Homogeneity of the 16S rDNA sequence among geographically disparate isolates of Taylorella equigenitalis

Background

At present, six accessible sequences of 16S rDNA from Taylorella equigenitalis (T. equigenitalis) are available, whose sequence differences occur at a few nucleotide positions. Thus it is important to determine these sequences from additional strains in other countries, if possible, in order to clarify any anomalies regarding 16S rDNA sequence heterogeneity. Here, we clone and sequence the approximate full-length 16S rDNA from additional strains of T. equigenitalis isolated in Japan, Australia and France and compare these sequences to the existing published sequences.

Results

Clarification of any anomalies regarding 16S rDNA sequence heterogeneity of T. equigenitalis was carried out. When cloning, sequencing and comparison of the approximate full-length 16S rDNA from 17 strains of T. equigenitalis isolated in Japan, Australia and France, nucleotide sequence differences were demonstrated at the six loci in the 1,469 nucleotide sequence. Moreover, 12 polymorphic sites occurred among 23 sequences of the 16S rDNA, including the six reference sequences.

Conclusion

High sequence similarity (99.5% or more) was observed throughout, except from nucleotide positions 138 to 501 where substitutions and deletions were noted.     

Membrane anchors effectively traffic recombinant human glucocerebrosidase to the protein storage vacuole of Arabidopsis seeds but do not adequately control N-glycan maturation

Key message

Human glucocerebrosidase with vacuolar anchoring domains was targeted to protein storage vacuoles (PSVs) of Arabidopsis seeds, but unexpectedly via the Golgi complex. PSV-targeting to effectively avoid problematic N-glycans is protein dependent.

Abstract

Plant-specific N-glycosylation patterns elaborated within the Golgi complex are a major limitation of using plants to produce biopharmaceuticals as the presence of β1,2 xylose and/or α1,3 fucose residues on the recombinant glycoprotein can render the product immunogenic if administrated parenterally. A reporter protein fused to a vacuolar membrane targeting motif comprised of the BP-80 transmembrane domain (TMD), and the cytoplasmic tail (CT) of α-tonoplast intrinsic protein (α-TIP) is delivered to protein storage vacuoles (PSVs) of tobacco seeds by ER-derived transport vesicles that bypass the Golgi complex. This prompted us to investigate whether a pharmaceutical glycoprotein is targeted to PSVs using the same targeting sequences, thus avoiding the unwanted plant-Golgi-specific complex N-glycan modifications. The human lysosomal acid β-glucosidase (glucocerebrosidase; GCase) (EC 3.2.1.45) fused to the BP-80 TMD and α-TIP CT was produced in Arabidopsis thaliana wild-type (Col-0) seeds. The chimeric GCase became localized in PSVs but transited through the Golgi complex, as indicated by biochemical analyses of the recombinant protein’s N-glycans. Our findings suggest that use of this PSV-targeting strategy to avoid problematic N-glycan maturation on recombinant therapeutic proteins is not consistently effective, as it is likely protein- and/or species-specific.     

Not all transmembrane helices are born equal: Towards the extension of the sequence homology concept to membrane proteins

Background

Sequence homology considerations widely used to transfer functional annotation to uncharacterized protein sequences require special precautions in the case of non-globular sequence segments including membrane-spanning stretches composed of non-polar residues. Simple, quantitative criteria are desirable for identifying transmembrane helices (TMs) that must be included into or should be excluded from start sequence segments in similarity searches aimed at finding distant homologues.

Results

We found that there are two types of TMs in membrane-associated proteins. On the one hand, there are so-called simple TMs with elevated hydrophobicity, low sequence complexity and extraordinary enrichment in long aliphatic residues. They merely serve as membrane-anchoring device. In contrast, so-called complex TMs have lower hydrophobicity, higher sequence complexity and some functional residues. These TMs have additional roles besides membrane anchoring such as intra-membrane complex formation, ligand binding or a catalytic role. Simple and complex TMs can occur both in single- and multi-membrane-spanning proteins essentially in any type of topology. Whereas simple TMs have the potential to confuse searches for sequence homologues and to generate unrelated hits with seemingly convincing statistical significance, complex TMs contain essential evolutionary information.

