@article {49595, title = {Developmental expression of chicken FOXN1 and putative target genes during feather development.}, volume = {58}, year = {2014}, month = {2014}, pages = {57-64}, abstract = {

FOXN1 is a member of the forkhead box family of transcription factors. FOXN1 is crucial for hair outgrowth and thymus differentiation in mammals. Unlike the thymus, which is found in all amniotes, hair is an epidermal appendage that arose after the last shared common ancestor between mammals and birds, and hair and feathers differ markedly in their differentiation and gene expression. Here, we show that FOXN1 is expressed in embryonic chicken feathers, nails and thymus, demonstrating an evolutionary conservation that goes beyond obvious homology. At embryonic day (ED) 12, FOXN1 is expressed in some feather buds and at ED13 expression extends along the length of the feather filament. At ED14 FOXN1 mRNA is restricted to the proximal feather filament and is not detectable in distal feather shafts. At the base of the feather, FOXN1 is expressed in the epithelium of the feather sheath and distal barb and marginal plate, whereas in the midsection FOXN1 transcripts are mainly detected in the barb plates of the feather filament. FOXN1 is also expressed in claws; however, no expression was detected in skin or scales. Despite expression of FOXN1 in developing feathers, examination of chick homologs of five putative mammalian FOXN1 target genes shows that, while these genes are expressed in feathers, there is little similarity to the FOXN1 expression pattern, suggesting that some gene regulatory networks may have diverged during evolution of epidermal appendages.

}, keywords = {Amino Acid Sequence, Animals, Biological Evolution, Blotting, Western, Cell Differentiation, Cells, Cultured, Chick Embryo, Chickens, Cloning, Molecular, Embryo, Nonmammalian, Epidermis, Feathers, Forkhead Transcription Factors, Gene Expression Regulation, Developmental, In Situ Hybridization, Molecular Sequence Data, Morphogenesis, Phylogeny, Real-Time Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction, RNA, Messenger, Sequence Homology, Amino Acid}, issn = {1696-3547}, doi = {10.1387/ijdb.130023sy}, author = {Darnell, Diana K and Zhang, Li S and Hannenhalli, Sridhar and Yaklichkin, Sergey Y} } @article {49736, title = {Stoichiometry of site-specific lysine acetylation in an entire proteome.}, journal = {J Biol Chem}, volume = {289}, year = {2014}, month = {2014 Aug 1}, pages = {21326-38}, abstract = {

Acetylation of lysine ϵ-amino groups influences many cellular processes and has been mapped to thousands of sites across many organisms. Stoichiometric information of acetylation is essential to accurately interpret biological significance. Here, we developed and employed a novel method for directly quantifying stoichiometry of site-specific acetylation in the entire proteome of Escherichia coli. By coupling isotopic labeling and a novel pairing algorithm, our approach performs an in silico enrichment of acetyl peptides, circumventing the need for immunoenrichment. We investigated the function of the sole NAD(+)-dependent protein deacetylase, CobB, on both site-specific and global acetylation. We quantified 2206 peptides from 899 proteins and observed a wide distribution of acetyl stoichiometry, ranging from less than 1\% up to 98\%. Bioinformatic analysis revealed that metabolic enzymes, which either utilize or generate acetyl-CoA, and proteins involved in transcriptional and translational processes displayed the highest degree of acetylation. Loss of CobB led to increased global acetylation at low stoichiometry sites and induced site-specific changes at high stoichiometry sites, and biochemical analysis revealed altered acetyl-CoA metabolism. Thus, this study demonstrates that sirtuin deacetylase deficiency leads to both site-specific and global changes in protein acetylation stoichiometry, affecting central metabolism.

}, keywords = {Acetylation, Amino Acid Sequence, Bacterial Proteins, Chromatography, High Pressure Liquid, Computational Biology, Escherichia coli, Lysine, Molecular Sequence Data, Proteome, Tandem Mass Spectrometry}, issn = {1083-351X}, doi = {10.1074/jbc.M114.581843}, author = {Baeza, Josue and Dowell, James A and Smallegan, Michael J and Fan, Jing and Amador-Noguez, Daniel and Khan, Zia and Denu, John M} } @article {38119, title = {Archaeosortases and exosortases are widely distributed systems linking membrane transit with posttranslational modification}, journal = {Journal of bacteriologyJournal of bacteriology}, volume = {194}, year = {2012}, note = {http://www.ncbi.nlm.nih.gov/pubmed/22037399?dopt=Abstract}, type = {10.1128/JB.06026-11}, abstract = {Multiple new prokaryotic C-terminal protein-sorting signals were found that reprise the tripartite architecture shared by LPXTG and PEP-CTERM: motif, TM helix, basic cluster. Defining hidden Markov models were constructed for all. PGF-CTERM occurs in 29 archaeal species, some of which have more than 50 proteins that share the domain. PGF-CTERM proteins include the major cell surface protein in Halobacterium, a glycoprotein with a partially characterized diphytanylglyceryl phosphate linkage near its C terminus. Comparative genomics identifies a distant exosortase homolog, designated archaeosortase A (ArtA), as the likely protein-processing enzyme for PGF-CTERM. Proteomics suggests that the PGF-CTERM region is removed. Additional systems include VPXXXP-CTERM/archeaosortase B in two of the same archaea and PEF-CTERM/archaeosortase C in four others. Bacterial exosortases often fall into subfamilies that partner with very different cohorts of extracellular polymeric substance biosynthesis proteins; several species have multiple systems. Variant systems include the VPDSG-CTERM/exosortase C system unique to certain members of the phylum Verrucomicrobia, VPLPA-CTERM/exosortase D in several alpha- and deltaproteobacterial species, and a dedicated (single-target) VPEID-CTERM/exosortase E system in alphaproteobacteria. Exosortase-related families XrtF in the class Flavobacteria and XrtG in Gram-positive bacteria mark distinctive conserved gene neighborhoods. A picture emerges of an ancient and now well-differentiated superfamily of deeply membrane-embedded protein-processing enzymes. Their target proteins are destined to transit cellular membranes during their biosynthesis, during which most undergo additional posttranslational modifications such as glycosylation.}, keywords = {Amino Acid Sequence, Aminoacyltransferases, Archaeal Proteins, Bacterial Proteins, Cell Membrane, Cysteine Endopeptidases, Gene Expression Regulation, Archaeal, Gene Expression Regulation, Bacterial, Gene Expression Regulation, Enzymologic, Molecular Sequence Data, Protein Processing, Post-Translational}, author = {Haft, Daniel H. and Payne, Samuel H. and J. Selengut} } @article {49775, title = {Archaeosortases and exosortases are widely distributed systems linking membrane transit with posttranslational modification.}, journal = {J Bacteriol}, volume = {194}, year = {2012}, month = {2012 Jan}, pages = {36-48}, abstract = {

Multiple new prokaryotic C-terminal protein-sorting signals were found that reprise the tripartite architecture shared by LPXTG and PEP-CTERM: motif, TM helix, basic cluster. Defining hidden Markov models were constructed for all. PGF-CTERM occurs in 29 archaeal species, some of which have more than 50 proteins that share the domain. PGF-CTERM proteins include the major cell surface protein in Halobacterium, a glycoprotein with a partially characterized diphytanylglyceryl phosphate linkage near its C terminus. Comparative genomics identifies a distant exosortase homolog, designated archaeosortase A (ArtA), as the likely protein-processing enzyme for PGF-CTERM. Proteomics suggests that the PGF-CTERM region is removed. Additional systems include VPXXXP-CTERM/archeaosortase B in two of the same archaea and PEF-CTERM/archaeosortase C in four others. Bacterial exosortases often fall into subfamilies that partner with very different cohorts of extracellular polymeric substance biosynthesis proteins; several species have multiple systems. Variant systems include the VPDSG-CTERM/exosortase C system unique to certain members of the phylum Verrucomicrobia, VPLPA-CTERM/exosortase D in several alpha- and deltaproteobacterial species, and a dedicated (single-target) VPEID-CTERM/exosortase E system in alphaproteobacteria. Exosortase-related families XrtF in the class Flavobacteria and XrtG in Gram-positive bacteria mark distinctive conserved gene neighborhoods. A picture emerges of an ancient and now well-differentiated superfamily of deeply membrane-embedded protein-processing enzymes. Their target proteins are destined to transit cellular membranes during their biosynthesis, during which most undergo additional posttranslational modifications such as glycosylation.

