mRNA Production of Gene 50 in JacoRen57

Mycobacteriophages are viruses that infect mycobacteria; little is known about their cis-regulatory transcriptional control elements. In previous work, we identified and characterized a regulatory region (40,670- 40,951 bp) in JacoRen57, an AB cluster mycobacteriophage. The regulatory region appears to play a role in regulating the expression of Gene 50. Utilizing qRT-PCR, we investigated further to see if Gene 50 mRNA is produced during an active infection of Mycobacterium smegmatis by JacoRen57

Population Genetics and Spawning Time of Lake Taupo Rainbow Trout

The rainbow trout (Oncorhynchus mykiss) of Lake Taupo, New Zealand provide an exceptional opportunity to explore the contemporary adaptation of an introduced aquatic species. Recently it has become evident that their spawning migration time has shifted to later in the season. I investigated the genetic basis of these changes in spawning time by (1) using genetic markers to determine the origins of Taupo trout in California, (2) determining the pattern and extent of spatial population genetic variation throughout the Lake Taupo catchment and in comparison to nearby Lake Tarawera in the Rotorua district, (3) analysing genetic variation at the OtsClock1b spawning time gene in temporal replicates from several sites from Taupo, and (4) comparing contemporary genetic variation at this gene and microsatellite markers to genetic variation from three Taupo tributaries in 1980s. I compared the ability of single nucleotide polymorphism (SNP) and microsatellite markers to determine the origins of Lake Taupo rainbow trout, translocated from California around 120 years ago. Data were collected from 15 microsatellite and 93 SNP markers, using samples from the Lake Taupo population and ten populations throughout California, which included all historically indicated populations of origin. Results revealed that the Lake Taupo population has significantly diverged from Californian populations at both microsatellite and SNP loci. These analyses also showed that the Lake Taupo population was probably derived from several sources in California (the most likely origins being the McCloud River and Lake Almanor), and an indeterminate California coastal population. This conclusion was supported with simulations of founder events, which suggested that the genetic patterns of a single source of introduction would still be detectable 100 years post-founding, but with multiple introductions exact source populations become more difficult to detect. Approximately 50 individuals from 10 locations throughout the catchment were then analysed using 15 microsatellite loci to determine if there was any spatial population genetic differentiation. There was no significant difference in genetic distance between locations within Lake Taupo, although there was a significant difference between these populations and Rotorua and Waipakihi, which are isolated by geographic barriers. Lake Taupo rainbow trout do appear to diverge at markers potentially under selection, though, because genotyping of the poly-Q region of the timing locus OtsClock1b shows significant differentiation between individuals sampled at different times in the Waipa River. Two other sites, however, did not show the same pattern of significant seasonal variation in OtsClock1b allele frequencies. This suggests that genotypes at this locus could be influencing spawning migration time, but that this variation could also be site specific, and therefore have a strong environmental component. Scale samples from the 1980s show no significant divergence at 5 microsatellites and OtsClock1b, indicating that allele frequencies have not changed significantly over the last 20 years at neutral markers or markers under selection. I therefore conclude that while Taupo rainbow trout have diverged from their origins in California, they have only slightly diverged within their new environment, and do not show a consistent pattern of genetic change over time. This information will contribute not only to the management of the Taupo fishery but also to the current understanding of the population genetic structuring of introduced salmonids

Exploring a Putative Promoter Region in Mycobacteriophage JacoRen57

Phages are abundant particles that infect bacteria. For the SEA-PHAGES program, students discover phages and annotate their genomes. Throughout the annotation process, genes are identified based on bioinformatics evidence; however, little is known about mycobacteriophage promoters as they are not annotated. Promoters are necessary for gene expression, and in mycobacteriophages, a promoter typically precedes a series of genes that are expressed as a single transcript from which multiple proteins are translated. JacoRen57 is a singleton mycobacteriophage with a siphoviridae morphotype that possesses forward and reverse genes with gaps located at the transitions from forward to reverse genes. We hypothesized that these gaps contain promoters. We used BPROM and PePPER, prokaryotic promoter predictor software, which yielded matches to promoter consensus sequences in one of the gap regions. We cloned the putative promoter region into pLO86, a vector containing the mCherry reporter gene, to determine if the cloned region functions as a promoter by inducing mCherryexpression in Mycobacterium smegmatis. The putative promoter region did not function as a promoter in vivo under standard M. smegmatisgrowth conditions