Conclusion

For extending the homology concept onto membrane proteins, we provide a necessary quantitative criterion to distinguish simple TMs (and a sufficient criterion for complex TMs) in query sequences prior to their usage in homology searches based on assessment of hydrophobicity and sequence complexity of the TM sequence segments.

Reviewers

This article was reviewed by Shamil Sunyaev, L. Aravind and Arcady Mushegian.     

Expression cartography of human tissues using self organizing maps

Background

Although homology-based methods are among the most widely used methods for predicting the structure and function of proteins, the question as to whether interface sequence conservation can be effectively exploited in predicting protein-protein interfaces has been a subject of debate.

Results

We studied more than 300,000 pair-wise alignments of protein sequences from structurally characterized protein complexes, including both obligate and transient complexes. We identified sequence similarity criteria required for accurate homology-based inference of interface residues in a query protein sequence. Based on these analyses, we developed HomPPI, a class of sequence homology-based methods for predicting protein-protein interface residues. We present two variants of HomPPI: (i) NPS-HomPPI (Non partner-specific HomPPI), which can be used to predict interface residues of a query protein in the absence of knowledge of the interaction partner; and (ii) PS-HomPPI (Partner-specific HomPPI), which can be used to predict the interface residues of a query protein with a specific target protein. Our experiments on a benchmark dataset of obligate homodimeric complexes show that NPS-HomPPI can reliably predict protein-protein interface residues in a given protein, with an average correlation coefficient (CC) of 0.76, sensitivity of 0.83, and specificity of 0.78, when sequence homologs of the query protein can be reliably identified. NPS-HomPPI also reliably predicts the interface residues of intrinsically disordered proteins. Our experiments suggest that NPS-HomPPI is competitive with several state-of-the-art interface prediction servers including those that exploit the structure of the query proteins. The partner-specific classifier, PS-HomPPI can, on a large dataset of transient complexes, predict the interface residues of a query protein with a specific target, with a CC of 0.65, sensitivity of 0.69, and specificity of 0.70, when homologs of both the query and the target can be reliably identified. The HomPPI web server is available at http://homppi.cs.iastate.edu/.

Conclusions

Sequence homology-based methods offer a class of computationally efficient and reliable approaches for predicting the protein-protein interface residues that participate in either obligate or transient interactions. For query proteins involved in transient interactions, the reliability of interface residue prediction can be improved by exploiting knowledge of putative interaction partners.     

A novel 9-bp insertion detected in steroid 21-hydroxylase gene (CYP21A2): prediction of its structural and functional implications by computational methods

Background

Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia (CAH). Detection of underlying mutations in CYP21A2 gene encoding steroid 21-hydroxylase enzyme is helpful both for confirmation of diagnosis and management of CAH patients. Here we report a novel 9-bp insertion in CYP21A2 gene and its structural and functional consequences on P450c21 protein by molecular modeling and molecular dynamics simulations methods.

Methods

A 30-day-old child was referred to our laboratory for molecular diagnosis of CAH. Sequencing of the entire CYP21A2 gene revealed a novel insertion (duplication) of 9-bp in exon 2 of one allele and a well-known mutation I172N in exon 4 of other allele. Molecular modeling and simulation studies were carried out to understand the plausible structural and functional implications caused by the novel mutation.

Results

Insertion of the nine bases in exon 2 resulted in addition of three valine residues at codon 71 of the P450c21 protein. Molecular dynamics simulations revealed that the mutant exhibits a faster unfolding kinetics and an overall destabilization of the structure due to the triple valine insertion was also observed.

Conclusion

The novel 9-bp insertion in exon 2 of CYP21A2 genesignificantly lowers the structural stability of P450c21 thereby leading to the probable loss of its function.     

A BAC-based integrated linkage map of the silkworm Bombyx mori

Background

In 2004, draft sequences of the model lepidopteran Bombyx mori were reported using whole-genome shotgun sequencing. Because of relatively shallow genome coverage, the silkworm genome remains fragmented, hampering annotation and comparative genome studies. For a more complete genome analysis, we developed extended scaffolds combining physical maps with improved genetic maps.