}, keywords = {Amino Acid Sequence, Aminoacyltransferases, Archaeal Proteins, Bacterial Proteins, Cell Membrane, Cysteine Endopeptidases, Gene Expression Regulation, Archaeal, Gene Expression Regulation, Bacterial, Gene Expression Regulation, Enzymologic, Molecular Sequence Data, Protein Processing, Post-Translational}, issn = {1098-5530}, doi = {10.1128/JB.06026-11}, author = {Haft, Daniel H and Payne, Samuel H and Selengut, Jeremy D} } @article {49531, title = {Plasmodium falciparum merozoite surface protein 1 blocks the proinflammatory protein S100P.}, volume = {109}, year = {2012}, month = {2012 Apr 3}, pages = {5429-34}, abstract = {

The malaria parasite, Plasmodium falciparum, and the human immune system have coevolved to ensure that the parasite is not eliminated and reinfection is not resisted. This relationship is likely mediated through a myriad of host-parasite interactions, although surprisingly few such interactions have been identified. Here we show that the 33-kDa fragment of P. falciparum merozoite surface protein 1 (MSP1(33)), an abundant protein that is shed during red blood cell invasion, binds to the proinflammatory protein, S100P. MSP1(33) blocks S100P-induced NFκB activation in monocytes and chemotaxis in neutrophils. Remarkably, S100P binds to both dimorphic alleles of MSP1, estimated to have diverged >27 Mya, suggesting an ancient, conserved relationship between these parasite and host proteins that may serve to attenuate potentially damaging inflammatory responses.

}, keywords = {Amino Acid Sequence, Animals, Calcium-Binding Proteins, Chromatography, Gel, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, HUMANS, Merozoite Surface Protein 1, Microscopy, Confocal, Molecular Sequence Data, Neoplasm Proteins, Plasmodium falciparum, Sequence Homology, Amino Acid, Surface Plasmon Resonance}, issn = {1091-6490}, doi = {10.1073/pnas.1202689109}, author = {Waisberg, Michael and Cerqueira, Gustavo C and Yager, Stephanie B and Francischetti, Ivo M B and Lu, Jinghua and Gera, Nidhi and Srinivasan, Prakash and Miura, Kazutoyo and Rada, Balazs and Lukszo, Jan and Barbian, Kent D and Leto, Thomas L and Porcella, Stephen F and Narum, David L and El-Sayed, Najib and Miller, Louis H and Pierce, Susan K} } @article {49744, title = {Accurate proteome-wide protein quantification from high-resolution 15N mass spectra.}, journal = {Genome Biol}, volume = {12}, year = {2011}, month = {2011}, pages = {R122}, abstract = {

In quantitative mass spectrometry-based proteomics, the metabolic incorporation of a single source of 15N-labeled nitrogen has many advantages over using stable isotope-labeled amino acids. However, the lack of a robust computational framework for analyzing the resulting spectra has impeded wide use of this approach. We have addressed this challenge by introducing a new computational methodology for analyzing 15N spectra in which quantification is integrated with identification. Application of this method to an Escherichia coli growth transition reveals significant improvement in quantification accuracy over previous methods.

}, keywords = {algorithms, Amino Acid Sequence, Bacterial Proteins, Escherichia coli, Isotope Labeling, Mass Spectrometry, Molecular Sequence Data, Nitrogen Isotopes, Proteome, proteomics, Sensitivity and Specificity, software}, issn = {1474-760X}, doi = {10.1186/gb-2011-12-12-r122}, author = {Khan, Zia and Amini, Sasan and Bloom, Joshua S and Ruse, Cristian and Caudy, Amy A and Kruglyak, Leonid and Singh, Mona and Perlman, David H and Tavazoie, Saeed} } @article {38506, title = {Sites Inferred by Metabolic Background Assertion Labeling (SIMBAL): adapting the Partial Phylogenetic Profiling algorithm to scan sequences for signatures that predict protein function}, journal = {BMC bioinformaticsBMC Bioinformatics}, volume = {11}, year = {2010}, note = {http://www.ncbi.nlm.nih.gov/pubmed/20102603?dopt=Abstract}, type = {10.1186/1471-2105-11-52}, abstract = {BACKGROUND: Comparative genomics methods such as phylogenetic profiling can mine powerful inferences from inherently noisy biological data sets. We introduce Sites Inferred by Metabolic Background Assertion Labeling (SIMBAL), a method that applies the Partial Phylogenetic Profiling (PPP) approach locally within a protein sequence to discover short sequence signatures associated with functional sites. The approach is based on the basic scoring mechanism employed by PPP, namely the use of binomial distribution statistics to optimize sequence similarity cutoffs during searches of partitioned training sets. RESULTS: Here we illustrate and validate the ability of the SIMBAL method to find functionally relevant short sequence signatures by application to two well-characterized protein families. In the first example, we partitioned a family of ABC permeases using a metabolic background property (urea utilization). Thus, the TRUE set for this family comprised members whose genome of origin encoded a urea utilization system. By moving a sliding window across the sequence of a permease, and searching each subsequence in turn against the full set of partitioned proteins, the method found which local sequence signatures best correlated with the urea utilization trait. Mapping of SIMBAL "hot spots" onto crystal structures of homologous permeases reveals that the significant sites are gating determinants on the cytosolic face rather than, say, docking sites for the substrate-binding protein on the extracellular face. In the second example, we partitioned a protein methyltransferase family using gene proximity as a criterion. In this case, the TRUE set comprised those methyltransferases encoded near the gene for the substrate RF-1. SIMBAL identifies sequence regions that map onto the substrate-binding interface while ignoring regions involved in the methyltransferase reaction mechanism in general. Neither method for training set construction requires any prior experimental characterization. CONCLUSIONS: SIMBAL shows that, in functionally divergent protein families, selected short sequences often significantly outperform their full-length parent sequence for making functional predictions by sequence similarity, suggesting avenues for improved functional classifiers. When combined with structural data, SIMBAL affords the ability to localize and model functional sites.}, keywords = {algorithms, Amino Acid Sequence, Gene Expression Profiling, Molecular Sequence Data, Phylogeny, Proteins, Sequence Analysis, Protein, Structure-Activity Relationship}, author = {J. Selengut and Rusch, Douglas B. and Haft, Daniel H.} } @article {49779, title = {Sites Inferred by Metabolic Background Assertion Labeling (SIMBAL): adapting the Partial Phylogenetic Profiling algorithm to scan sequences for signatures that predict protein function.}, journal = {BMC Bioinformatics}, volume = {11}, year = {2010}, month = {2010}, pages = {52}, abstract = {

BACKGROUND: Comparative genomics methods such as phylogenetic profiling can mine powerful inferences from inherently noisy biological data sets. We introduce Sites Inferred by Metabolic Background Assertion Labeling (SIMBAL), a method that applies the Partial Phylogenetic Profiling (PPP) approach locally within a protein sequence to discover short sequence signatures associated with functional sites. The approach is based on the basic scoring mechanism employed by PPP, namely the use of binomial distribution statistics to optimize sequence similarity cutoffs during searches of partitioned training sets.

RESULTS: Here we illustrate and validate the ability of the SIMBAL method to find functionally relevant short sequence signatures by application to two well-characterized protein families. In the first example, we partitioned a family of ABC permeases using a metabolic background property (urea utilization). Thus, the TRUE set for this family comprised members whose genome of origin encoded a urea utilization system. By moving a sliding window across the sequence of a permease, and searching each subsequence in turn against the full set of partitioned proteins, the method found which local sequence signatures best correlated with the urea utilization trait. Mapping of SIMBAL "hot spots" onto crystal structures of homologous permeases reveals that the significant sites are gating determinants on the cytosolic face rather than, say, docking sites for the substrate-binding protein on the extracellular face. In the second example, we partitioned a protein methyltransferase family using gene proximity as a criterion. In this case, the TRUE set comprised those methyltransferases encoded near the gene for the substrate RF-1. SIMBAL identifies sequence regions that map onto the substrate-binding interface while ignoring regions involved in the methyltransferase reaction mechanism in general. Neither method for training set construction requires any prior experimental characterization.

