Showing papers by "Applied Biosystems published in 2008"

Analyzing real-time PCR data by the comparative C(T) method.

Abstract: Two different methods of presenting quantitative gene expression exist: absolute and relative quantification. Absolute quantification calculates the copy number of the gene usually by relating the PCR signal to a standard curve. Relative gene expression presents the data of the gene of interest relative to some calibrator or internal control gene. A widely used method to present relative gene expression is the comparative C(T) method also referred to as the 2 (-DeltaDeltaC(T)) method. This protocol provides an overview of the comparative C(T) method for quantitative gene expression studies. Also presented here are various examples to present quantitative gene expression data using this method.

Stem cell transcriptome profiling via massive-scale mRNA sequencing.

Abstract: We developed a massive-scale RNA sequencing protocol, short quantitative random RNA libraries or SQRL, to survey the complexity, dynamics and sequence content of transcriptomes in a near-complete fashion. This method generates directional, random-primed, linear cDNA libraries that are optimized for next-generation short-tag sequencing. We surveyed the poly(A)+ transcriptomes of undifferentiated mouse embryonic stem cells (ESCs) and embryoid bodies (EBs) at an unprecedented depth (10 Gb), using the Applied Biosystems SOLiD technology. These libraries capture the genomic landscape of expression, state-specific expression, single-nucleotide polymorphisms (SNPs), the transcriptional activity of repeat elements, and both known and new alternative splicing events. We investigated the impact of transcriptional complexity on current models of key signaling pathways controlling ESC pluripotency and differentiation, highlighting how SQRL can be used to characterize transcriptome content and dynamics in a quantitative and reproducible manner, and suggesting that our understanding of transcriptional complexity is far from complete.

The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus)

Abstract: Papaya, a fruit crop cultivated in tropical and subtropical regions, is known for its nutritional benefits and medicinal applications. Here we report a 3x draft genome sequence of 'SunUp' papaya, the first commercial virus-resistant transgenic fruit tree to be sequenced. The papaya genome is three times the size of the Arabidopsis genome, but contains fewer genes, including significantly fewer disease-resistance gene analogues. Comparison of the five sequenced genomes suggests a minimal angiosperm gene set of 13,311. A lack of recent genome duplication, atypical of other angiosperm genomes sequenced so far, may account for the smaller papaya gene number in most functional groups. Nonetheless, striking amplifications in gene number within particular functional groups suggest roles in the evolution of tree-like habit, deposition and remobilization of starch reserves, attraction of seed dispersal agents, and adaptation to tropical daylengths. Transgenesis at three locations is closely associated with chloroplast insertions into the nuclear genome, and with topoisomerase I recognition sites. Papaya offers numerous advantages as a system for fruit-tree functional genomics, and this draft genome sequence provides the foundation for revealing the basis of Carica's distinguishing morpho-physiological, medicinal and nutritional properties.

miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme cells and induce differentiation of brain tumor stem cells.

