Padmanabhan Mahadevan

Evidence for a retroviral insertion in TRPM1 as the cause of congenital stationary night blindness and leopard complex spotting in the horse.

Leopard complex spotting is a group of white spotting patterns in horses caused by an incompletely dominant gene (LP) where homozygotes (LP/LP) are also affected with congenital stationary night blindness. Previous studies implicated Transient Receptor Potential Cation Channel, Subfamily M, Member 1 (TRPM1) as the best candidate gene for both CSNB and LP. RNA-Seq data pinpointed a 1378 bp insertion in intron 1 of TRPM1 as the potential cause. This insertion, a long terminal repeat (LTR) of an endogenous retrovirus, was completely associated with LP, testing 511 horses (χ2=1022.00, p<<0.0005), and CSNB, testing 43 horses (χ2=43, p<<0.0005). The LTR was shown to disrupt TRPM1 transcription by premature poly-adenylation. Furthermore, while deleterious transposable element insertions should be quickly selected against the identification of this insertion in three ancient DNA samples suggests it has been maintained in the horse gene pool for at least 17,000 years. This study represents the first description of an LTR insertion being associated with both a pigmentation phenotype and an eye disorder.

CoreGenes3.5: a webserver for the determination of core genes from sets of viral and small bacterial genomes

BackgroundCoreGenes3.5 is a webserver that determines sets of core genes from viral and small bacterial genomes as an automated batch process. Previous versions of CoreGenes have been used to classify bacteriophage genomes and mine data from pathogen genomes.FindingsCoreGenes3.5 accepts as input GenBank accession numbers of genomes and performs iterative BLASTP analyses to output a set of core genes. After completion of the program run, the results can be either displayed in a new window for one pair of reference and query genomes or emailed to the user for multiple pairs of small genomes in tabular format.ConclusionsWith the number of genomes sequenced increasing daily and interest in determining phylogenetic relationships, CoreGenes3.5 provides a user-friendly web interface for wet-bench biologists to process multiple small genomes for core gene determinations. CoreGenes3.5 is available at http://binf.gmu.edu:8080/CoreGenes3.5.

In Sffamily Identification of Genes in Bacteriophage DNA

One of the most satisfying aspects of a genome sequencing project is the identification of the genes contained within it.These are of two types: those which encode tRNAs and those which produce proteins. After a general introduction on the properties of protein-encoding genes and the utility of the Basic Local Alignment Search Tool (BLASTX) to identify genes through homologs, a variety of tools are discussed by their creators. These include for genome annotation: GeneMark, Artemis, and BASys; and, for genome comparisons: Artemis Comparison Tool (ACT), Mauve, CoreGenes, and GeneOrder.

Natural variants of human adenovirus type 3 provide evidence for relative genome stability across time and geographic space

Human adenovirus type 3 (HAdV-B3) has an apparently stable genome yet remains a major circulating and problematic respiratory pathogen. Comparisons of the prototype genome to genomes from three current field strains, including two isolated from epidemics, and a laboratory strain, yielded small-scale nucleotide variations across 50 years of time and space (U.S. and China). This is in contrast to the recombination events that have been reported recently for HAdV genomes. Recombinant genomes have been identified in emergent HAdV pathogens and is a pathway for the molecular evolution of types. These two contrasting views of HAdV genome stability have repercussions in the development and use of vaccines for countering HAdV-B3, as well as in the continued effectiveness of vaccines developed against earlier and current circulating types of HAdV.

Integration of genomic and proteomic analyses in the classification of the Siphoviridae family

Using a variety of genomic (BLASTN, ClustalW) and proteomic (Phage Proteomic Tree, CoreGenes) tools we have tackled the taxonomic status of members of the largest bacteriophage family, the Siphoviridae. In all over 400 phages were examined and we were able to propose 39 new genera, comprising 216 phage species, and add 62 species to two previously defined genera (Phic3unalikevirus; L5likevirus) grouping, in total, 390 fully sequenced phage isolates. Many of the remainders are orphans which the Bacterial and Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) chooses not to ascribe genus status at the time being.

Genomic and bioinformatics analyses of HAdV-14p, reference strain of a re-emerging respiratory pathogen and analysis of B1/B2

Unlike other human adenovirus (HAdV) species, B is divided into subspecies B1 and B2. Originally this was partly based on restriction enzyme (RE) analysis. B1 members, except HAdV-50, are commonly associated with respiratory diseases while B2 members are rarely associated with reported respiratory diseases. Recently two members of B2 have been identified in outbreaks of acute respiratory disease (ARD). One, HAdV-14, has re-emerged after an apparent 52-year absence. Genomic analysis and bioinformatics data are reported for HAdV-14 prototype for use as a reference and to understand and counter its re-emergence. The data complement and extend the original criteria for subspecies designation, unique amongst the adenoviruses, and highlight differences between B1 and B2, representing the first comprehensive analysis of this division. These data also provide finer granularity into the pathoepidemiology of the HAdVs. Whole genome analysis uncovers heterogeneous identity structures of the hexon and fiber genes amongst the HAdV-14 and the B1/B2 subspecies, which may be important in prescient vaccine development. Analysis of cell surface proteins provides insight into HAdV-14 tropism, accounting for its role as a respiratory pathogen. This HAdV-14 prototype genome is also a reference for applications of B2 adenoviruses as vectors for vaccine development and gene therapy.

