Investigating Gene Functions in Mycobacteriophage Island3

Island3 is a temperate Cluster I1 mycobacteriophage that infects Mycobacterium smegmatis mc2155. Its genome consists of 76 protein-coding genes, only 17 of which have known functions. Towards the goal of identifying additional gene functions, we amplified, cloned, and assayed 14 genes for host cytotoxicity and the ability to render the host resistant to infection by other phages (defense). We analyzed genes 10, 11, 12, 13, 14, 15, 21, 22, 25, 50, 51, 57, 60, and 61 and concluded that none of these genes exhibited either host cytotoxicity or defense against phage infection. We are in the process of assaying the remaining genes of Island3

Elucidating Antiproliferative Mechanisms of Grapeseed, Guava, and Juniper Berry Extracts

Plant extracts are an untapped source of medicinal potential. Even today they are used as standalone treatments and applied alongside conventional therapies. The focus of our laboratory is to identify plant extracts exhibiting antiproliferative activity in vitro, to determine which chemicals are responsible for this activity, and to elucidate mechanism(s) by which growth is slowed/inhibited by plant extracts. Specifically, we exposed five cell lines/strains to twenty-two plant extracts and measured cell proliferation. Extracts from Vinca, Juniper Berry, Guava, Grapeseed, and Yew slowed the growth of all five lines/strains in a dose dependent fashion. We are working to understand the mechanism of antiproliferation by measuring induction of apoptosis, effects on microtubule assembly, and wound healing

Analysis of a Putative Promoter in Mycobacteriophage JacoRen57

JacoRen57 is a cluster AB mycobacteriophage that infects Mycobacterium smegmatis mc²155. We recently reported on the characterization of a putative promoter in JacoRen57 using an mCherry reporter construct. This promoter is present in a gap upstream of a gene that is present in all AB phages. In all cases, these are forward genes immediately following a long series of reverse genes. The genes are most frequently identified as a RecA-like DNA recombinases but also as RepA by bioinformatics. To further analyze this putative promoter and gene product, NWC Molecular Genetics students cloned the RecA-like DNA recombinase into an E. coli expression vector with a TVMV removable N-terminal His-tag. They expressed and we purified the tagged protein and are using it to immunize Balb/c mice. We plan to use the antiserum to confirm RecA-like DNA recombinase expression patterns when JacoRen57 infects M. smegmatis

Exploring Gene Functions and Phage-Host Protein Interactions in Mycobacteriophage Island3

Island3 is an I1 mycobacteriophage that infects Mycobacterium smegmatis mc²155. It has a total of 76 protein coding genes, but only 17 of these genes have functions assigned by bioinformatics. To discover the functions of the additional genes, we cloned 72 of Island3’s genes and are assaying each gene product for two functions when expressed in the host M. smegmatis: the ability to reduce growth of the host (cytotoxicity) and the ability to protect the host from infection by Island3 or another phage (defense). So far, we have assayed more than 60 of Island3’s genes and found 14 genes that exhibited cytotoxicity but none that exhibited definitive defense against phage infection. We are currently analyzing the remaining genes for cytotoxicity and defense. In addition, we are moving forward with bacterial two-hybrid assays on two of the genes that exhibited cytotoxicity, seeking to identify host proteins that interact with the cytotoxic phage gene products in an attempt to understand the mechanism of cytotoxicity.

Genomic Annotation of Bacteriophages Clayda5, GShelby23, Santhid, and Wrigley

We annotated the genomes of four recently discovered Actinobacteriophages. Clayda5 and GShelby23 were isolated on Microbacterium foliorum NRRL B-24224. Clayda5 is a lytic, cluster EB phage, one of only 47 discovered to date. It has 10 base pair 3’ sticky overhanging ends and a GC content is 67.2%. It has 70 protein-coding genes and two tRNA genes in its 39,894 bp genome. Clayda5 was purified from soil collected in Hull, IA. GShelby23 was isolated from soil collected in Storm Lake, IA. It is a cluster EM phage, one of only six discovered to date. Its genome is circularly permuted and 53,603 bp long. Its GC content is 64.8%. Santhid and Wrigley are phages that infect Gordonia terrae 3612. Santhid is a cluster DY phage, one of only five discovered to date. It was isolated from soil collected in Orange City, IA. Its genome is 39,295 bp long and includes 60 protein-coding genes. Its GC content is 67.7% and has 10 base pair 3’ sticky overhanging ends. Wrigley was isolated using an enrichment protocol from soil collected in Johnston, IA. It is a cluster CY phage, one of only 17 discovered to date. It is a temperate phage whose genome is 51,878 bp long and includes 81 protein-coding genes. It has 10 base pair 3’ sticky overhanging ends and a GC content of 66.3%.

How Families’ Lifestyles are Impacted When There is a Child or Parent with a Cognitive Disability

Today, millions of people have been diagnosed with a disability. Ultimately, this leaves an impact, whether it be a positive one or not, on the families and/or caregivers in people’s lives (Trivettte, 2010 & Vohra, 2013). In this review of the literature, there are several analyzed studies that focus on the level of involvement and what kind of impact involvement may have on the loved one with a disability. Looking more thoroughly into these studies, there becomes a trend of what roles the family member with the disability play and to what extent their disability may impact the home. Through these studies it can be proven that the higher the level of involvement, the higher lever of impact and the lower level of involvement can lead negative and consequential lifestyles. This study is on the topic of how having a child or parent with a cognitive disability impacts a family\u27s’ lifestyle and involvement based on influence in this family member\u27s life