Exploring a Transcriptional Regulatory Region in Mycobacteriophage JacoRen57

Mycobacteriophages are infectious particles that infect mycobacteria, and little is known about cis-regulatory elements that control their gene expression. In phage genomes, cis-regulatory elements commonly precede a series of genes that are expressed as an operon. JacoRen57 is a cluster AB mycobacteriophage that possesses forward and reverse genes with non-coding gaps interspersed throughout its genome. We assayed one of the gap regions of JacoRen57 (40644-40974 bps) for regulatory activity in the downstream direction when present in its host, Mycobacterium smegmatis, by cloning the region into pLO86, a vector containing the mCherry reporter gene. The putative regulatory region induced the expression of mCherry in vivo, indicating the presence of a promoter in this region of the JacoRen57 genome. Utilizing 5’ deletions analysis, we identified promoter and repressor elements within this regulatory region. We are conducting further experiments to understand the characteristics of the repressor region and which sigma factor/s binds to this promoter

Concept Design for Optical Tweezers to be used in DNA Research

Optical tweezers are a Nobel Prize-winning technology capable of trapping microscopic and sub-microscopic particles using a laser beam. There are several new and useful applications available with the use of optical tweezers. A single optical tweezers set up can cost upwards of two hundred thousand dollars; however, we have designed a cost effective set up to study damaged DNA for under thirty thousand dollars. Using this design, we applied for a grant that would give us the necessary funds to build this set up. The building process itself will be very useful hands-on time learning about the laser set up. In addition, our optical tweezers would be integrated into undergraduate classes

Investigating the Putative RecA-Like Recombinase Gene

Our Biochemistry: Molecular Genetics class has partnered with the Immunology class to investigate the expression of JacoRen57’s gene 50. The bacteriophage JacoRen57 – found in Sioux Center, Iowa (accession: MK279840). JacoRen57’s genome has sequenced by Pittsburg SEA-PHAGES Institute and fully annotated by Northwestern College students in 2018. A region between gene 49 and 50 caught our attention as there is a large gap between these genes. Almail et al., investigated if this is a transcription regulatory region for genes 49 and/or 50 (2021). This work demonstrated the region has a regulatory function in the direction of gene 50. Based on comparison genomics, gene 50 is a putative RecA-like recombinase (Almail et al., 2019). This protein has several functions including guiding the recombination of DNA within a gene. RecA-like recombinase allows the virus to evolve into new variants which can improve infection and replication. This is crucial for creating diversity in the genome and DNA repair mechanisms (Galletto and Kowalczykowski, 2007). To continue examination of gene 50 expression, we are working towards developing antibodies for this protein. To do this, the first step is to create an expression construct (Figure 1), express the protein in bacteria, purify the protein, and then use the purified protein to inoculate mice. This poster describes the construction of the expression vector. This work will provide valuable insight into the expression of gene 50, the RecA-like recombinase