CONCLUSIONS: SIMBAL shows that, in functionally divergent protein families, selected short sequences often significantly outperform their full-length parent sequence for making functional predictions by sequence similarity, suggesting avenues for improved functional classifiers. When combined with structural data, SIMBAL affords the ability to localize and model functional sites.

}, keywords = {algorithms, Amino Acid Sequence, Gene Expression Profiling, Molecular Sequence Data, Phylogeny, Proteins, Sequence Analysis, Protein, Structure-Activity Relationship}, issn = {1471-2105}, doi = {10.1186/1471-2105-11-52}, author = {Selengut, Jeremy D and Rusch, Douglas B and Haft, Daniel H} } @article {49778, title = {Unexpected abundance of coenzyme F(420)-dependent enzymes in Mycobacterium tuberculosis and other actinobacteria.}, journal = {J Bacteriol}, volume = {192}, year = {2010}, month = {2010 Nov}, pages = {5788-98}, abstract = {

Regimens targeting Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), require long courses of treatment and a combination of three or more drugs. An increase in drug-resistant strains of M. tuberculosis demonstrates the need for additional TB-specific drugs. A notable feature of M. tuberculosis is coenzyme F(420), which is distributed sporadically and sparsely among prokaryotes. This distribution allows for comparative genomics-based investigations. Phylogenetic profiling (comparison of differential gene content) based on F(420) biosynthesis nominated many actinobacterial proteins as candidate F(420)-dependent enzymes. Three such families dominated the results: the luciferase-like monooxygenase (LLM), pyridoxamine 5{\textquoteright}-phosphate oxidase (PPOX), and deazaflavin-dependent nitroreductase (DDN) families. The DDN family was determined to be limited to F(420)-producing species. The LLM and PPOX families were observed in F(420)-producing species as well as species lacking F(420) but were particularly numerous in many actinobacterial species, including M. tuberculosis. Partitioning the LLM and PPOX families based on an organism{\textquoteright}s ability to make F(420) allowed the application of the SIMBAL (sites inferred by metabolic background assertion labeling) profiling method to identify F(420)-correlated subsequences. These regions were found to correspond to flavonoid cofactor binding sites. Significantly, these results showed that M. tuberculosis carries at least 28 separate F(420)-dependent enzymes, most of unknown function, and a paucity of flavin mononucleotide (FMN)-dependent proteins in these families. While prevalent in mycobacteria, markers of F(420) biosynthesis appeared to be absent from the normal human gut flora. These findings suggest that M. tuberculosis relies heavily on coenzyme F(420) for its redox reactions. This dependence and the cofactor{\textquoteright}s rarity may make F(420)-related proteins promising drug targets.

}, keywords = {Actinobacteria, Amino Acid Sequence, Binding Sites, Coenzymes, Flavonoids, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Genome, Bacterial, molecular biology, Molecular Sequence Data, Molecular Structure, Mycobacterium tuberculosis, Phylogeny, Protein Conformation, Riboflavin}, issn = {1098-5530}, doi = {10.1128/JB.00425-10}, author = {Selengut, Jeremy D and Haft, Daniel H} } @article {38556, title = {Unexpected abundance of coenzyme F(420)-dependent enzymes in Mycobacterium tuberculosis and other actinobacteria}, journal = {Journal of bacteriologyJournal of bacteriology}, volume = {192}, year = {2010}, note = {http://www.ncbi.nlm.nih.gov/pubmed/20675471?dopt=Abstract}, type = {10.1128/JB.00425-10}, abstract = {Regimens targeting Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), require long courses of treatment and a combination of three or more drugs. An increase in drug-resistant strains of M. tuberculosis demonstrates the need for additional TB-specific drugs. A notable feature of M. tuberculosis is coenzyme F(420), which is distributed sporadically and sparsely among prokaryotes. This distribution allows for comparative genomics-based investigations. Phylogenetic profiling (comparison of differential gene content) based on F(420) biosynthesis nominated many actinobacterial proteins as candidate F(420)-dependent enzymes. Three such families dominated the results: the luciferase-like monooxygenase (LLM), pyridoxamine 5{\textquoteright}-phosphate oxidase (PPOX), and deazaflavin-dependent nitroreductase (DDN) families. The DDN family was determined to be limited to F(420)-producing species. The LLM and PPOX families were observed in F(420)-producing species as well as species lacking F(420) but were particularly numerous in many actinobacterial species, including M. tuberculosis. Partitioning the LLM and PPOX families based on an organism{\textquoteright}s ability to make F(420) allowed the application of the SIMBAL (sites inferred by metabolic background assertion labeling) profiling method to identify F(420)-correlated subsequences. These regions were found to correspond to flavonoid cofactor binding sites. Significantly, these results showed that M. tuberculosis carries at least 28 separate F(420)-dependent enzymes, most of unknown function, and a paucity of flavin mononucleotide (FMN)-dependent proteins in these families. While prevalent in mycobacteria, markers of F(420) biosynthesis appeared to be absent from the normal human gut flora. These findings suggest that M. tuberculosis relies heavily on coenzyme F(420) for its redox reactions. This dependence and the cofactor{\textquoteright}s rarity may make F(420)-related proteins promising drug targets.}, keywords = {Actinobacteria, Amino Acid Sequence, Binding Sites, Coenzymes, Flavonoids, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Genome, Bacterial, molecular biology, Molecular Sequence Data, Molecular Structure, Mycobacterium tuberculosis, Phylogeny, Protein Conformation, Riboflavin}, author = {J. Selengut and Haft, Daniel H.} } @article {49644, title = {Genomic organization and expression profile of the mucin-associated surface protein (masp) family of the human pathogen Trypanosoma cruzi.}, journal = {Nucleic Acids Res}, volume = {37}, year = {2009}, month = {2009 Jun}, pages = {3407-17}, abstract = {

A novel large multigene family was recently identified in the human pathogen Trypanosoma cruzi, causative agent of Chagas disease, and corresponds to approximately 6\% of the parasite diploid genome. The predicted gene products, mucin-associated surface proteins (MASPs), are characterized by highly conserved N- and C-terminal domains and a strikingly variable and repetitive central region. We report here an analysis of the genomic organization and expression profile of masp genes. Masps are not randomly distributed throughout the genome but instead are clustered with genes encoding mucin and other surface protein families. Masp transcripts vary in size, are preferentially expressed during the trypomastigote stage and contain highly conserved 5{\textquoteright} and 3{\textquoteright} untranslated regions. A sequence analysis of a trypomastigote cDNA library reveals the expression of multiple masp variants with a bias towards a particular masp subgroup. Immunofluorescence assays using antibodies generated against a MASP peptide reveals that the expression of particular MASPs at the cell membrane is limited to subsets of the parasite population. Western blots of phosphatidylinositol-specific phospholipase C (PI-PLC)-treated parasites suggest that MASP may be GPI-anchored and shed into the medium culture, thus contributing to the large repertoire of parasite polypeptides that are exposed to the host immune system.

