ends is crucial for promoter activity. The role of the intergenic region (IGR) located between the two open reading frames of the ambisense UUK virus S segment was analyzed by inserting this sequence element downstream of the reporter genes. The presence of the IGR was found to enhance reporter expression, demonstrating that efficient transcription termination, regulated by the IGR, is important for optimal minigenome mRNA translation. Finally, genome packaging efficacy varied for different NCRs and was strongest for L followed by M and S. Strong reporter gene activity was still observed after seven consecutive cell culture passages, indicating a selective rather than random genome-packaging mechanism. In summary, our results demonstrate that the NCRs from all three segments contain the necessary signals to initiate transcription and replication as well as packaging. Based on promoter strength, M-segment NCRs may be the preferred choice for the development of reverse genetics and minigenome rescue systems for bunyaviruses.

Norepinephrine transporter splice variants and their interaction with substrates and blockers

Norepinephrine transporter (NET), a member of the Na +/Cl −-dependent neurotransmitter transporter family, displays species-specific isoforms produced by alternative RNA splicing. This occurs at 3′-flanking coding and noncoding regions, resulting in different carboxy-terminals. When these NET splice variants were expressed in cultured cell lines, the characteristics of substrate transport and the sensitivity to uptake inhibitors differed between isoforms. The different functional expression suggests the physiological importance of the action and interaction of NET splice variants in synaptic transmission.

Inherited BRCA2 mutations in African Americans with breast and/or ovarian cancer: a study of familial and early onset cases.

In order to identify the spectrum of BRCA2 mutations in African Americans, breast or ovarian cancer patients from 74 independent families at elevated risk of germline mutations were investigated. The entire coding regions and flanking introns of BRCA2 were screened for germline mutations by single-stranded conformation polymorphism, protein truncation test, or denaturing high performance liquid chromatography followed by DNA sequencing. Eight distinct protein-truncating mutations were detected in six female patients (average age of onset of breast cancer: 37.6 years) and two male patients, but not in 163 unrelated disease-free controls. Two (1993delAA, 8643delAT) of the eight pathogenic mutations observed in African Americans have not been previously described. The other six pathogenic mutations (1882delT, 1991delATAA, 2001delTTAT, 2816insA, 4075delGT, 4088delA) have been detected in Caucasians; only the 2816insA mutation has been reported previously in African Americans. There were no significant differences in the frequency of deleterious BRCA2 mutations in African Americans compared with Caucasians. Six rare variations, not previously reported, were identified in five breast cancer patients but not in 163 disease-free control subjects. Of 11 different polymorphisms identified in high-risk African-American breast cancer patients, four may be unique to African Americans. An intron 10 polymorphism observed in patients was not detected in 163 disease-free African-American control subjects; this difference is statistically significant. Since many different pathogenic mutations and variants of unknown significance are observed in African Americans, BRCA2 genetic testing in high-risk African-American families must include the entire coding and flanking non-coding regions of the gene.