Browsing by Subject "gene"

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    A computational screen for site selective A-to-I editing detects novel sites in neuron specific Hu proteins
    (2010) Ensterö, Mats; Akerborg, Orjan; Lundin, Daniel; Wang, Bei; Furey, Terrence S; Ohman, Marie; Lagergren, Jens
    Background: Several bioinformatic approaches have previously been used to find novel sites of ADAR mediated A-to-I RNA editing in human. These studies have discovered thousands of genes that are hyper-edited in their non-coding intronic regions, especially in alu retrotransposable elements, but very few substrates that are site-selectively edited in coding regions. Known RNA edited substrates suggest, however, that site selective A-to-I editing is particularly important for normal brain development in mammals. Results: We have compiled a screen that enables the identification of new sites of site-selective editing, primarily in coding sequences. To avoid hyper-edited repeat regions, we applied our screen to the alu-free mouse genome. Focusing on the mouse also facilitated better experimental verification. To identify candidate sites of RNA editing, we first performed an explorative screen based on RNA structure and genomic sequence conservation. We further evaluated the results of the explorative screen by determining which transcripts were enriched for A-G mismatches between the genomic template and the expressed sequence since the editing product, inosine (I), is read as guanosine (G) by the translational machinery. For expressed sequences, we only considered coding regions to focus entirely on re-coding events. Lastly, we refined the results from the explorative screen using a novel scoring scheme based on characteristics for known A-to-I edited sites. The extent of editing in the final candidate genes was verified using total RNA from mouse brain and 454 sequencing. Conclusions: Using this method, we identified and confirmed efficient editing at one site in the Gabra3 gene. Editing was also verified at several other novel sites within candidates predicted to be edited. Five of these sites are situated in genes coding for the neuron-specific RNA binding proteins HuB and HuD.
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    Changes in Bni4 localization induced by cell stress in Saccharomyces cerevisiae
    (2010) Larson, Jennifer R; Kozubowski, Lukasz; Tatchell, Kelly
    Septin complexes at the bud neck in Saccharomyces cerevisiae serve as a scaffold for proteins involved in signaling, cell cycle control, and cell wall synthesis. Many of these bind asymmetrically, associating with either the mother-or daughter-side of the neck. Septin structures are inherently apolar so the basis for the asymmetric binding remains unknown. Bni4, a regulatory subunit of yeast protein phosphatase type 1, Glc7, binds to the outside of the septin ring prior to bud formation and remains restricted to the mother-side of the bud neck after bud emergence. Bni4 is responsible for targeting Glc7 to the mother-side of the bud neck for proper deposition of the chitin ring. We show here that Bni4 localizes symmetrically, as two distinct rings on both sides of the bud neck following energy depletion or activation of cell cycle checkpoints. Our data indicate that loss of Bni4 asymmetry can occur via at least two different mechanisms. Furthermore, we show that Bni4 has a Swe1-dependent role in regulating the cell morphogenesis checkpoint in response to hydroxyurea, which suggests that the change in localization of Bni4 following checkpoint activation may help stabilize the cell cycle regulator Swe1 during cell cycle arrest.
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    Detection of Alternative Splice Variants at the Proteome Level in Aspergillus flavus
    (2010) Chang, Kung-Yen; Georgianna, D Ryan; Heber, Steffen; Payne, Gary A; Muddiman, David C
    Identification of proteins from proteolytic peptides or intact proteins plays an essential role in proteomics. Researchers use search engines to match the acquired peptide sequences to the target proteins. However, search engines depend on protein databases to provide candidates for consideration. Alternative splicing (AS), the mechanism where the exon of pre-mRNAs can be spliced and rearranged to generate distinct mRNA and therefore protein variants, enable higher eukaryotic organisms, with only a limited number of genes, to have the requisite complexity and diversity at the proteome level. Multiple alternative isoforms from one gene often share common segments of sequences. However, many protein databases only include a limited number of isoforms to keep minimal redundancy. As a result, the database search might not identify a target protein even with high quality tandem MS data and accurate intact precursor ion mass. We computationally predicted an exhaustive list of putative isoforms of Aspergillus flavus proteins from 20 371 expressed sequence tags to investigate whether an alternative splicing protein database can assign a greater proportion of mass spectrometry data. The newly constructed AS database provided 9807 new alternatively spliced variants in addition to 12 832 previously annotated proteins. The searches of the existing tandem MS spectra data set using the AS database identified 29 new proteins encoded by 26 genes. Nine fungal genes appeared to have multiple protein isoforms. In addition to the discovery of splice variants, AS database also showed potential to improve genome annotation. In summary, the introduction of an alternative splicing database helps identify more proteins and unveils more information about a proteome.
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    Interchangeable Domains in the Kdo Transferases of Escherichia coli and Haemophilus influenzae
    (2010) Chung, Hak Suk; Raetz, Christian RH
    Kdo(2)-lipid A, a conserved substructure of lipopolysaccharide, plays critical roles in Gram-negative bacterial survival and interaction with host organisms. Inhibition of Kdo biosynthesis in Escherichia coli results in cell death and accumulation of the tetra-acylated precursor lipid IVA. E. coil KdtA (EcKdtA) is a bifunctional enzyme that transfers two Kdo units from two CMP-Kdo molecules to lipid IVA. In contrast, Haemophilia influenzae KdtA (HiKdtA) transfers only one Kdo unit. E. coil CMR300, which lacks Kdo transferase because of a deletion in kdtA, can be rescued to grow in broth at 37 degrees C if multiple copies of msbA are provided in trans. MsbA, the inner membrane transporter for nascent lipopolysaccharide, prefers hexa-acylated to tetra-acylated lipid A, but with the excess MsbA present in CMR300, lipid IVA is efficiently exported to the outer membrane. CMR300 is hypersensitive to hydrophobic antibiotics and bile salts and does not grow at 42 degrees C. Expressing HiKdtA in CMR300 results in the accumulation of Kdo-lipid IVA in place of lipid IVA without suppression of its growth phenotypes at 30 degrees C. EcKdtA restores intact lipopolysaccharide, together with normal antibiotic resistance, detergent resistance, and growth at 42 degrees C. To determine which residues are important for the mono- or bifunctional character of KdtA, protein chimeras were constructed using EcKdtA and HiKdtA. These chimeras, which are catalytically active, were characterized by in vitro assays and in vivo complementation. The N-terminal half of KdtA, especially the first 30 amino acid residues, specifies whether one or two Kdo units are transferred to lipid IVA.