}, keywords = {3{\textquoteright} Flanking Region, 5{\textquoteright} Flanking Region, Amino Acid Sequence, Animals, Base Sequence, Conserved Sequence, Gene Expression Profiling, Genes, Protozoan, Genome, Protozoan, Membrane Proteins, Molecular Sequence Data, Mucins, Multigene Family, Protozoan Proteins, RNA, Messenger, Trypanosoma cruzi}, issn = {1362-4962}, doi = {10.1093/nar/gkp172}, author = {Bartholomeu, Daniella C and Cerqueira, Gustavo C and Le{\~a}o, Ana Carolina A and daRocha, Wanderson D and Pais, Fabiano S and Macedo, Camila and Djikeng, Appolinaire and Teixeira, Santuza M R and El-Sayed, Najib M} } @article {49675, title = {Two alternatively spliced isoforms of the Arabidopsis SR45 protein have distinct roles during normal plant development.}, journal = {Plant Physiol}, volume = {150}, year = {2009}, month = {2009 Jul}, pages = {1450-8}, abstract = {

The serine-arginine-rich (SR) proteins constitute a conserved family of pre-mRNA splicing factors. In Arabidopsis (Arabidopsis thaliana), they are encoded by 19 genes, most of which are themselves alternatively spliced. In the case of SR45, the use of alternative 3{\textquoteright} splice sites 21 nucleotides apart generates two alternatively spliced isoforms. Isoform 1 (SR45.1) has an insertion relative to isoform 2 (SR45.2) that replaces a single arginine with eight amino acids (TSPQRKTG). The biological implications of SR45 alternative splicing have been unclear. A previously described loss-of-function mutant affecting both isoforms, sr45-1, shows several developmental defects, including defects in petal development and root growth. We found that the SR45 promoter is highly active in regions with actively growing and dividing cells. We also tested the ability of each SR45 isoform to complement the sr45-1 mutant by overexpression of isoform-specific green fluorescent protein (GFP) fusion proteins. As expected, transgenic plants overexpressing either isoform displayed both nuclear speckles and GFP fluorescence throughout the nucleoplasm. We found that SR45.1-GFP complements the flower petal phenotype, but not the root growth phenotype. Conversely, SR45.2-GFP complements root growth but not floral morphology. Mutation of a predicted phosphorylation site within the alternatively spliced segment, SR45.1-S219A-GFP, does not affect complementation. However, a double mutation affecting both serine-219 and the adjacent threonine-218 (SR45.1-T218A + S219A-GFP) behaves like isoform 2, complementing the root but not the floral phenotype. In conclusion, our study provides evidence that the two alternatively spliced isoforms of SR45 have distinct biological functions.

}, keywords = {Alternative Splicing, Amino Acid Sequence, Arabidopsis, Arabidopsis Proteins, Carrier Proteins, Flowers, Molecular Sequence Data, Mutation, Plant Roots, Protein Isoforms, Ribonucleoproteins, RNA-Binding Proteins, sequence alignment}, issn = {0032-0889}, doi = {10.1104/pp.109.138180}, author = {Zhang, Xiao-Ning and Mount, Stephen M} } @article {38247, title = {Exopolysaccharide-associated protein sorting in environmental organisms: the PEP-CTERM/EpsH system. Application of a novel phylogenetic profiling heuristic}, journal = {BMC biologyBMC biology}, volume = {4}, year = {2006}, note = {http://www.ncbi.nlm.nih.gov/pubmed/16930487?dopt=Abstract}, type = {10.1186/1741-7007-4-29}, abstract = {BACKGROUND: Protein translocation to the proper cellular destination may be guided by various classes of sorting signals recognizable in the primary sequence. Detection in some genomes, but not others, may reveal sorting system components by comparison of the phylogenetic profile of the class of sorting signal to that of various protein families. RESULTS: We describe a short C-terminal homology domain, sporadically distributed in bacteria, with several key characteristics of protein sorting signals. The domain includes a near-invariant motif Pro-Glu-Pro (PEP). This possible recognition or processing site is followed by a predicted transmembrane helix and a cluster rich in basic amino acids. We designate this domain PEP-CTERM. It tends to occur multiple times in a genome if it occurs at all, with a median count of eight instances; Verrucomicrobium spinosum has sixty-five. PEP-CTERM-containing proteins generally contain an N-terminal signal peptide and exhibit high diversity and little homology to known proteins. All bacteria with PEP-CTERM have both an outer membrane and exopolysaccharide (EPS) production genes. By a simple heuristic for screening phylogenetic profiles in the absence of pre-formed protein families, we discovered that a homolog of the membrane protein EpsH (exopolysaccharide locus protein H) occurs in a species when PEP-CTERM domains are found. The EpsH family contains invariant residues consistent with a transpeptidase function. Most PEP-CTERM proteins are encoded by single-gene operons preceded by large intergenic regions. In the Proteobacteria, most of these upstream regions share a DNA sequence, a probable cis-regulatory site that contains a sigma-54 binding motif. The phylogenetic profile for this DNA sequence exactly matches that of three proteins: a sigma-54-interacting response regulator (PrsR), a transmembrane histidine kinase (PrsK), and a TPR protein (PrsT). CONCLUSION: These findings are consistent with the hypothesis that PEP-CTERM and EpsH form a protein export sorting system, analogous to the LPXTG/sortase system of Gram-positive bacteria, and correlated to EPS expression. It occurs preferentially in bacteria from sediments, soils, and biofilms. The novel method that led to these findings, partial phylogenetic profiling, requires neither global sequence clustering nor arbitrary similarity cutoffs and appears to be a rapid, effective alternative to other profiling methods.}, keywords = {Amino Acid Motifs, Amino Acid Sequence, bacteria, Bacterial Proteins, Biofilms, Genome, Bacterial, Markov chains, Molecular Sequence Data, Phylogeny, Polysaccharides, Bacterial, Protein Sorting Signals, Protein Transport, Seawater, sequence alignment, Soil Microbiology}, author = {Haft, Daniel H. and Paulsen, Ian T. and Ward, Naomi and J. Selengut} } @article {49640, title = {Trypanosoma cruzi mitochondrial maxicircles display species- and strain-specific variation and a conserved element in the non-coding region.}, journal = {BMC Genomics}, volume = {7}, year = {2006}, month = {2006}, pages = {60}, abstract = {

BACKGROUND: The mitochondrial DNA of kinetoplastid flagellates is distinctive in the eukaryotic world due to its massive size, complex form and large sequence content. Comprised of catenated maxicircles that contain rRNA and protein-coding genes and thousands of heterogeneous minicircles encoding small guide RNAs, the kinetoplast network has evolved along with an extreme form of mRNA processing in the form of uridine insertion and deletion RNA editing. Many maxicircle-encoded mRNAs cannot be translated without this post-transcriptional sequence modification.

RESULTS: We present the complete sequence and annotation of the Trypanosoma cruzi maxicircles for the CL Brener and Esmeraldo strains. Gene order is syntenic with Trypanosoma brucei and Leishmania tarentolae maxicircles. The non-coding components have strain-specific repetitive regions and a variable region that is unique for each strain with the exception of a conserved sequence element that may serve as an origin of replication, but shows no sequence identity with L. tarentolae or T. brucei. Alternative assemblies of the variable region demonstrate intra-strain heterogeneity of the maxicircle population. The extent of mRNA editing required for particular genes approximates that seen in T. brucei. Extensively edited genes were more divergent among the genera than non-edited and rRNA genes. Esmeraldo contains a unique 236-bp deletion that removes the 5{\textquoteright}-ends of ND4 and CR4 and the intergenic region. Esmeraldo shows additional insertions and deletions outside of areas edited in other species in ND5, MURF1, and MURF2, while CL Brener has a distinct insertion in MURF2.

CONCLUSION: The CL Brener and Esmeraldo maxicircles represent two of three previously defined maxicircle clades and promise utility as taxonomic markers. Restoration of the disrupted reading frames might be accomplished by strain-specific RNA editing. Elements in the non-coding region may be important for replication, transcription, and anchoring of the maxicircle within the kinetoplast network.