Abstract: Glioblastoma multiforme (GBM) is an invariably fatal central nervous system tumor despite treatment with surgery, radiation, and chemotherapy. Further insights into the molecular and cellular mechanisms that drive GBM formation are required to improve patient outcome. MicroRNAs are emerging as important regulators of cellular differentiation and proliferation, and have been implicated in the etiology of a variety of cancers, yet the role of microRNAs in GBM remains poorly understood. In this study, we investigated the role of microRNAs in regulating the differentiation and proliferation of neural stem cells and glioblastoma-multiforme tumor cells. We used quantitative RT-PCR to assess microRNA expression in high-grade astrocytomas and adult mouse neural stem cells. To assess the function of candidate microRNAs in high-grade astrocytomas, we transfected miR mimics to cultured-mouse neural stem cells, -mouse oligodendroglioma-derived stem cells, -human glioblastoma multiforme-derived stem cells and -glioblastoma multiforme cell lines. Cellular differentiation was assessed by immunostaining, and cellular proliferation was determined using fluorescence-activated cell sorting. Our studies revealed that expression levels of microRNA-124 and microRNA-137 were significantly decreased in anaplastic astrocytomas (World Health Organization grade III) and glioblastoma multiforme (World Health Organization grade IV) relative to non-neoplastic brain tissue (P < 0.01), and were increased 8- to 20-fold during differentiation of cultured mouse neural stem cells following growth factor withdrawal. Expression of microRNA-137 was increased 3- to 12-fold in glioblastoma multiforme cell lines U87 and U251 following inhibition of DNA methylation with 5-aza-2'-deoxycytidine (5-aza-dC). Transfection of microRNA-124 or microRNA-137 induced morphological changes and marker expressions consistent with neuronal differentiation in mouse neural stem cells, mouse oligodendroglioma-derived stem cells derived from S100β-v-erbB tumors and cluster of differentiation 133+ human glioblastoma multiforme-derived stem cells (SF6969). Transfection of microRNA-124 or microRNA-137 also induced G1 cell cycle arrest in U251 and SF6969 glioblastoma multiforme cells, which was associated with decreased expression of cyclin-dependent kinase 6 and phosphorylated retinoblastoma (pSer 807/811) proteins. microRNA-124 and microRNA-137 induce differentiation of adult mouse neural stem cells, mouse oligodendroglioma-derived stem cells and human glioblastoma multiforme-derived stem cells and induce glioblastoma multiforme cell cycle arrest. These results suggest that targeted delivery of microRNA-124 and/or microRNA-137 to glioblastoma multiforme tumor cells may be therapeutically efficacious for the treatment of this disease.

Identification of miRNA Changes in Alzheimer's Disease Brain and CSF Yields Putative Biomarkers and Insights into Disease Pathways

Abstract: MicroRNAs have essential functional roles in brain development and neuronal specification but their roles in neurode- generative diseases such as Alzheimer's disease (AD) is unknown. Using a sensitive qRT-PCR platform we identified regional and stage-specific deregulation of miRNA expression in AD patient brains. We used experimental validation in addition to literature to reveal how the deregulated brain microRNAs are biomarkers for known and novel pathways in AD pathogenesis related to amyloid processing, neurogenesis, insulin resistance, and innate immunity. We additionally recovered miRNAs from cerebrospinal fluid and discovered AD-specific miRNA changes consistent with their role as potential biomarkers of disease.

A recurrent 15q13.3 microdeletion syndrome associated with mental retardation and seizures.

Abstract: We report a recurrent microdeletion syndrome causing mental retardation, epilepsy and variable facial and digital dysmorphisms. We describe nine affected individuals, including six probands: two with de novo deletions, two who inherited the deletion from an affected parent and two with unknown inheritance. The proximal breakpoint of the largest deletion is contiguous with breakpoint 3 (BP3) of the Prader-Willi and Angelman syndrome region, extending 3.95 Mb distally to BP5. A smaller 1.5-Mb deletion has a proximal breakpoint within the larger deletion (BP4) and shares the same distal BP5. This recurrent 1.5-Mb deletion contains six genes, including a candidate gene for epilepsy (CHRNA7) that is probably responsible for the observed seizure phenotype. The BP4-BP5 region undergoes frequent inversion, suggesting a possible link between this inversion polymorphism and recurrent deletion. The frequency of these microdeletions in mental retardation cases is approximately 0.3% (6/2,082 tested), a prevalence comparable to that of Williams, Angelman and Prader-Willi syndromes.

MicroRNA biogenesis is required for mouse primordial germ cell development and spermatogenesis.

Abstract: Background MicroRNAs (miRNAs) are critical regulators of transcriptional and post-transcriptional gene silencing, which are involved in multiple developmental processes in many organisms. Apart from miRNAs, mouse germ cells express another type of small RNA, piwi-interacting RNAs (piRNAs). Although it has been clear that piRNAs play a role in repression of retrotransposons during spermatogenesis, the function of miRNA in mouse germ cells has been unclear.