Applying Genomic and Bioinformatic Resources to Human Adenovirus Genomes for Use in Vaccine Development and for Applications in Vector Development for Gene Delivery

Technological advances and increasingly cost-effect methodologies in DNA sequencing and computational analysis are providing genome and proteome data for human adenovirus research. Applying these tools, data and derived knowledge to the development of vaccines against these pathogens will provide effective prophylactics. The same data and approaches can be applied to vector development for gene delivery in gene therapy and vaccine delivery protocols. Examination of several field strain genomes and their analyses provide examples of data that are available using these approaches. An example of the development of HAdV-B3 both as a vaccine and also as a vector is presented.

Depletion of Olig2 in oligodendrocyte progenitor cells infected by Theiler's murine encephalomyelitis virus.

Theiler’s murine encephalomyelitis virus (TMEV) infects the central nervous system of mice and causes a demyelinating disease that is a model for multiple sclerosis. During the chronic phase of the disease, TMEV persists in oligodendrocytes and macrophages. Lack of remyelination has been attributed to insufficient proliferation and differentiation of oligodendrocyte progenitor cells (OPCs), but the molecular mechanisms remain unknown. Here, we employed pluripotent stem cell technologies to generate pure populations of mouse OPCs to study the temporal and molecular effects of TMEV infection. Global transcriptome analysis of RNA sequencing data revealed that TMEV infection of OPCs caused significant up-regulation of 1926 genes, whereas 1853 genes were significantly down-regulated compared to uninfected cells. Pathway analysis revealed that TMEV disrupted many genes required for OPC growth and maturation. Down-regulation of Olig2, a transcription factor necessary for OPC proliferation, was confirmed by real-time PCR, immunofluorescence microscopy, and western blot analysis. Depletion of Olig2 was not found to be specific to viral strain and did not require expression of the leader (L) protein, which is a multifunctional protein important for persistence, modulation of gene expression, and cell death. These data suggest that direct infection of OPCs by TMEV may inhibit remyelination during the chronic phase of TMEV-induced demyelinating disease.

Prokaryotic Autolysin Database Construction

Databases are valuable resources that are capable of collecting and disseminating sizeable quantities of data with the purpose of furthering research done in that particular field. Currently, there are databases for organismal genomes and classes of proteins, but currently there is no database for bacterial autolysins in particular. Autolysins such as LytA in Streptococcus pneumoniae are of vital importance as it allows the bacterium to evade antimicrobial peptides (Kietzman et. al, 2015). Additionally autolysins have been found to promote horizontal gene transfer in S. pneumoniae, as well as promoting sporulation in Bacillus subtilis (Allocati et. al, 2015). While some methods currently exist to manipulate autolysin expression, such as a broth with 2% choline, a large scale computational analysis of these proteins may provide more insight to these mechanisms (Balachandran et al, 2001). 11,458 proteomes encompassing all available Streptococcus (n=9,341), Bacillus (n=1,003), and Listeria (n=1,114) were obtained from GenBank, and 5 autolytic domains including amidase 3 and glucosaminidase were detected using hmmer with an E-value threshold of 10^-5. Resulting 68,748 output files were analyzed indicating 35,319 positive results containing at least one detected domain. This revealed that certain domains such as Glucosaminidase and LysM are highly conserved having been detected in 98.6%, 93.6%, and 99.9% of Streptococcus, Bacillus, and Listeria proteomes respectively, whereas others were noticeably more prevalent in a particular genus such as amidase 2 (75.5% Streptococcus, 92.1% Bacillus, 76.5% Listeria). Additionally, the proteomic locus of each detected domain has been recorded. Currently we aim to organize and compile data into a useful database using SQLite. Additionally, more genera are expected to be added to eventually encompass all bacterial proteomes. Moreover we aim to determine associated domains using hmm scan.

In silico bioinformatic tools for determining core genes from sets of genomes

The set of genes common to a group of organisms is known as the “core” set and the set of genes common to one or more of a group of organisms, but not to all of them is known as the “dispensable” set. Collectively, these two sets comprise the “pan‐genome” of a set of organisms. While there are many software tools available for ortholog detection and clustering, there are far fewer tools to determine the core set of genes as well as the dispensable set. Continued development of these computational and data mining tools is essential to further our understanding of the pan‐genomes of organisms and to make use of them. Drug Dev Res 72: 147–152, 2011.