}, keywords = {Amino Acid Sequence, Animals, Animals, Inbred Strains, Base Composition, Conserved Sequence, DNA, Kinetoplast, Frameshifting, Ribosomal, Gene Deletion, Gene Order, Genetic Variation, Leishmania, Models, Biological, Molecular Sequence Data, Muscle Proteins, NADH Dehydrogenase, Open Reading Frames, Regulatory Elements, Transcriptional, RNA Editing, Sequence Homology, Amino Acid, Species Specificity, Trypanosoma brucei brucei, Trypanosoma cruzi, Ubiquitin-Protein Ligases, Untranslated Regions}, issn = {1471-2164}, doi = {10.1186/1471-2164-7-60}, author = {Westenberger, Scott J and Cerqueira, Gustavo C and El-Sayed, Najib M and Zingales, Bianca and Campbell, David A and Sturm, Nancy R} } @article {38287, title = {Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: implications for the microbial "pan-genome"}, journal = {Proceedings of the National Academy of Sciences of the United States of AmericaProceedings of the National Academy of Sciences of the United States of America}, volume = {102}, year = {2005}, note = {http://www.ncbi.nlm.nih.gov/pubmed/16172379?dopt=Abstract}, type = {10.1073/pnas.0506758102}, abstract = {The development of efficient and inexpensive genome sequencing methods has revolutionized the study of human bacterial pathogens and improved vaccine design. Unfortunately, the sequence of a single genome does not reflect how genetic variability drives pathogenesis within a bacterial species and also limits genome-wide screens for vaccine candidates or for antimicrobial targets. We have generated the genomic sequence of six strains representing the five major disease-causing serotypes of Streptococcus agalactiae, the main cause of neonatal infection in humans. Analysis of these genomes and those available in databases showed that the S. agalactiae species can be described by a pan-genome consisting of a core genome shared by all isolates, accounting for approximately 80\% of any single genome, plus a dispensable genome consisting of partially shared and strain-specific genes. Mathematical extrapolation of the data suggests that the gene reservoir available for inclusion in the S. agalactiae pan-genome is vast and that unique genes will continue to be identified even after sequencing hundreds of genomes.}, keywords = {Amino Acid Sequence, Bacterial Capsules, Base Sequence, Gene expression, Genes, Bacterial, Genetic Variation, Genome, Bacterial, Molecular Sequence Data, Phylogeny, sequence alignment, Sequence Analysis, DNA, Streptococcus agalactiae, virulence}, author = {Tettelin, Herv{\'e} and Masignani, Vega and Cieslewicz, Michael J. and Donati, Claudio and Medini, Duccio and Ward, Naomi L. and Angiuoli, Samuel V. and Crabtree, Jonathan and Jones, Amanda L. and Durkin, A. Scott and DeBoy, Robert T. and Davidsen, Tanja M. and Mora, Marirosa and Scarselli, Maria and Margarit y Ros, Immaculada and Peterson, Jeremy D. and Hauser, Christopher R. and Sundaram, Jaideep P. and Nelson, William C. and Madupu, Ramana and Brinkac, Lauren M. and Dodson, Robert J. and Rosovitz, Mary J. and Sullivan, Steven A. and Daugherty, Sean C. and Haft, Daniel H. and J. Selengut and Gwinn, Michelle L. and Zhou, Liwei and Zafar, Nikhat and Khouri, Hoda and Radune, Diana and Dimitrov, George and Watkins, Kisha and O{\textquoteright}Connor, Kevin J. B. and Smith, Shannon and Utterback, Teresa R. and White, Owen and Rubens, Craig E. and Grandi, Guido and Madoff, Lawrence C. and Kasper, Dennis L. and Telford, John L. and Wessels, Michael R. and Rappuoli, Rino and Fraser, Claire M.} } @article {49636, title = {Telomere and subtelomere of Trypanosoma cruzi chromosomes are enriched in (pseudo)genes of retrotransposon hot spot and trans-sialidase-like gene families: the origins of T. cruzi telomeres.}, journal = {Gene}, volume = {346}, year = {2005}, month = {2005 Feb 14}, pages = {153-61}, abstract = {

Here, we sequenced two large telomeric regions obtained from the pathogen protozoan Trypanosoma cruzi. These sequences, together with in silico assembled contigs, allowed us to establish the general features of telomeres and subtelomeres of this parasite. Our findings can be summarized as follows: We confirmed the presence of two types of telomeric ends; subtelomeric regions appeared to be enriched in (pseudo)genes of RHS (retrotransposon hot spot), TS (trans-sialidase)-like proteins, and putative surface protein DGF-1 (dispersed gene family-1). Sequence analysis of the ts-like genes located at the telomeres suggested that T. cruzi chromosomal ends could have been the site for generation of new gp85 variants, an important adhesin molecule involved in the invasion of mammalian cells by T. cruzi. Finally, a mechanism for generation of T. cruzi telomere by chromosome breakage and telomere healing is proposed.

}, keywords = {Amino Acid Sequence, Animals, Base Sequence, Chromosomes, Chromosomes, Artificial, Bacterial, DNA, Protozoan, Genes, Protozoan, Glycoproteins, Molecular Sequence Data, Multigene Family, Neuraminidase, Pseudogenes, Retroelements, Sequence Homology, Amino Acid, Sequence Homology, Nucleic Acid, Telomere, Trypanosoma cruzi}, issn = {0378-1119}, doi = {10.1016/j.gene.2004.10.014}, author = {Kim, Dong and Chiurillo, Miguel Angel and El-Sayed, Najib and Jones, Kristin and Santos, M{\'a}rcia R M and Porcile, Patricio E and Andersson, Bj{\"o}rn and Myler, Peter and da Silveira, Jose Franco and Ram{\'\i}rez, Jos{\'e} Luis} } @article {49684, title = {The Drosophila U1-70K protein is required for viability, but its arginine-rich domain is dispensable.}, journal = {Genetics}, volume = {168}, year = {2004}, month = {2004 Dec}, pages = {2059-65}, abstract = {

The conserved spliceosomal U1-70K protein is thought to play a key role in RNA splicing by linking the U1 snRNP particle to regulatory RNA-binding proteins. Although these protein interactions are mediated by repeating units rich in arginines and serines (RS domains) in vitro, tests of this domain{\textquoteright}s importance in intact multicellular organisms have not been carried out. Here we report a comprehensive genetic analysis of U1-70K function in Drosophila. Consistent with the idea that U1-70K is an essential splicing factor, we find that loss of U1-70K function results in lethality during embryogenesis. Surprisingly, and contrary to the current view of U1-70K function, animals carrying a mutant U1-70K protein lacking the arginine-rich domain, which includes two embedded sets of RS dipeptide repeats, have no discernible mutant phenotype. Through double-mutant studies, however, we show that the U1-70K RS domain deletion no longer supports viability when combined with a viable mutation in another U1 snRNP component. Together our studies demonstrate that while the protein interactions mediated by the U1-70K RS domain are not essential for viability, they nevertheless contribute to an essential U1 snRNP function.

}, keywords = {Amino Acid Sequence, Animals, Animals, Genetically Modified, Arginine, Drosophila, Drosophila Proteins, Molecular Sequence Data, Mutation, Protein Structure, Tertiary, Ribonucleoprotein, U1 Small Nuclear, RNA-Binding Proteins}, issn = {0016-6731}, doi = {10.1534/genetics.104.032532}, author = {Salz, Helen K and Mancebo, Ricardo S Y and Nagengast, Alexis A and Speck, Olga and Psotka, Mitchell and Mount, Stephen M} } @article {49634, title = {The ingi and RIME non-LTR retrotransposons are not randomly distributed in the genome of Trypanosoma brucei.}, journal = {Mol Biol Evol}, volume = {21}, year = {2004}, month = {2004 Mar}, pages = {520-8}, abstract = {

The ingi (long and autonomous) and RIME (short and nonautonomous) non--long-terminal repeat retrotransposons are the most abundant mobile elements characterized to date in the genome of the African trypanosome Trypanosoma brucei. These retrotransposons were thought to be randomly distributed, but a detailed and comprehensive analysis of their genomic distribution had not been performed until now. To address this question, we analyzed the ingi/RIME sequences and flanking sequences from the ongoing T. brucei genome sequencing project (TREU927/4 strain). Among the 81 ingi/RIME elements analyzed, 60\% are complete, and 7\% of the ingi elements (approximately 15 copies per haploid genome) appear to encode for their own transposition. The size of the direct repeat flanking the ingi/RIME retrotransposons is conserved (i.e., 12-bp), and a strong 11-bp consensus pattern precedes the 5{\textquoteright}-direct repeat. The presence of a consensus pattern upstream of the retroelements was confirmed by the analysis of the base occurrence in 294 GSS containing 5{\textquoteright}-adjacent ingi/RIME sequences. The conserved sequence is present upstream of ingis and RIMEs, suggesting that ingi-encoded enzymatic activities are used for retrotransposition of RIMEs, which are short nonautonomous retroelements. In conclusion, the ingi and RIME retroelements are not randomly distributed in the genome of T. brucei and are preceded by a conserved sequence, which may be the recognition site of the ingi-encoded endonuclease.