The balance of reproducibility, sensitivity, and specificity of lists of differentially expressed genes in microarray studies

Abstract: Background Reproducibility is a fundamental requirement in scientific experiments. Some recent publications have claimed that microarrays are unreliable because lists of differentially expressed genes (DEGs) are not reproducible in similar experiments. Meanwhile, new statistical methods for identifying DEGs continue to appear in the scientific literature. The resultant variety of existing and emerging methods exacerbates confusion and continuing debate in the microarray community on the appropriate choice of methods for identifying reliable DEG lists.

Nonlinear Fitting Method for Determining Local False Discovery Rates from Decoy Database Searches

Abstract: False discovery rate (FDR) analyses of protein and peptide identification results using decoy database searching conventionally report aggregate or global FDRs for a whole set of identifications, which are often not very informative about the error rates of individual members in the set. We describe a nonlinear curve fitting method for calculating the local FDR, which estimates the chance that an individual protein (or peptide) is incorrect, and present a simple tool that implements this analysis. The goal of this method is to offer a simple extension to the now commonplace decoy database searching, providing additional valuable information.

Rapid whole-genome mutational profiling using next-generation sequencing technologies

Abstract: Forward genetic mutational studies, adaptive evolution, and phenotypic screening are powerful tools for creating new variant organisms with desirable traits. However, mutations generated in the process cannot be easily identified with traditional genetic tools. We show that new high-throughput, massively parallel sequencing technologies can completely and accurately characterize a mutant genome relative to a previously sequenced parental (reference) strain. We studied a mutant strain of Pichia stipitis, a yeast capable of converting xylose to ethanol. This unusually efficient mutant strain was developed through repeated rounds of chemical mutagenesis, strain selection, transformation, and genetic manipulation over a period of seven years. We resequenced this strain on three different sequencing platforms. Surprisingly, we found fewer than a dozen mutations in open reading frames. All three sequencing technologies were able to identify each single nucleotide mutation given at least 10–15-fold nominal sequence coverage. Our results show that detecting mutations in evolved and engineered organisms is rapid and cost-effective at the whole-genome level using new sequencing technologies. Identification of specific mutations in strains with altered phenotypes will add insight into specific gene functions and guide further metabolic engineering efforts.

Heterogeneous dysregulation of microRNAs across the autism spectrum

Abstract: microRNAs (miRNAs) are ~21 nt transcripts capable of regulating the expression of many mRNAs and are abundant in the brain. miRNAs have a role in several complex diseases including cancer as well as some neurological diseases such as Tourette’s syndrome and Fragile x syndrome. As a genetically complex disease, dysregulation of miRNA expression might be a feature of autism spectrum disorders (ASDs). Using multiplex quantitative polymerase chain reaction (PCR), we compared the expression of 466 human miRNAs from postmortem cerebellar cortex tissue of individuals with ASD (n = 13) and a control set of non-autistic cerebellar samples (n = 13). While most miRNAs levels showed little variation across all samples suggesting that autism does not induce global dysfunction of miRNA expression, some miRNAs among the autistic samples were expressed at significantly different levels compared to the mean control value. Twenty-eight miRNAs were expressed at significantly different levels compared to the non-autism control set in at least one of the autism samples. To validate the finding, we reversed the analysis and compared each non-autism control to a single mean value for each miRNA across all autism cases. In this analysis, the number of dysregulated miRNAs fell from 28 to 9 miRNAs. Among the predicted targets of dysregulated miRNAs are genes that are known genetic causes of autism such Neurexin and SHANK3. This study finds that altered miRNA expression levels are observed in postmortem cerebellar cortex from autism patients, a finding which suggests that dysregulation of miRNAs may contribute to autism spectrum phenotype.