Results

We mapped 1,755 single nucleotide polymorphism (SNP) markers from bacterial artificial chromosome (BAC) end sequences onto 28 linkage groups using a recombining male backcross population, yielding an average inter-SNP distance of 0.81 cM (about 270 kilobases). We constructed 6,221 contigs by fingerprinting clones from three BAC libraries digested with different restriction enzymes, and assigned a total of 724 single copy genes to them by BLAST (basic local alignment search tool) search of the BAC end sequences and high-density BAC filter hybridization using expressed sequence tags as probes. We assigned 964 additional expressed sequence tags to linkage groups by restriction fragment length polymorphism analysis of a nonrecombining female backcross population. Altogether, 361.1 megabases of BAC contigs and singletons were integrated with a map containing 1,688 independent genes. A test of synteny using Oxford grid analysis with more than 500 silkworm genes revealed six versus 20 silkworm linkage groups containing eight or more orthologs of Apis versus Tribolium, respectively.

Conclusion

The integrated map contains approximately 10% of predicted silkworm genes and has an estimated 76% genome coverage by BACs. This provides a new resource for improved assembly of whole-genome shotgun data, gene annotation and positional cloning, and will serve as a platform for comparative genomics and gene discovery in Lepidoptera and other insects.     

The N-Terminal Transmembrane Domain of λ S Is Required for Holin but Not Antiholin Function

The λ S gene encodes a holin, S105, and an antiholin, S107, which differs by its Met-Lys N-terminal extension. The model for the lysis-defective character of S107 stipulates that the additional N-terminal basic residue keeps S107 from assuming the topology of S105, which is N-out, C-in, with three transmembrane domains (TMDs). Here we show that the N terminus of S105 retains its fMet residue but that the N terminus of S107 is fully deformylated. This supports the model that in S105, TMD1 inserts into the membrane very rapidly but that in S107, it is retained in the cytoplasm. Further, it reveals that, compared to S105, S107 has two extra positively charged moieties, Lys2 and the free N-terminal amino group, to hinder its penetration into an energized membrane. Moreover, an allele, S105_ΔTMD1, with TMD1 deleted, was found to be defective in lysis, insensitive to membrane depolarization, and dominant to the wild-type allele, indicating that the lysis-defective, antiholin character of S107 is due to the absence of TMD1 from the bilayer rather than to its ectopic localization at the inner face of the cytoplasmic membrane. Finally, the antiholin function of the deletion protein was compromised by the substitution of early-lysis missense mutations in either the deletion protein or parental S105 but restored when both S105_ΔTMD1 and holin carried the substitution.In general, holins control the length of the infection cycle of double-stranded DNA phages (37). During late gene expression, the holin protein accumulates harmlessly in the bilayer until suddenly and spontaneously triggering the formation of holes in the membrane at an allele-specific time (13, 15). Holin genes are extremely diverse, but most can be grouped into two main classes based on the number of predicted transmembrane domains (TMDs): class I, with three TMDs and a predicted N-out, C-in topology, and class II, with two TMDs and a predicted N-in, C-in topology (38). Holin genes and function are subject to several levels of regulation, among which a particularly striking feature is the common occurrence of two potential translational starts, or dual-start motifs (5, 37), separated by only a few codons. Dual-start motifs are found in many holins of both of the two major classes; in nearly every case, the two starts are separated by at least one basic residue. The first dual-start motif to be characterized was that of λ S, the prototype class I holin gene (Fig. 1A and B). Translation initiation events occur at codons 1 and 3, giving rise to two products, S107 and S105, each named because of the length of its amino acid sequence; in the wild-type (wt) allele, two RNA structures define the ratio of initiations at the two start codons, resulting in an S105/S107 ratio of ∼2:1.Open in a separate windowFIG. 1.Gene, topology, and sequence of λ S. (A, top) The λ lysis cassette, including genes S, R, Rz, and Rz1, is shown, along with its promoter p_R′, and Q, encoding the late gene activator. The 5′ end of the class I holin gene S has two start codons, Met1, the start for S107, and Met3, the start for S105, and two RNA structures that regulate initiations at these codons. The S105 and S107 alleles have Leu (CUG) codons in place of the Met3 and Met1 codons, respectively. (B) Primary structure of S proteins. Missense changes relevant to the text are shown. Starts for S107 and S105 are indicated by asterisks. The three TMDs are boxed (13), and the extent of the ΔTMD1 deletion is indicated. (C) Model for the membrane topology of S105, S107, and S105_ΔTMD1. Topology and boundary residues for TMD1, -2, and -3 are based on Graschopf and Blasi (11) and Gründling et al. (13), respectively.Although they differ only by the Met-Lys N-terminal extension of S107, the two proteins have opposing functions; S105 is the holin and S107 the antiholin. The antiholin function is reflected by four principal features: first, when the Met3 start is inactivated, the mutant allele, designated S107 (Fig. ​(Fig.1A),1A), is lysis defective (26); second, the S107 protein binds and inhibits S105 specifically (3, 16); third, when S107 is produced in stoichiometric excess over S105, lysis is blocked for several times the length of the normal infection cycle (3, 4, 7, 16); and fourth, S107 antiholin function, i.e., inhibition of S105 hole formation, can be instantly subverted by collapsing the proton motive force, most easily done by addition of energy poisons to the medium (3). The predicted N-out, C-in topology and the requirement for the energized membrane led to a model in which S107 is initially inserted in the membrane with only two TMDs, with TMD1 being blocked from insertion by the presence of the positively charged residue, Lys2, whereas S105 has three TMDs (Fig. ​(Fig.1C)1C) (39). From this perspective, S105-S107 complexes, which are approximately twice as numerous as the S105 homodimers, are defective in triggering hole formation. An appealing feature of this model is that when an S105-mediated hole formation event does occur in a cell, the resultant collapse of the membrane potential allows insertion of TMD1 of S107 into the membrane, instantly tripling the amount of active holin by making the previously inactive pool of S105-S107 complexes functional (38).Some genetic and physiological evidence for the topology of the λ S proteins has been obtained using gene fusions. First, a fusion of the S gene at codon 105 with lacZ generates a functional, membrane-inserted β-galactosidase chimera, indicating, as expected, the cytoplasmic disposition of the highly charged C terminus of the S protein (40). Second, Graschopf and Bläsi (12) demonstrated that S-mediated hole formation could be obtained with constructs where a secretory signal sequence was fused to the N termini of both S105 and S107. Lysis required the cleavage of the signal sequence by leader peptidase, and export of the signal-S107 form was slower than for the signal-S105 form. However, evidence for the topology of native forms of S has not been available. Moreover, no basis for the inhibitory character of S107 has been established. In the simplest view, the antiholin function could be due to the absence of TMD1 from the bilayer or the ectopic localization of TMD1 in the cytoplasm, or both. Here, we report studies directed at dissecting the precise role of topology in S107 function and correlating antiholin activity with its ability to heterodimerize with S105. The results are discussed in terms of a general model for the formation of the holin lesion and the role of dynamic membrane topology in its temporal regulation.     

