A reverse transcriptase-related protein mediates phage resistance and polymerizes untemplated DNA in vitro

Reverse transcriptases (RTs) are RNA-dependent DNA polymerases that usually function in the replication of selfish DNAs such as retrotransposons and retroviruses. Here, we have biochemically characterized a RT-related protein, AbiK, which is required for abortive phage infection in the Grampositive bacterium Lactococcus lactis. In vitro, AbiK does not exhibit the properties expected for an RT, but polymerizes long DNAs of ‘random’ sequence, analogous to a terminal transferase. Moreover, the polymerized DNAs appear to be covalently attached to the AbiK protein, presumably because an amino acid serves as a primer. Mutagenesis experiments indicate that the polymerase activity resides in the RT motifs and is essential for phage resistance in vivo. These results establish a novel biochemical property and a non-replicative biological role for a polymerase

Writing in Britain and Ireland, c. 400 to c. 800

No abstract available

Characterization of a Group II Intron and its Intron-Encoded Protein

Group II introns are a class of mobile genetic elements found in the genomes of bacteria, archaea and in the organellar genomes of some eukaryotes. They are comprised of a large structural RNA with ribozyme activity and a multi-functional intron-encoded protein. The IEP encodes four domains: reverse-transcriptase, maturase, DNA-binding and endonuclease domains. The RNA and IEP interact to form a functional ribonucleoprotien that facilitates the two activities of group II introns, splicing and mobility. The interaction between group IIA introns and their IEPs has been well characterized; however, many of the details regarding the IIC intron-IEP interaction are poorly understood. This dissertation examines the interaction between the B.h.I1 group II intron and its intron-encoded protein. The B.h.I1 intron is a class IIC intron encoding an IEP that lacks the endonuclease domain. The intron is found in the organism Bacillus halodurans and exhibits clear evidence of retroelement behaviour, inserting downstream of intrinsic transcriptional terminators. In order to better characterize the intron-IEP interaction, it was determined that significant improvement to the IEP purification was required. Through a combination of optimization of expression and FPLC the yield and purity of the IEP was improved significantly. With purified components in sufficient quantity, the hypothesis that IIA and IIC IEPs recognize different high-affinity binding sites was examined using an in vitro mobility assay. Ultimately it was determined that IIC IEPs recognize a structurally similar but functionally different high-affinity binding site than is recognized by IIA-encoded IEPs. During binding site experiments, it was determined that the B.h.I1 intron uses the same IEP binding-site for splicing and mobility, reconciling a previously observed inconsistency. A kinetic characterization found that the B.h.I1-IEP interaction occurs with lower affinity compared to the IIA intron and that the B.h.I1 intron-IEP interaction relies more heavily on secondary contacts made outside the high-affinity binding site. Finally, data obtained from cross-linking experiments implicate the IEP in recognition of the 3’ exon, a previously undiscovered function of the IEP in intron mobility. Though subtle, the differences observed for the IIC intron-IEP interaction appear to facilitate the specific requirements of its genomic niche

Evolution of group II introns

Present in the genomes of bacteria and eukaryotic organelles, group II introns are an ancient class of ribozymes and retroelements that are believed to have been the ancestors of nuclear pre-mRNA introns. Despite long-standing speculation, there is limited understanding about the actual pathway by which group II introns evolved into eukaryotic introns. In this review, we focus on the evolution of group II introns themselves. We describe the different forms of group II introns known to exist in nature and then address how these forms may have evolved to give rise to spliceosomal introns and other genetic elements. Finally, we summarize the structural and biochemical parallels between group II introns and the spliceosome, including recent data that strongly support their hypothesized evolutionary relationship.Ye

Characterizing non-mammalian mitogenic growth factors for use in serum free media

Commercially available growth factors are primarily of mammalian origin, leaving cell culture applications using non-mammalian cell lines with sub-optimal options for serum free medium. We plan to develop a cost effective microbial expression system to facilitate production of non-mammalian mitogenic growth factors for use in serum free cell culture medium

Modality of Classes and Burnout in College Students

The Covid-19 pandemic has changed how students learn. Many students had to adapt to an all-online class modality. With the increased use of online modality of classes, will burnout scores increase in college students? In previous studies, researchers have mainly focused on burnout in the workplace. There is a gap in the literature in student burnout which we plan to explore. Research about the academic workforce found that indicators of burnout have increased sharply. The current study investigated if a student\u27s class modality influences burnout scores. Students will receive a survey asking about their class modalities (online, in-person, hybrid) and will also be asked questions from the Maslach Burnout Inventory. We found that the Maslach Burnout Inventory has been the leading burnout indication measurement. This inventory will give three different scores in three different categories: occupational exhaustion, loss of empathy, and personal accomplishment. With these questions answered, we hypothesized that students taking more online classes will score higher on the Maslach Burnout Inventory in occupational exhaustion and loss of empathy. We also hypothesize that students taking more online classes will show a lower score in the personal accomplishment category

A pipeline of programs for collecting and analyzing group II intron retroelement sequences from GenBank

Article deposited according to publisher policies: http://www.biomedcentral.com/about/copyright June 12, 2014YesFunding provided by the Open Access Authors Fun