A comprehensive functional analysis of tissue specificity of human gene expression

Abstract: In recent years, the maturation of microarray technology has allowed the genome-wide analysis of gene expression patterns to identify tissue-specific and ubiquitously expressed ('housekeeping') genes. We have performed a functional and topological analysis of housekeeping and tissue-specific networks to identify universally necessary biological processes, and those unique to or characteristic of particular tissues. We measured whole genome expression in 31 human tissues, identifying 2374 housekeeping genes expressed in all tissues, and genes uniquely expressed in each tissue. Comprehensive functional analysis showed that the housekeeping set is substantially larger than previously thought, and is enriched with vital processes such as oxidative phosphorylation, ubiquitin-dependent proteolysis, translation and energy metabolism. Network topology of the housekeeping network was characterized by higher connectivity and shorter paths between the proteins than the global network. Ontology enrichment scoring and network topology of tissue-specific genes were consistent with each tissue's function and expression patterns clustered together in accordance with tissue origin. Tissue-specific genes were twice as likely as housekeeping genes to be drug targets, allowing the identification of tissue 'signature networks' that will facilitate the discovery of new therapeutic targets and biomarkers of tissue-targeted diseases. A comprehensive functional analysis of housekeeping and tissue-specific genes showed that the biological function of housekeeping and tissue-specific genes was consistent with tissue origin. Network analysis revealed that tissue-specific networks have distinct network properties related to each tissue's function. Tissue 'signature networks' promise to be a rich source of targets and biomarkers for disease treatment and diagnosis.

The genomic analysis of erythrocyte microRNA expression in sickle cell diseases.

Abstract: Background Since mature erythrocytes are terminally differentiated cells without nuclei and organelles, it is commonly thought that they do not contain nucleic acids. In this study, we have re-examined this issue by analyzing the transcriptome of a purified population of human mature erythrocytes from individuals with normal hemoglobin (HbAA) and homozygous sickle cell disease (HbSS). Methods and Findings Using a combination of microarray analysis, real-time RT-PCR and Northern blots, we found that mature erythrocytes, while lacking ribosomal and large-sized RNAs, contain abundant and diverse microRNAs. MicroRNA expression of erythrocytes was different from that of reticulocytes and leukocytes, and contributed the majority of the microRNA expression in whole blood. When we used microRNA microarrays to analyze erythrocytes from HbAA and HbSS individuals, we noted a dramatic difference in their microRNA expression pattern. We found that miR-320 played an important role for the down-regulation of its target gene, CD71 during reticulocyte terminal differentiation. Further investigation revealed that poor expression of miR-320 in HbSS cells was associated with their defective downregulation CD71 during terminal differentiation. Conclusions In summary, we have discovered significant microRNA expression in human mature erythrocytes, which is dramatically altered in HbSS erythrocytes and their defect in terminal differentiation. Thus, the global analysis of microRNA expression in circulating erythrocytes can provide mechanistic insights into the disease phenotypes of erythrocyte diseases.

Radiation modulation of microRNA in prostate cancer cell lines.

Abstract: Background MicroRNAs (miRNA) are gene regulators and play an important role in response to cellular stress.

Development and validation of the AmpFlSTR MiniFiler PCR Amplification Kit: a MiniSTR multiplex for the analysis of degraded and/or PCR inhibited DNA.

Abstract: DNA typing of degraded DNA samples can be a challenging task when using the current commercially available multiplex short tandem repeat (STR) analysis kits. However, the ability to type degraded DNA specimens improves by redesigning current STR marker amplicons such that smaller sized polymerase chain reaction (PCR) products are generated. In an effort to increase the amount of information derived from these types of DNA samples, the AmpFlSTR MiniFiler PCR Amplification Kit has been developed. The kit contains reagents for the amplification of eight miniSTRs which are the largest sized loci in the AmpFlSTR Identifiler PCR Amplification Kit (D7S820, D13S317, D16S539, D21S11, D2S1338, D18S51, CSF1PO, and FGA). Five of these STR loci (D16S539, D21S11, D2S1338, D18S51, and FGA) also are some of the largest loci in the AmpFlSTR SGM Plus kit. This informative nine-locus multiplex, which includes the gender-identification locus Amelogenin, has been validated according to the FBI/National Standards and SWGDAM guidelines. Our results demonstrate significant performance improvements in models of DNA degradation, PCR inhibition, and nonprobative samples when compared to the AmpFlSTR Identifiler and SGM Plus kits. These data support that the MiniFiler kit will increase the likelihood of obtaining additional STR information from forensic samples in situations in which standard STR chemistries fail to produce complete profiles.