Genomic analysis of the relationship between gene expression variation and DNA polymorphism in Drosophila simulans

Background

Understanding how DNA sequence polymorphism relates to variation in gene expression is essential to connecting genotypic differences with phenotypic differences among individuals. Addressing this question requires linking population genomic data with gene expression variation.

Results

Using whole genome expression data and recent light shotgun genome sequencing of six Drosophila simulans genotypes, we assessed the relationship between expression variation in males and females and nucleotide polymorphism across thousands of loci. By examining sequence polymorphism in gene features, such as untranslated regions and introns, we find that genes showing greater variation in gene expression between genotypes also have higher levels of sequence polymorphism in many gene features. Accordingly, X-linked genes, which have lower sequence polymorphism levels than autosomal genes, also show less expression variation than autosomal genes. We also find that sex-specifically expressed genes show higher local levels of polymorphism and divergence than both sex-biased and unbiased genes, and that they appear to have simpler regulatory regions.

Conclusion

The gene-feature-based analyses and the X-to-autosome comparisons suggest that sequence polymorphism in cis-acting elements is an important determinant of expression variation. However, this relationship varies among the different categories of sex-biased expression, and trans factors might contribute more to male-specific gene expression than cis effects. Our analysis of sex-specific gene expression also shows that female-specific genes have been overlooked in analyses that only point to male-biased genes as having unusual patterns of evolution and that studies of sexually dimorphic traits need to recognize that the relationship between genetic and expression variation at these traits is different from the genome as a whole.     

