Characterization of 5 UTR splicing and the CGI in HIV-2 RNA

HIV-2 tightly regulates several steps of its replication cycle via regulatory elements found within the 5 untranslated region of HIV-2 genomic RNA. Two elements of interest are the 5 UTR intron and the proposed long distance base pairing interaction between the C-box and G-box termed the CGI. This research focuses on the effects of 5 UTR splicing and the CGI in HIV-2 translation and replication. The central hypothesis is that both 5 UTR splicing and the CGI modulate HIV-2 translation and replication. This hypothesis was tested and supported by employing in vitro translation assays, SELEX, and cell culture studies. Results of these experiments demonstrate that splicing of the 5 UTR intron produces an isoform of gag mRNA that is specialized for high translational efficiency. Further, evidence is provided for a novel branched secondary structure within the CGI that is important for HIV-2 replication. In addition, we show that mutation of the C-box alone can enhance RNA encapsidation and mutation of the G-box can alter levels of Gag protein isoforms. These studies suggest coordinated regulation of RNA translation, dimerization, and encapsidation during HIV-2 replication. This research provides new insight into HIV translational mechanisms, in turn identifying potential antiviral targets that may be exploited for antiviral therapeutic strategies

Complete Genome Sequences of Mycobacterium smegmatis Phages NihilNomen and Carlyle, Isolated in Las Vegas, Nevada

We present the complete genomes of the Mycobacterium smegmatis phages Carlyle and NihilNomen, isolated from soil in Las Vegas, Nevada. The phages were isolated and annotated by undergraduate students enrolled in the Phage Discovery course offered by the School of Life Sciences at the University of Nevada Las Vegas

Complete Genome Sequences of Mycobacterium smegmatis Phages Chewbacca, Reptar3000, and Riparian, Isolated in Las Vegas, Nevada

Here, we present the complete genome sequences of Mycobacterium smegmatis phages Chewbacca, Reptar3000, and Riparian, isolated from soil in Las Vegas, NV. The phages were isolated and annotated by undergraduate students enrolled in the Phage Discovery course offered by the School of Life Sciences at the University of Nevada, Las Vega

Complete Genome Sequences of Cluster A6 and Cluster G1 Mycobacterium Smegmatis Phages Hoot and Jolene

We present the complete genome sequences of Mycobacterium smegmatis phages Hoot and Jolene, isolated in Las Vegas, NV. The phages were isolated and annotated by students enrolled in an undergraduate research course at the University of Nevada, Las Vegas. Hoot is a cluster A6 mycobacteriophage, while Jolene is in cluster G1

Complete Genome Sequences of Cluster P1 and Cluster C1 Mycobacterium smegmatis Phages Jung and Ronan

We present the complete genome sequences of Mycobacterium smegmatis phages Jung and Ronan, isolated from soil in Las Vegas, Nevada. The phages were isolated and annotated by students enrolled in a course for undergraduate research experience (CURE). Jung is a cluster P1 mycobacteriophage, while Ronan is in cluster C1

Models of classroom assessment for course-based research experiences

Course-based research pedagogy involves positioning students as contributors to authentic research projects as part of an engaging educational experience that promotes their learning and persistence in science. To develop a model for assessing and grading students engaged in this type of learning experience, the assessment aims and practices of a community of experienced course-based research instructors were collected and analyzed. This approach defines four aims of course-based research assessment—(1) Assessing Laboratory Work and Scientific Thinking; (2) Evaluating Mastery of Concepts, Quantitative Thinking and Skills; (3) Appraising Forms of Scientific Communication; and (4) Metacognition of Learning—along with a set of practices for each aim. These aims and practices of assessment were then integrated with previously developed models of course-based research instruction to reveal an assessment program in which instructors provide extensive feedback to support productive student engagement in research while grading those aspects of research that are necessary for the student to succeed. Assessment conducted in this way delicately balances the need to facilitate students’ ongoing research with the requirement of a final grade without undercutting the important aims of a CRE education

Flavonoid Glucosyltranferases: Cloning and Sequencing of Putative Glucosyltranferases from \u3cem\u3eCitrus paradisi\u3c/em\u3e (Grapefruit) Leaves.

Flavonoids are chemically modified by glucosylation, hydroxylation, methylation, etc. During glucosylation, the sugar moiety from UDP-sugar is transferred to aglycone flavonoid substrates by glucosyltransferases (GTs). Grapefruit contains 5 different glucosyltransferases that demonstrate differences in not only substrate but also position specificity. Previous research obtained 3 putative 5’ grapefruit GT clones using SMART RACE RT-PCR with a degenerate gene specific primer based on a highly conserved sequence area in the Plant Secondary Product Glucosyltransferase box. The objective of this research was to use clone specific primers to obtain 3’ clones of the 3 previously mentioned 5’ clones as well as verify putative GT candidacy based on sequence data. Two of the 3 putative GT candidates were designated non-GTs following 3’end sequencing. During pursuit of sequence for the remaining 5’ clone, 1 full-length clone and 1 partial putative GT clone were obtained. To verify GT status, the clones must undergo expression/biochemical characterization

Viral SELEX reveals individual and cooperative roles of the C-box and G-box in HIV-2 replication

The 5′ UTR of HIV-2 genomic RNA contains signaling motifs that regulate specific steps of the replication cycle. Two motifs of interest are the C-box and the G-box. The C-box is found in the 5′ untranslated region upstream of the primer binding site, while the G-box is found downstream from the major splice donor site, encompassing the gag start codon and flanking nucleotides. Together the C-box and the G-box form a long-range base-pairing interaction called the CGI. We and others have previously shown that formation of the CGI affects RNA dimerization in vitro and the positions of the C-box and the G-box are suggestive of potential roles of the CGI in other steps of HIV-2 replication. Therefore, we attempted to elucidate the role of the CGI using a viral SELEX approach. We constructed proviral DNA libraries containing randomized regions of the C-box or G-box paired with wild-type or mutant base-pairing partners. These proviral DNA libraries were transfected into COS-7 cells to produce viral libraries that were then used to infect permissive C8166 cells. The “winner” viruses were sequenced and further characterized. Our results demonstrate that there is strong selective pressure favoring viruses that can form a branched CGI. In addition, we show that the mutation of the C-box alone can enhance RNA encapsidation, and mutation of the G-box can alter the levels of Gag protein isoforms. These results suggest coordinated regulation of RNA translation, dimerization, and encapsidation during HIV-2 replication