Further characterization of the first seminoma cell line TCam-2.

Abstract: Testicular germ cell tumors of adolescents and adults (TGCTs) can be classified into seminomatous and nonseminomatous tumors. Various nonseminomatous cell lines, predominantly embryonal carcinoma, have been established and proven to be valuable for pathobiological and clinical studies. So far, no cell lines have been derived from seminoma which constitutes more than 50% of invasive TGCTs. Such a cell line is essential for experimental investigation of biological characteristics of the cell of origin of TGCTs, i.e., carcinoma in situ of the testis, which shows characteristics of a seminoma cell. Before a cell line can be used as model, it must be verified regarding its origin and characteristics. Therefore, a multidisciplinary approach was undertaken on TCam-2 cells, supposedly the first seminoma cell line. Fluorescence in situ hybridization, array comparative genomic hybridization, and spectral karyotyping demonstrated an aneuploid DNA content, with gain of 12p, characteristic for TGCTs. Genome wide mRNA and microRNA expression profiling supported the seminoma origin, in line with the biallelic expression of imprinted genes IGF2/H19 and associated demethylation of the imprinting control region. Moreover, the presence of specific markers, demonstrated by immunohistochemistry, including (wild type) KIT, stem cell factor, placental alkaline phosphatase, OCT3/4 (also demonstrated by a specific Q-PCR) and NANOG, and the absence of CD30, SSX2-4, and SOX2, confirms that TCam-2 is a seminoma cell line. Although mutations in oncogenes and tumor suppressor genes are rather rare in TGCTs, TCam-2 had a mutated BRAF gene (V600E), which likely explains the fact that these cells could be propagated in vitro. In conclusion, TCam-2 is the first well-characterized seminoma-derived cell line, with an exceptional mutation, rarely found in TGCTs.

Identifying selected regions from heterozygosity and divergence using a light-coverage genomic dataset from two human populations.

Abstract: When a selective sweep occurs in the chromosomal region around a target gene in two populations that have recently separated, it produces three dramatic genomic consequences: 1) decreased multi-locus heterozygosity in the region; 2) elevated or diminished genetic divergence (FST) of multiple polymorphic variants adjacent to the selected locus between the divergent populations, due to the alternative fixation of alleles; and 3) a consequent regional increase in the variance of FST (S2FST) for the same clustered variants, due to the increased alternative fixation of alleles in the loci surrounding the selection target. In the first part of our study, to search for potential targets of directional selection, we developed and validated a resampling-based computational approach; we then scanned an array of 31 different-sized moving windows of SNP variants (5–65 SNPs) across the human genome in a set of European and African American population samples with 183,997 SNP loci after correcting for the recombination rate variation. The analysis revealed 180 regions of recent selection with very strong evidence in either population or both. In the second part of our study, we compared the newly discovered putative regions to those sites previously postulated in the literature, using methods based on inspecting patterns of linkage disequilibrium, population divergence and other methodologies. The newly found regions were cross-validated with those found in nine other studies that have searched for selection signals. Our study was replicated especially well in those regions confirmed by three or more studies. These validated regions were independently verified, using a combination of different methods and different databases in other studies, and should include fewer false positives. The main strength of our analysis method compared to others is that it does not require dense genotyping and therefore can be used with data from population-based genome SNP scans from smaller studies of humans or other species.