}, keywords = {Amino Acid Sequence, Animals, Base Sequence, Consensus Sequence, Genome, Protozoan, Molecular Sequence Data, Retroelements, Sequence Analysis, Trypanosoma brucei brucei}, issn = {0737-4038}, doi = {10.1093/molbev/msh045}, author = {Bringaud, Frederic and Biteau, Nicolas and Zuiderwijk, Eduard and Berriman, Matthew and El-Sayed, Najib M and Ghedin, Elodie and Melville, Sara E and Hall, Neil and Baltz, Th{\'e}o} } @article {38578, title = {X-ray crystal structure of the hypothetical phosphotyrosine phosphatase MDP-1 of the haloacid dehalogenase superfamily}, journal = {BiochemistryBiochemistry}, volume = {43}, year = {2004}, note = {http://www.ncbi.nlm.nih.gov/pubmed/15461449?dopt=Abstract}, type = {10.1021/bi0490688}, abstract = {The haloacid dehalogenase (HAD) superfamily is comprised of structurally homologous enzymes that share several conserved sequence motifs (loops I-IV) in their active site. The majority of HAD members are phosphohydrolases and may be divided into three subclasses depending on domain organization. In classes I and II, a mobile "cap" domain reorients upon substrate binding, closing the active site to bulk solvent. Members of the third class lack this additional domain. Herein, we report the 1.9 A X-ray crystal structures of a member of the third subclass, magnesium-dependent phosphatase-1 (MDP-1) both in its unliganded form and with the product analogue, tungstate, bound to the active site. The secondary structure of MDP-1 is similar to that of the "core" domain of other type I and type II HAD members with the addition of a small, 28-amino acid insert that does not close down to exclude bulk solvent in the presence of ligand. In addition, the monomeric oligomeric state of MDP-1 does not allow the participation of a second subunit in the formation and solvent protection of the active site. The binding sites for the phosphate portion of the substrate and Mg(II) cofactor are also similar to those of other HAD members, with all previously observed contacts conserved. Unlike other subclass III HAD members, MDP-1 appears to be equally able to dephosphorylate phosphotyrosine and closed-ring phosphosugars. Modeling of possible substrates in the active site of MDP-1 reveals very few potential interactions with the substrate leaving group. The mapping of conserved residues in sequences of MDP-1 from different eukaryotic organisms reveals that they colocalize to a large region on the surface of the protein outside the active site. This observation combined with the modeling studies suggests that the target of MDP-1 is most likely a phosphotyrosine in an unknown protein rather than a small sugar-based substrate.}, keywords = {Amino Acid Sequence, Animals, Binding Sites, Crystallography, X-Ray, HUMANS, Hydrogen-Ion Concentration, Hydrolases, Magnesium, Mice, Models, Molecular, Molecular Sequence Data, Phosphoprotein Phosphatases, Phosphotyrosine, Protein Phosphatase 1, Protein Structure, Quaternary, Protein Structure, Tertiary, sequence alignment, Solvents, Substrate Specificity}, author = {Peisach, Ezra and J. Selengut and Dunaway-Mariano, Debra and Allen, Karen N.} } @article {49686, title = {Sex-lethal splicing autoregulation in vivo: interactions between SEX-LETHAL, the U1 snRNP and U2AF underlie male exon skipping.}, journal = {Development}, volume = {130}, year = {2003}, month = {2003 Feb}, pages = {463-71}, abstract = {

Alternative splicing of the Sex-lethal pre-mRNA has long served as a model example of a regulated splicing event, yet the mechanism by which the female-specific SEX-LETHAL RNA-binding protein prevents inclusion of the translation-terminating male exon is not understood. Thus far, the only general splicing factor for which there is in vivo evidence for a regulatory role in the pathway leading to male-exon skipping is sans-fille (snf), a protein component of the spliceosomal U1 and U2 snRNPs. Its role, however, has remained enigmatic because of questions about whether SNF acts as part of an intact snRNP or a free protein. We provide evidence that SEX-LETHAL interacts with SANS-FILLE in the context of the U1 snRNP, through the characterization of a point mutation that interferes with both assembly into the U1 snRNP and complex formation with SEX-LETHAL. Moreover, we find that SEX-LETHAL associates with other integral U1 snRNP components, and we provide genetic evidence to support the biological relevance of these physical interactions. Similar genetic and biochemical approaches also link SEX-LETHAL with the heterodimeric splicing factor, U2AF. These studies point specifically to a mechanism by which SEX-LETHAL represses splicing by interacting with these key splicing factors at both ends of the regulated male exon. Moreover, because U2AF and the U1 snRNP are only associated transiently with the pre-mRNA during the course of spliceosome assembly, our studies are difficult to reconcile with the current model that proposes that the SEX-LETHAL blocks splicing at the second catalytic step, and instead argue that the SEX-LETHAL protein acts after splice site recognition, but before catalysis begins.

}, keywords = {Alternative Splicing, Amino Acid Sequence, Animals, Animals, Genetically Modified, Drosophila melanogaster, Drosophila Proteins, Exons, Female, Gene Expression Regulation, Developmental, Genes, Insect, Homeostasis, Male, Models, Genetic, Molecular Sequence Data, Nuclear Proteins, Point Mutation, Ribonucleoprotein, U1 Small Nuclear, Ribonucleoproteins, RNA Splicing, RNA-Binding Proteins, Sequence Homology, Amino Acid, Sex Differentiation}, issn = {0950-1991}, author = {Nagengast, Alexis A and Stitzinger, Shane M and Tseng, Chin-Hsiu and Mount, Stephen M and Salz, Helen K} } @article {38536, title = {The transcription factor Eyes absent is a protein tyrosine phosphatase}, journal = {NatureNature}, volume = {426}, year = {2003}, note = {http://www.ncbi.nlm.nih.gov/pubmed/14628053?dopt=Abstract}, type = {10.1038/nature02097}, abstract = {Post-translational modifications provide sensitive and flexible mechanisms to dynamically modulate protein function in response to specific signalling inputs. In the case of transcription factors, changes in phosphorylation state can influence protein stability, conformation, subcellular localization, cofactor interactions, transactivation potential and transcriptional output. Here we show that the evolutionarily conserved transcription factor Eyes absent (Eya) belongs to the phosphatase subgroup of the haloacid dehalogenase (HAD) superfamily, and propose a function for it as a non-thiol-based protein tyrosine phosphatase. Experiments performed in cultured Drosophila cells and in vitro indicate that Eyes absent has intrinsic protein tyrosine phosphatase activity and can autocatalytically dephosphorylate itself. Confirming the biological significance of this function, mutations that disrupt the phosphatase active site severely compromise the ability of Eyes absent to promote eye specification and development in Drosophila. Given the functional importance of phosphorylation-dependent modulation of transcription factor activity, this evidence for a nuclear transcriptional coactivator with intrinsic phosphatase activity suggests an unanticipated method of fine-tuning transcriptional regulation.}, keywords = {Amino Acid Motifs, Amino Acid Sequence, Animals, Antibodies, Phospho-Specific, Drosophila melanogaster, Drosophila Proteins, Embryonic Induction, eye, Eye Proteins, Gene Expression Regulation, Kinetics, Mice, Models, Molecular, Molecular Sequence Data, Mutation, Phosphorylation, Protein Conformation, Protein Tyrosine Phosphatases, Substrate Specificity, Transcription Factors}, author = {Tootle, Tina L. and Silver, Serena J. and Davies, Erin L. and Newman, Victoria and Latek, Robert R. and Mills, Ishara A. and J. Selengut and Parlikar, Beth E. W. and Rebay, Ilaria} } @article {49688, title = {Evidence for a plastid origin of plant ethylene receptor genes.}, journal = {Plant Physiol}, volume = {130}, year = {2002}, month = {2002 Sep}, pages = {10-4}, keywords = {Amino Acid Sequence, Anabaena, Arabidopsis, Cyanobacteria, Molecular Sequence Data, Plant Proteins, Plastids, Protein Kinases, Receptors, Cell Surface, Sequence Homology, Amino Acid}, issn = {0032-0889}, doi = {10.1104/pp.005397}, author = {Mount, Stephen M and Chang, Caren} } @article {49631, title = {A new, expressed multigene family containing a hot spot for insertion of retroelements is associated with polymorphic subtelomeric regions of Trypanosoma brucei.}, journal = {Eukaryot Cell}, volume = {1}, year = {2002}, month = {2002 Feb}, pages = {137-51}, abstract = {