Hydration studies on the archaeal protein Sso7d using NMR measurements and MD simulations

Background

Cytoplasmic class XI myosins are the fastest processive motors known. This class functions in high-velocity cytoplasmic streaming in various plant cells from algae to angiosperms. The velocities at which they process are ten times faster than its closest class V homologues.

Results

To provide sequence determinants and structural rationale for the molecular mechanism of this fast pace myosin, we have compared the sequences from myosin class V and XI through Evolutionary Trace (ET) analysis. The current study identifies class-specific residues of myosin XI spread over the actin binding site, ATP binding site and light chain binding neck region. Sequences for ET analysis were accumulated from six plant genomes, using literature based text search and sequence searches, followed by triple validation viz. CDD search, string-based searches and phylogenetic clustering. We have identified nine myosin XI genes in sorghum and seven in grape by sequence searches. Both the plants possess one gene product each belonging to myosin type VIII as well. During this process, we have re-defined the gene boundaries for three sorghum myosin XI genes using fgenesh program.

Conclusion

Molecular modelling and subsequent analysis of putative interactions involving these class-specific residues suggest a structural basis for the molecular mechanism behind high velocity of plant myosin XI. We propose a model of a more flexible switch I region that contributes to faster ADP release leading to high velocity movement of the algal myosin XI.     

Sympatric Plasmodium falciparum isolates from Venezuela have structured var gene repertoires

Background

Plasmodium falciparum antigenic diversity and polymorphism confuses the issue of antimalarial vaccine development. Merozoite surface protein (MSP)-1 and -2 are two highly polymorphic vaccine candidates. Characterisation of their precise polymorphism in endemic regions may facilitate the design of an effective vaccine.

Methods

Isolates obtained in 52 Gabonese children presenting with uncomplicated malaria were genotyped by nested-PCR of msp -1 block 2, and msp -2 block 3, to analyze both parasite population polymorphism and clone fluctuations.

Results

Twenty-five and 19 different alleles were respectively obtained for msp-1 and msp-2 loci, the RO33 family of msp-1 being poorly polymorphic. Four cases of non-random distribution of alleles were reported of the FC27, and/or 3D7 families of msp-2. All but two isolates were composed of more than one genotype, and the multiplicity of infection (MOI) was 4.0. Neither parasite density nor age was related to MOI. Clone fluctuations were studied for ten subjects who were sampled again at reappearance of parasites in blood. Disappearance and reappearance of alleles were observed following treatment, suggesting difficulties in assessing polymorphism and in distinguishing reinfection from recrudescence.

Conclusion

P. falciparum polymorphism is extensive in Southeast Gabon, and most of infections are composed of multiple clones. The fluctuation of clones contributes to parasite diversity.     

The identification of allergen proteins in sugar beet (Beta vulgaris) pollen causing occupational allergy in greenhouses

Background

During production of sugar beet (Beta vulgaris) seeds in greenhouses, workers frequently develop allergic symptoms. The aim of this study was to identify and characterize possible allergens in sugar beet pollen.

Methods

Sera from individuals at a local sugar beet seed producing company, having positive SPT and specific IgE to sugar beet pollen extract, were used for immunoblotting. Proteins in sugar beet pollen extracts were separated by 1- and 2-dimensional electrophoresis, and IgE-reactive proteins analyzed by liquid chromatography tandem mass spectrometry.

Results

A 14 kDa protein was identified as an allergen, since IgE-binding was inhibited by the well-characterized allergen Che a 2, profilin, from the related species Chenopodium album. The presence of 17 kDa and 14 kDa protein homologues to both the allergens Che a 1 and Che a 2 were detected in an extract from sugar beet pollen, and partial amino acid sequences were determined, using inclusion lists for tandem mass spectrometry based on homologous sequences.

Conclusion

Two occupational allergens were identified in sugar beet pollen showing sequence similarity with Chenopodium allergens. Sequence data were obtained by mass spectrometry (70 and 25%, respectively for Beta v 1 and Beta v 2), and can be used for cloning and recombinant expression of the allergens. As for treatment of Chenopodium pollinosis, immunotherapy with sugar beet pollen extracts may be feasible.     