Second-Tier Test for Quantification of Alloisoleucine and Branched-Chain Amino Acids in Dried Blood Spots to Improve Newborn Screening for Maple Syrup Urine Disease (MSUD)

Abstract: Background: Newborn screening for maple syrup urine disease (MSUD) relies on finding increased concentrations of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine by tandem mass spectrometry (MS/MS). d-Alloisoleucine (allo-Ile) is the only pathognomonic marker of MSUD, but it cannot be identified by existing screening methods because it is not differentiated from isobaric amino acids. Furthermore, newborns receiving total parenteral nutrition often have increased concentrations of BCAAs. To improve the specificity of newborn screening for MSUD and to reduce the number of diet-related false-positive results, we developed a LC-MS/MS method for quantifying allo-Ile. Methods: Allo-Ile and other BCAAs were extracted from a 3/16-inch dried blood spot punch with methanol/H 2 O, dried under nitrogen, and reconstituted into mobile phase. Quantitative LC-MS/MS analysis of allo-Ile, its isomers, and isotopically labeled internal standards was achieved within 15 min. To determine a reference interval for BCAAs including allo-Ile, we analyzed 541 dried blood spots. We also measured allo-Ile in blinded samples from 16 MSUD patients and 21 controls and compared results to an HPLC method. Results: Intra- and interassay imprecision (mean CVs) for allo-Ile, leucine, isoleucine, and valine ranged from 1.8% to 7.4%, and recovery ranged from 91% to 129%. All 16 MSUD patients were correctly identified. Conclusions: The LC-MS/MS method can reliably measure allo-Ile in dried blood spots for the diagnosis of MSUD. Applied to newborn screening as a second-tier test, it will reduce false-positive results, which produce family anxiety and increase follow-up costs. The assay also appears suitable for use in monitoring treatment of MSUD patients.

Gene Profiling in the Livers of Wild-type and PPARα-Null Mice Exposed to Perfluorooctanoic Acid

Abstract: Health concerns have been raised because perfluorooctanoic acid (PFOA) is commonly found in the environment and can be detected in humans. In rodents, PFOA is a carcinogen and a developmental toxicant. PFOA is a peroxisome proliferator-activated receptor alpha (PPARalpha) activator; however, PFOA is capable of inducing heptomegaly in the PPARalpha-null mouse. To study the mechanism associated with PFOA toxicity, wild-type and PPARalpha-null mice were orally dosed for 7 days with PFOA (1 or 3 mg/kg) or the PPARalpha agonist Wy14,643 (50 mg/kg). Gene expression was evaluated using commercial microarrays. In wild-type mice, PFOA and Wy14,643 induced changes consistent with activation of PPARalpha. PFOA-treated wild-type mice deviated from Wy14,643-exposed mice with respect to genes involved in xenobiotic metabolism. In PFOA-treated null mice, changes were observed in transcripts related to fatty acid metabolism, inflammation, xenobiotic metabolism, and cell cycle regulation. Hence, a component of the PFOA response was found to be independent of PPARalpha. Although the signaling pathways responsible for these effects are not readily apparent, overlapping gene regulation by additional PPAR isoforms could account for changes related to fatty acid metabolism and inflammation, whereas regulation of xenobiotic metabolizing genes is suggestive of constitutive androstane receptor activation.

Improved RNA quality and TaqMan ® Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials

Abstract: Archival formalin-fixed paraffin-embedded (FFPE) tissues represent an abundant source of clinical specimens; however their use is limited in applications involving analysis of gene expression due to RNA degradation and modification during fixation and processing. This study improved the quality of RNA extracted from FFPE by introducing a heating step into the selected extraction protocols. Further, it evaluated a novel pre-amplification system (PreAmp) designed to enhance expression analysis from tissue samples using assays with a range of amplicon size (62–164 bp). Results from the Bioanalyzer and TaqMan® data showed improvement of RNA quality extracted using the modified protocols from FFPE. Incubation at 70°C for 20 minutes was determined to be the best condition of those tested to disrupt cross-links while not compromising RNA integrity. TaqMan® detection was influenced by master mix, amplicon size and the incorporation of a pre-amplification step. TaqMan® PreAmp consistently achieved decreased CT values in both snap frozen and FFPE aliquots compared with no pre-amplification. Modification to extraction protocols has facilitated procurement of RNA that may be successfully amplified using QRT-PCR. TaqMan® PreAmp system is a robust and practical solution to limited quantities of RNA from FFPE extracts.