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

Population Genetics and Spawning Time of Lake Taupo Rainbow Trout

The rainbow trout (Oncorhynchus mykiss) of Lake Taupo, New Zealand provide an exceptional opportunity to explore the contemporary adaptation of an introduced aquatic species. Recently it has become evident that their spawning migration time has shifted to later in the season. I investigated the genetic basis of these changes in spawning time by (1) using genetic markers to determine the origins of Taupo trout in California, (2) determining the pattern and extent of spatial population genetic variation throughout the Lake Taupo catchment and in comparison to nearby Lake Tarawera in the Rotorua district, (3) analysing genetic variation at the OtsClock1b spawning time gene in temporal replicates from several sites from Taupo, and (4) comparing contemporary genetic variation at this gene and microsatellite markers to genetic variation from three Taupo tributaries in 1980s. I compared the ability of single nucleotide polymorphism (SNP) and microsatellite markers to determine the origins of Lake Taupo rainbow trout, translocated from California around 120 years ago. Data were collected from 15 microsatellite and 93 SNP markers, using samples from the Lake Taupo population and ten populations throughout California, which included all historically indicated populations of origin. Results revealed that the Lake Taupo population has significantly diverged from Californian populations at both microsatellite and SNP loci. These analyses also showed that the Lake Taupo population was probably derived from several sources in California (the most likely origins being the McCloud River and Lake Almanor), and an indeterminate California coastal population. This conclusion was supported with simulations of founder events, which suggested that the genetic patterns of a single source of introduction would still be detectable 100 years post-founding, but with multiple introductions exact source populations become more difficult to detect. Approximately 50 individuals from 10 locations throughout the catchment were then analysed using 15 microsatellite loci to determine if there was any spatial population genetic differentiation. There was no significant difference in genetic distance between locations within Lake Taupo, although there was a significant difference between these populations and Rotorua and Waipakihi, which are isolated by geographic barriers. Lake Taupo rainbow trout do appear to diverge at markers potentially under selection, though, because genotyping of the poly-Q region of the timing locus OtsClock1b shows significant differentiation between individuals sampled at different times in the Waipa River. Two other sites, however, did not show the same pattern of significant seasonal variation in OtsClock1b allele frequencies. This suggests that genotypes at this locus could be influencing spawning migration time, but that this variation could also be site specific, and therefore have a strong environmental component. Scale samples from the 1980s show no significant divergence at 5 microsatellites and OtsClock1b, indicating that allele frequencies have not changed significantly over the last 20 years at neutral markers or markers under selection. I therefore conclude that while Taupo rainbow trout have diverged from their origins in California, they have only slightly diverged within their new environment, and do not show a consistent pattern of genetic change over time. This information will contribute not only to the management of the Taupo fishery but also to the current understanding of the population genetic structuring of introduced salmonids

Renal Nerve Stimulation-Induced Blood Pressure Changes Predict Ambulatory Blood Pressure Response After Renal Denervation

Blood pressure (BP) response to renal denervation (RDN) is highly variable and its effectiveness debated. A procedural end point for RDN may improve consistency of response. The objective of the current analysis was to look for the association between renal nerve stimulation (RNS)-induced BP increase before and after RDN and changes in ambulatory BP monitoring (ABPM) after RDN. Fourteen patients with drug-resistant hypertension referred for RDN were included. RNS was performed under general anesthesia at 4 sites in the right and left renal arteries, both before and immediately after RDN. RNS-induced BP changes were monitored and correlated to changes in ambulatory BP at a follow-up of 3 to 6 months after RDN. RNS resulted in a systolic BP increase of 50±27 mm Hg before RDN and systolic BP increase of 13±16 mm Hg after RDN (P<0.001). Average systolic ABPM was 153±11 mm Hg before RDN and decreased to 137±10 mm Hg at 3- to 6-month follow-up (P=0.003). Changes in RNS-induced BP increase before versus immediately after RDN and changes in ABPM before versus 3 to 6 months after RDN were correlated, both for systolic BP (R=0.77, P=0.001) and diastolic BP (R=0.79, P=0.001). RNS-induced maximum BP increase before RDN had a correlation of R=0.61 (P=0.020) for systolic and R=0.71 (P=0.004) for diastolic ABPM changes. RNS-induced BP changes before versus after RDN were correlated with changes in 24-hour ABPM 3 to 6 months after RDN. RNS should be tested as an acute end point to assess the efficacy of RDN and predict BP response to RDN.status: publishe