Population diversity and antibody selective pressure to Plasmodium falciparum MSP1 block2 locus in an African malaria-endemic setting

Background

Genetic evidence for diversifying selection identified the Merozoite Surface Protein1 block2 (PfMSP1 block2) as a putative target of protective immunity against Plasmodium falciparum. The locus displays three family types and one recombinant type, each with multiple allelic forms differing by single nucleotide polymorphism as well as sequence, copy number and arrangement variation of three amino acid repeats. The family-specific antibody responses observed in endemic settings support immune selection operating at the family level. However, the factors contributing to the large intra-family allelic diversity remain unclear. To address this question, population allelic polymorphism and sequence variant-specific antibody responses were studied in a single Senegalese rural community where malaria transmission is intense and perennial.

Results

Family distribution showed no significant temporal fluctuation over the 10 y period surveyed. Sequencing of 358 PCR fragments identified 126 distinct alleles, including numerous novel alleles in each family and multiple novel alleles of recombinant types. The parasite population consisted in a large number of low frequency alleles, alongside one high-frequency and three intermediate frequency alleles. Population diversity tests supported positive selection at the family level, but showed no significant departure from neutrality when considering intra-family allelic sequence diversity and all families combined. Seroprevalence, analysed using biotinylated peptides displaying numerous sequence variants, was moderate and increased with age. Reactivity profiles were individual-specific, mapped to the family-specific flanking regions and to repeat sequences shared by numerous allelic forms within a family type. Seroreactivity to K1-, Mad20- and R033 families correlated with the relative family genotype distribution within the village. Antibody specificity remained unchanged with cumulated exposure to an increasingly large number of alleles.

Conclusion

The Pfmsp1 block2 locus presents a very large population sequence diversity. The lack of stable acquisition of novel antibody specificities despite exposure to novel allelic forms is reminiscent of clonal imprinting. The locus appears under antibody-mediated diversifying selection in a variable environment that maintains a balance between the various family types without selecting for sequence variant allelic forms. There is no evidence of positive selection for intra-family sequence diversity, consistent with the observed characteristics of the antibody response.     

Novel and unexpected bacterial diversity in an arsenic-rich ecosystem revealed by culture-dependent approaches

Background

BLAST is a commonly-used software package for comparing a query sequence to a database of known sequences; in this study, we focus on protein sequences. Position-specific-iterated BLAST (PSI-BLAST) iteratively searches a protein sequence database, using the matches in round i to construct a position-specific score matrix (PSSM) for searching the database in round i?+?1. Biegert and S?ding developed Context-sensitive BLAST (CS-BLAST), which combines information from searching the sequence database with information derived from a library of short protein profiles to achieve better homology detection than PSI-BLAST, which builds its PSSMs from scratch.

Results

We describe a new method, called domain enhanced lookup time accelerated BLAST (DELTA-BLAST), which searches a database of pre-constructed PSSMs before searching a protein-sequence database, to yield better homology detection. For its PSSMs, DELTA-BLAST employs a subset of NCBI??s Conserved Domain Database (CDD). On a test set derived from ASTRAL, with one round of searching, DELTA-BLAST achieves a ROC₅₀₀₀ of 0.270 vs. 0.116 for CS-BLAST. The performance advantage diminishes in iterated searches, but DELTA-BLAST continues to achieve better ROC scores than CS-BLAST.

Conclusions

DELTA-BLAST is a useful program for the detection of remote protein homologs. It is available under the ??Protein BLAST?? link at http://blast.ncbi.nlm.nih.gov.

Reviewers

This article was reviewed by Arcady Mushegian, Nick V. Grishin, and Frank Eisenhaber.     

The lp13.3 genomic region -rs599839- is associated with endothelial dysfunction in patients with rheumatoid arthritis

Introduction

Rheumatoid arthritis (RA) is an inflammatory disease associated with accelerated atherosclerosis and high risk of cardiovascular (CV) disease. Since genome-wide association studies demonstrated association between rs599839 polymorphism and coronary artery disease, in the present study we assessed the potential association of this polymorphism with endothelial dysfunction, an early step in atherogenesis.