Guidelines for reporting the use of mass spectrometry in proteomics

Abstract: Joeri Borstlap1, Glyn Stacey2, Andreas Kurtz3, Anja Elstner3, Alexander Damaschun1, Begoña Arán4 & Anna Veiga4,5 1CellNet Initiative, Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité– Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. 2The UK Stem Cell Bank, National Institute for Biological Standards and Control, Blanch Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK. 3Cell Therapy Group, BerlinBrandenburg Center for Regenerative Therapies (BCRT), Charité–Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. 4Banc de Linies Cellulars, Centre de Medicina Regenerativa de Barcelona (CMRB), C/Dr. Aiguader 88, 08003-Barcelona, Spain. 5Institut Universitari Dexeus, Passeig de la Bonanova 67, 08017-Barcelona, Spain. e-mail: joeri.borstlap@b-crt.de

The prevalence of folate-remedial MTHFR enzyme variants in humans

Abstract: Studies of rare, inborn metabolic diseases establish that the phenotypes of some mutations in vitamin-dependent enzymes can be suppressed by supplementation of the cognate vitamin, which restores function of the defective enzyme. To determine whether polymorphisms exist that more subtly affect enzymes yet are augmentable in the same way, we sequenced the coding region of a prototypical vitamin-dependent enzyme, methylenetetrahydrofolate reductase (MTHFR), from 564 individuals of diverse ethnicities. All nonsynonymous changes were evaluated in functional in vivo assays in Saccharomyces cerevisiae to identify enzymatic defects and folate remediability of impaired alleles. We identified 14 nonsynonymous changes: 11 alleles with minor allele frequencies <1% and 3 common alleles (A222V, E429A, and R594Q). Four of 11 low-frequency alleles affected enzyme function, as did A222V. Of the five impaired alleles, four could be restored to normal functionality by elevating intracellular folate levels. All five impaired alleles mapped to the N-terminal catalytic domain of the enzyme, whereas changes in the C-terminal regulatory domain had little effect on activity. Impaired activity correlated with the phosphorylation state of MTHFR, with more severe mutations resulting in lower abundance of the phosphorylated protein. Significantly, diploid yeast heterozygous for mutant alleles were impaired for growth, particularly with lower folate supplementation. These results suggested that multiple less-frequent alleles, in aggregate, might significantly contribute to metabolic dysfunction. Furthermore, vitamin remediation of mutant enzymes may be a common phenomenon in certain domains of proteins.

Guidelines for reporting the use of mass spectrometry informatics in proteomics.

Abstract: volume 26 number 8 august 2008 nature biotechnology are addressed in the MIAPE-MS (mass spectrometry) module, the latest version of which can be obtained from the MIAPE home page. Note also that these guidelines do not cover all the available features of a protein and peptide identification and characterization tool (e.g., some of the less frequently used parameters, types of spectra or other experimental data); subsequent versions may have expanded coverage, as will almost certainly be the case for all MIAPE modules. These guidelines will evolve in step with progress in research. The most recent version of MIAPE-MSI is available at http://www.psidev.info/miape/msi/ and the content is replicated here as supplementary information (Supplementary Guidelines and Supplementary Table 1). To contribute or to track the process to remain ‘MIAPE compliant’, browse the website at http:// www.psidev.info/miape/.

Alternative nucleic acid sequencing methods

Abstract: Embodiments are provided that provide for parallel sequencing of nucleic acid segments. In some embodiments, a single sequence is sequenced by at least two different sequencing techniques and the results compared, allowing for deficiencies or strengths of one technique to be complemented by the second technique.