We describe a novel gene family that forms clusters in subtelomeric regions of Trypanosoma brucei chromosomes and partially accounts for the observed clustering of retrotransposons. The ingi and ribosomal inserted mobile element (RIME) non-LTR retrotransposons share 250 bp at both extremities and are the most abundant putatively mobile elements, with about 500 copies per haploid genome. From cDNA clones and subsequently in the T. brucei genomic DNA databases, we identified 52 homologous gene and pseudogene sequences, 16 of which contain a RIME and/or ingi retrotransposon inserted at exactly the same relative position. Here these genes are called the RHS family, for retrotransposon hot spot. Comparison of the protein sequences encoded by RHS genes (21 copies) and pseudogenes (24 copies) revealed a conserved central region containing an ATP/GTP-binding motif and the RIME/ingi insertion site. The RHS proteins share between 13 and 96\% identity, and six subfamilies, RHS1 to RHS6, can be defined on the basis of their divergent C-terminal domains. Immunofluorescence and Western blot analyses using RHS subfamily-specific immune sera show that RHS proteins are constitutively expressed and occur mainly in the nucleus. Analysis of Genome Survey Sequence databases indicated that the Trypanosoma brucei diploid genome contains about 280 RHS (pseudo)genes. Among the 52 identified RHS (pseudo)genes, 48 copies are in three RHS clusters located in subtelomeric regions of chromosomes Ia and II and adjacent to the active bloodstream form expression site in T. brucei strain TREU927/4 GUTat10.1. RHS genes comprise the remaining sequence of the size-polymorphic "repetitive region" described for T. brucei chromosome I, and a homologous gene family is present in the Trypanosoma cruzi genome.

}, keywords = {Amino Acid Sequence, Animals, Base Sequence, Cloning, Molecular, DNA Primers, DNA, Protozoan, Escherichia coli, Genes, Protozoan, Molecular Sequence Data, Multigene Family, Mutagenesis, Insertional, Phylogeny, Polymorphism, Genetic, Protozoan Proteins, Pseudogenes, Retroelements, sequence alignment, Sequence Homology, Amino Acid, Telomere, Trypanosoma brucei brucei, Trypanosoma cruzi}, issn = {1535-9778}, author = {Bringaud, Frederic and Biteau, Nicolas and Melville, Sara E and Hez, St{\'e}phanie and El-Sayed, Najib M and Leech, Vanessa and Berriman, Matthew and Hall, Neil and Donelson, John E and Baltz, Th{\'e}o} } @article {38368, title = {MDP-1 is a new and distinct member of the haloacid dehalogenase family of aspartate-dependent phosphohydrolases}, journal = {BiochemistryBiochemistry}, volume = {40}, year = {2001}, note = {http://www.ncbi.nlm.nih.gov/pubmed/11601995?dopt=Abstract}, abstract = {MDP-1 is a eukaryotic magnesium-dependent acid phosphatase with little sequence homology to previously characterized phosphatases. The presence of a conserved motif (Asp-X-Asp-X-Thr) in the N terminus of MDP-1 suggested a relationship to the haloacid dehalogenase (HAD) superfamily, which contains a number of magnesium-dependent acid phosphatases. These phosphatases utilize an aspartate nucleophile and contain a number of conserved active-site residues and hydrophobic patches, which can be plausibly aligned with conserved residues in MDP-1. Seven site-specific point mutants of MDP-1 were produced by modifying the catalytic aspartate, serine, and lysine residues to asparagine or glutamate, alanine, and arginine, respectively. The activity of these mutants confirms the assignment of MDP-1 as a member of the HAD superfamily. Detailed comparison of the sequence of the 15 MDP-1 sequences from various organisms with other HAD superfamily sequences suggests that MDP-1 is not closely related to any particular member of the superfamily. The crystal structures of several HAD family enzymes identify a domain proximal to the active site responsible for important interactions with low molecular weight substrates. The absence of this domain or any other that might perform the same function in MDP-1 suggests an "open" active site capable of interactions with large substrates such as proteins. This suggestion was experimentally confirmed by demonstration that MDP-1 is competent to catalyze the dephosphorylation of tyrosine-phosphorylated proteins.}, keywords = {Amino Acid Motifs, Amino Acid Sequence, Animals, Aspartic Acid, Catalytic Domain, HUMANS, Hydrolases, Mice, Molecular Sequence Data, Multigene Family, Mutagenesis, Site-Directed, Phosphoprotein Phosphatases, Protein Structure, Tertiary, Protein Tyrosine Phosphatases, Rats, Saccharomyces cerevisiae, sequence alignment, Sequence Homology, Amino Acid}, author = {J. Selengut} } @article {38369, title = {MDP-1: A novel eukaryotic magnesium-dependent phosphatase}, journal = {BiochemistryBiochemistry}, volume = {39}, year = {2000}, note = {http://www.ncbi.nlm.nih.gov/pubmed/10889041?dopt=Abstract}, abstract = {We report here the purification, cloning, expression, and characterization of a novel phosphatase, MDP-1. In the course of investigating the reported acid phosphatase activity of carbonic anhydrase III preparations, several discrete phosphatases were discerned. One of these, a magnesium-dependent species of 18.6 kDa, was purified to homogeneity and yielded several peptide sequences from which the parent gene was identified by database searching. Although orthologous genes were identified in fungi and plants as well as mammalian species, there was no apparent homology to any known family of phosphatases. The enzyme was expressed in Escherichia coli with a fusion tag and purified by affinity methods. The recombinant enzyme showed magnesium-dependent acid phosphatase activity comparable to the originally isolated rabbit protein. The enzyme catalyzes the rapid hydrolysis of p-nitrophenyl phosphate, ribose-5-phosphate, and phosphotyrosine. The selectivity for phosphotyrosine over phosphoserine or phosphothreonine is considerable, but the enzyme did not show activity toward five phosphotyrosine-containing peptides. None of the various substrates assayed (including various nucleotide, sugar, amino acid and peptide phosphates, phosphoinositides, and phosphodiesters) exhibited K(M) values lower than 1 mM, and many showed negligible rates of hydrolysis. The enzyme is inhibited by vanadate and fluoride but not by azide, cyanide, calcium, lithium, or tartaric acid. Chemical labeling, refolding, dialysis, and mutagenesis experiments suggest that the enzymatic mechanism is not dependent on cysteine, histidine, or nonmagnesium metal ions. In recognition of these observations, the enzyme has been given the name magnesium-dependent phosphatase-1 (MDP-1).}, keywords = {Amino Acid Sequence, Animals, Catalysis, Cations, Chromatography, Affinity, Cloning, Molecular, Cysteine, Enzyme Inhibitors, Histidine, Hydrogen-Ion Concentration, Magnesium, Mice, Molecular Sequence Data, Phosphoprotein Phosphatases, Protein Phosphatase 1, Rabbits, Sequence Analysis, Protein, Sequence Homology, Amino Acid, Substrate Specificity}, author = {J. Selengut and Levine, R. L.} } @article {49693, title = {Genetic depletion reveals an essential role for an SR protein splicing factor in vertebrate cells.}, journal = {Bioessays}, volume = {19}, year = {1997}, month = {1997 Mar}, pages = {189-92}, abstract = {

SR proteins are essential for the splicing of messenger RNA precursors in vitro, where they also alter splice site selection in a concentration-dependent manner. Although experiments involving overexpression or dominant mutations have confirmed that these proteins can influence RNA processing decisions in vivo, similar results with loss-of-function mutations have been lacking. Now, a system for genetic depletion of the chicken B cell line DT40 has revealed that the SR protein ASF/SF2 (alternative splicing factor/splicing factor 2) is essential for viability in these cells(1). This study opens the way for a complete functional dissection of this protein, and other SR proteins, in vivo.