Methods

A total of 128 RA patients without history of CV events were genotyped for rs599839 A/G polymorphism. The presence of endothelial dysfunction was assessed by brachial ultrasonography (brachial flow-mediated endothelium-dependent (FMD)).

Results

Patients carrying the allele G exhibited more severe endothelial dysfunction (FMD%: 4.61 ± 3.94%) than those carrying the wild allele A (FMD%: 6.01 ± 5.15%) (P = 0.08). Adjustment for gender, age at the time of study, follow-up time and classic CV risk factors disclosed a significant association between the rs599839 polymorphism and FMD (G vs. A: P = 0.0062).

Conclusions

Our results confirm an association of the rs599839 polymorphism with endothelial dysfunction in RA.     

Structure-Function Analysis of Diacylglycerol Acyltransferase Sequences from 70 Organisms

Background

Diacylglycerol acyltransferase families (DGATs) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Understanding the roles of DGATs will help to create transgenic plants with value-added properties and provide clues for therapeutic intervention for obesity and related diseases. The objective of this analysis was to identify conserved sequence motifs and amino acid residues for better understanding of the structure-function relationship of these important enzymes.

Results

117 DGAT sequences from 70 organisms including plants, animals, fungi and human are obtained from database search using tung tree DGATs. Phylogenetic analysis separates these proteins into DGAT1 and DGAT2 subfamilies. These DGATs are integral membrane proteins with more than 40% of the total amino acid residues being hydrophobic. They have similar properties and amino acid composition except that DGAT1s are approximately 20 kDa larger than DGAT2s. DGAT1s and DGAT2s have 41 and 16 completely conserved amino acid residues, respectively, although only two of them are shared by all DGATs. These residues are distributed in 7 and 6 sequence blocks for DGAT1s and DGAT2s, respectively, and located at the carboxyl termini, suggesting the location of the catalytic domains. These conserved sequence blocks do not contain the putative neutral lipid-binding domain, mitochondrial targeting signal, or ER retrieval motif. The importance of conserved residues has been demonstrated by site-directed and natural mutants.

Conclusions

This study has identified conserved sequence motifs and amino acid residues in all 117 DGATs and the two subfamilies. None of the completely conserved residues in DGAT1s and DGAT2s is present in recently reported isoforms in the multiple sequences alignment, raising an important question how proteins with completely different amino acid sequences could perform the same biochemical reaction. The sequence analysis should facilitate studying the structure-function relationship of DGATs with the ultimate goal to identify critical amino acid residues for engineering superb enzymes in metabolic engineering and selecting enzyme inhibitors in therapeutic application for obesity and related diseases.     

MulSatDB: a first online database for mulberry microsatellites

Key message

Simple sequence repeat motifs were mined from the genome and EST sequences of Morus notabilis and archived in MulSatDB. Bioinformatics tools were integrated with the database for the analysis of genomic datasets.

Abstract

Mulberry is a crop of economic importance in sericulture, which shapes the lives of millions of rural people among different Eurasian and Latin American countries. Limited availability of genomic resources has constrained the molecular breeding efforts in mulberry, a poorly studied crop. Microsatellite or simple sequence repeat (SSR) has revolutionized the plant breeding and is used in linkage mapping, association studies, diversity, and parentage analysis, etc. Recent availability of mulberry whole genome assembly provided an opportunity for the development of mulberry-specific DNA markers. In this study, we mined a total of 217,312 microsatellites from whole genome and 961 microsatellites from EST sequences of Morus notabilis. Mono-repeats were predominant among both whole genome and EST sequences. The SSR containing EST sequences were functionally annotated, and SSRs mined from whole genome were mapped on chromosomes of the phylogenetically related genus—Fragaria vesca, to aid the selection of markers based on the function and location. All the mined markers were archived in the mulberry microsatellite database (MulSatDB), and the markers can be retrieved based on different criteria like marker location, repeat kind, motif type and size. Primer3plus and CMap tools are integrated with the database to design primers for PCR amplification and to visualize markers on F. vesca chromosomes, respectively. A blast tool is also integrated to collate new markers with the database. MulSatDB is the first and complete destination for mulberry researchers to browse SSR markers, design primers, and locate markers on strawberry chromosomes. MulSatDB is freely accessible at http://btismysore.in/mulsatdb.