Genomic Analyses of the Novel Mycobacteriophage KashFlow

Eighteen new mycobacteriophages were isolated from soil samples collected around the state of Michigan and parts of the United States. All phages were capable of infecting Mycobacterium smegmatis and were isolated through either enrichment or direct plating at 32°C or 37°C. A variety of plaque morphologies were produced based on size, shape, and clarity; both lytic and temperate phages appear represented in this collection. The mycobacteriophage, KashFlow, was chosen as one of three phages for complete genome sequencing and comparative genomic analyses. The predominant plaque produced by KashFlow at 37°C was circular and 2 mm in diameter. The complete genome sequence for KashFlow revealed relationships to members of the Cluster J, which contains 30 sequenced members to date. The genome of KashFlow is 111.6 Kb, 60.2% GC, and contains 243 genes, including 2 tRNA(Lys-TTT) genes. Upon initial analysis, KashFlow shares a number of gene products with several other phages belonging to the J cluster. In a majority of these instances, these gene products are nearly identical to one another and are almost always perfectly aligned. This trend is predicted to continue throughout the entirety of the KashFlow genome as a number of cluster J phages share these same attributes. A detailed analysis of the complete genome sequences and comparison with sequenced mycobacteriophages is the subject of the second semester of this yearlong course and is presented

VPS35 Mutations in Parkinson Disease

The identification of genetic causes for Mendelian disorders has been based on the collection of multi-incident families, linkage analysis, and sequencing of genes in candidate intervals. This study describes the application of next-generation sequencing technologies to a Swiss kindred presenting with autosomal-dominant, late-onset Parkinson disease (PD). The family has tremor-predominant dopa-responsive parkinsonism with a mean onset of 50.6 ± 7.3 years. Exome analysis suggests that an aspartic-acid-to-asparagine mutation within vacuolar protein sorting 35 (VPS35 c.1858G>A; p.Asp620Asn) is the genetic determinant of disease. VPS35 is a central component of the retromer cargo-recognition complex, is critical for endosome-trans-golgi trafficking and membrane-protein recycling, and is evolutionarily highly conserved. VPS35 c.1858G>A was found in all affected members of the Swiss kindred and in three more families and one patient with sporadic PD, but it was not observed in 3,309 controls. Further sequencing of familial affected probands revealed only one other missense variant, VPS35 c.946C>T; (p.Pro316Ser), in a pedigree with one unaffected and two affected carriers, and thus the pathogenicity of this mutation remains uncertain. Retromer-mediated sorting and transport is best characterized for acid hydrolase receptors. However, the complex has many types of cargo and is involved in a diverse array of biologic pathways from developmental Wnt signaling to lysosome biogenesis. Our study implicates disruption of VPS35 and retromer-mediated trans-membrane protein sorting, rescue, and recycling in the neurodegenerative process leading to PD