}, keywords = {Amino Acid Sequence, Animals, Molecular Sequence Data, Nuclear Proteins, RNA Splicing, RNA-Binding Proteins, Serine-Arginine Splicing Factors, Vertebrates}, issn = {0265-9247}, doi = {10.1002/bies.950190302}, author = {Mount, S M} } @article {49696, title = {Genetic enhancement of RNA-processing defects by a dominant mutation in B52, the Drosophila gene for an SR protein splicing factor.}, journal = {Mol Cell Biol}, volume = {15}, year = {1995}, month = {1995 Nov}, pages = {6273-82}, abstract = {

SR proteins are essential for pre-mRNA splicing in vitro, act early in the splicing pathway, and can influence alternative splice site choice. Here we describe the isolation of both dominant and loss-of-function alleles of B52, the gene for a Drosophila SR protein. The allele B52ED was identified as a dominant second-site enhancer of white-apricot (wa), a retrotransposon insertion in the second intron of the eye pigmentation gene white with a complex RNA-processing defect. B52ED also exaggerates the mutant phenotype of a distinct white allele carrying a 5{\textquoteright} splice site mutation (wDR18), and alters the pattern of sex-specific splicing at doublesex under sensitized conditions, so that the male-specific splice is favored. In addition to being a dominant enhancer of these RNA-processing defects, B52ED is a recessive lethal allele that fails to complement other lethal alleles of B52. Comparison of B52ED with the B52+ allele from which it was derived revealed a single change in a conserved amino acid in the beta 4 strand of the first RNA-binding domain of B52, which suggests that altered RNA binding is responsible for the dominant phenotype. Reversion of the B52ED dominant allele with X rays led to the isolation of a B52 null allele. Together, these results indicate a critical role for the SR protein B52 in pre-mRNA splicing in vivo.

}, keywords = {Alleles, Amino Acid Sequence, Animals, Base Sequence, DNA Primers, Drosophila melanogaster, Drosophila Proteins, Frameshift Mutation, Genes, Dominant, Genes, Insect, Molecular Sequence Data, Nuclear Proteins, Phosphoproteins, Point Mutation, Protein Structure, Tertiary, Proteins, RNA Splicing, RNA-Binding Proteins, Sequence Deletion, Sex Determination Analysis}, issn = {0270-7306}, author = {Peng, X and Mount, S M} } @article {38336, title = {Identification of the calcium-binding protein calgranulin in the matrix of struvite stones}, journal = {Journal of endourology / Endourological SocietyJournal of endourology / Endourological Society}, volume = {8}, year = {1994}, note = {http://www.ncbi.nlm.nih.gov/pubmed/8061680?dopt=Abstract}, abstract = {The identification of calcium-binding proteins in urine and kidney stones has led to a closer look at the role of matrix proteins in urolithiasis. We analyzed five struvite stones for protein content and identified two bands (8 and 14 KDa) that were confirmed by gel electrophoresis and amino acid sequencing to be calgranulin. This protein, which is known by several other names, has bacteriostatic antifungal activity. Its role in the formation of struvite stones warrants further investigation.}, keywords = {Amino Acid Sequence, Calcium-Binding Proteins, Cell Adhesion Molecules, Neuronal, Electrophoresis, Enzyme-Linked Immunosorbent Assay, HUMANS, Kidney Calculi, Leukocyte L1 Antigen Complex, Magnesium Compounds, Molecular Sequence Data, Phosphates}, author = {Bennett, J. and Dretler, S. P. and J. Selengut and Orme-Johnson, W. H.} } @article {49707, title = {Sequence of a cDNA from the Drosophila melanogaster white gene.}, journal = {Nucleic Acids Res}, volume = {18}, year = {1990}, month = {1990 Mar 25}, pages = {1633}, keywords = {Amino Acid Sequence, Animals, Base Sequence, DNA, Drosophila melanogaster, Eye Color, genes, Molecular Sequence Data}, issn = {0305-1048}, author = {Pepling, M and Mount, S M} } @article {49706, title = {Structure and expression of the Drosophila melanogaster gene for the U1 small nuclear ribonucleoprotein particle 70K protein.}, journal = {Mol Cell Biol}, volume = {10}, year = {1990}, month = {1990 Jun}, pages = {2492-502}, abstract = {

A genomic clone encoding the Drosophila U1 small nuclear ribonucleoprotein particle 70K protein was isolated by hybridization with a human U1 small nuclear ribonucleoprotein particle 70K protein cDNA. Southern blot and in situ hybridizations showed that this U1 70K gene is unique in the Drosophila genome, residing at cytological position 27D1,2. Polyadenylated transcripts of 1.9 and 3.1 kilobases were observed. While the 1.9-kilobase mRNA is always more abundant, the ratio of these two transcripts is developmentally regulated. Analysis of cDNA and genomic sequences indicated that these two RNAs encode an identical protein with a predicted molecular weight of 52,879. Comparison of the U1 70K proteins predicted from Drosophila, human, and Xenopus cDNAs revealed 68\% amino acid identity in the most amino-terminal 214 amino acids, which include a sequence motif common to many proteins which bind RNA. The carboxy-terminal half is less well conserved but is highly charged and contains distinctive arginine-rich regions in all three species. These arginine-rich regions contain stretches of arginine-serine dipeptides like those found in transformer, transformer-2, and suppressor-of-white-apricot proteins, all of which have been identified as regulators of mRNA splicing in Drosophila melanogaster.

}, keywords = {Amino Acid Sequence, Animals, Base Sequence, Blotting, Northern, Blotting, Southern, Cloning, Molecular, DNA, Drosophila melanogaster, Gene expression, Gene Library, genes, HUMANS, Molecular Sequence Data, Molecular Weight, Oligonucleotide Probes, Poly A, Ribonucleoproteins, Ribonucleoproteins, Small Nuclear, RNA, RNA, Messenger, Sequence Homology, Nucleic Acid, Xenopus}, issn = {0270-7306}, author = {Mancebo, R and Lo, P C and Mount, S M} } @article {49709, title = {Sequence similarity.}, journal = {Nature}, volume = {325}, year = {1987}, month = {1987 Feb 5-11}, pages = {487}, keywords = {Adenosine Triphosphate, Amino Acid Sequence, Animals, Bacterial Proteins, Biological Transport, Active, Carrier Proteins, Drosophila melanogaster, genes, HUMANS, Pigments, Biological, Sequence Homology, Nucleic Acid}, issn = {0028-0836}, doi = {10.1038/325487c0}, author = {Mount, S M} } @article {49710, title = {Complete nucleotide sequence of the Drosophila transposable element copia: homology between copia and retroviral proteins.}, journal = {Mol Cell Biol}, volume = {5}, year = {1985}, month = {1985 Jul}, pages = {1630-8}, abstract = {

We have determined the complete nucleotide sequence of the copia element present at the white-apricot allele of the white locus in Drosophila melanogaster. This transposable element is 5,146 nucleotides long and contains a single long open reading frame of 4,227 nucleotides. Analysis of the coding potential of the large open reading frame, which appears to encode a polyprotein, revealed weak homology to a number of retroviral proteins, including a protease, nucleic acid-binding protein, and reverse transcriptase. Better homology existed between another part of the copia open reading frame and a region of the retroviral pol gene recently shown to be distinct from reverse transcriptase and required for the integration of circular DNA forms of the retroviral genome to form proviruses. Comparison of the copia sequence with those of the Saccharomyces cerevisiae transposable element Ty, several vertebrate retroviruses, and the D. melanogaster copia-like element 17.6 showed that Ty was most similar to copia, sharing amino acid sequence homology and organizational features not found in the other genetic elements.

}, keywords = {Amino Acid Sequence, Animals, Base Sequence, Codon, DNA Helicases, DNA Transposable Elements, Drosophila melanogaster, Gene Expression Regulation, Gene Products, gag, Integrases, Repetitive Sequences, Nucleic Acid, Retroviridae, RNA-Directed DNA Polymerase, Viral Envelope Proteins, Viral Proteins}, issn = {0270-7306}, author = {Mount, S M and Rubin, G M} }