Browsing by Author "Randall, Stephen"

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    20S proteasome assembly: alternative pathways and complexes
    (2017) Hammack, Lindsay J.; Kusmierczyk, Andrew R.; Mosley, Amber L.; Randall, Stephen; Baucum, AJ
    The ubiquitin-proteasome system is responsible for the targeted degradation of proteins within the cell. The 26S proteasome, which is the protease of this system, is a high molecular weight complex consisting of 33 subunits that arrange to form two smaller complexes the 19S regulatory particle (RP) and the 20S core particle (CP). The 19S RP can bind one or both ends of the 20S CP and is responsible for recognizing the ubiquitinated substrates. After recognition, the 19S RP will subsequently deubiquitinate, unfold, and translocate the substrates into the proteolytic 20S CP. The 20S CP consists of seven unique alpha and seven unique beta subunits that arrange into four stacked rings, with two alpha rings capping two beta rings. Assembly of the alpha(1-7)beta(1-7)beta(1-7)alpha(1-7) structure begins with the formation of an alpha ring and proceeds through specific assembly intermediates. This process is assisted by assembly chaperone proteins that promote on pathway interactions to efficiently construct the 20S CP. In this dissertation, three new findings are described which further characterize the proteasome assembly pathway. First, novel non-canonical complexes comprised of proteasome subunit alpha4 were identified in vivo, revealing proteasome subunits can assemble into complexes outside of the proteasome. Second, Hsp70 proteins, Ssa1/2, were shown to assist in the assembly of 20S CPs, adding to the growing list of proteins guiding proteasome assembly. Third, a novel complex was identified which is believed to represent a new proteasome assembly intermediate.
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    Accumulation Dynamics of Transcripts and Proteins of Cold-Responsive Genes in Fragaria vesca Genotypes of Differing Cold Tolerance
    (MDPI, 2021-06-07) Fattash, Isam; Deitch, Zachary; Njah, Relindis; Osuagwu, Nelson; Mageney, Vera; Wilson, Robert C.; Davik, Jahn; Alsheikh, Muath; Randall, Stephen; Biology, School of Science
    Identifying and characterizing cold responsive genes in Fragaria vesca associated with or responsible for low temperature tolerance is a vital part of strawberry cultivar development. In this study we have investigated the transcript levels of eight genes, two dehydrin genes, three putative ABA-regulated genes, two cold–inducible CBF genes and the alcohol dehydrogenase gene, extracted from leaf and crown tissues of three F. vesca genotypes that vary in cold tolerance. Transcript levels of the CBF/DREB1 transcription factor FvCBF1E exhibited stronger cold up-regulation in comparison to FvCBF1B.1 in all genotypes. Transcripts of FvADH were highly up-regulated in both crown and leaf tissues from all three genotypes. In the ‘ALTA’ genotype, FvADH transcripts were significantly higher in leaf than crown tissues and more than 10 to 20-fold greater than in the less cold-tolerant ‘NCGR1363’ and ‘FDP817’ genotypes. FvGEM, containing the conserved ABRE promoter element, transcript was found to be cold-regulated in crowns. Direct comparison of the kinetics of transcript and protein accumulation of dehydrins was scrutinized. In all genotypes and organs, the changes of XERO2 transcript levels generally preceded protein changes, while levels of COR47 protein accumulation preceded the increases in COR47 RNA in ‘ALTA’ crowns.
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    Advancements in forensic DNA-based identification
    (2017) Dembinski, Gina M.; Picard, Christine; Christie, Mark; Walsh, Susan; Randall, Stephen; Goodpaster, John
    Modern DNA profiling techniques have increased in sensitivity allowing for higher success in producing a DNA profile from limited evidence sources. However, this can lead to the amplification of more DNA profiles that do not get a hit on a suspect or DNA database and more mixture profiles. The work here aims to address or improve these consequences of current DNA profiling techniques. Based on allele-specific PCR and quantitative color measurements, a 24-SNP forensic phenotypic profile (FPP) assay was designed to simultaneously predict eye color, hair color, skin color, and ancestry, with the potential for age marker incorporation. Bayesian Networks (BNs) were built for model predictions based on a U.S sample population of 200 individuals. For discrete pigmentation traits using an ancestry influenced pigmentation prediction model, AUC values were greater than 0.65 for the eye, hair, and skin color categories considered. For ancestry using an all SNPs prediction model, AUC values were greater than 0.88 for the 5 continental ancestry categories considered. Quantitative pigmentation models were also built with prediction output as RGB values; the average amount of error was approximately 7% for eye color, 12% for hair color, and 8% for skin color. A novel sequencing method, methyl-RADseq, was developed to aid in the discovery of candidate age-informative CpG sites to incorporate into the FPP assay. There were 491 candidate CpG sites found that either increased or decreased with age in three forensically relevant xii fluids with greater than 70% correlation: blood, semen, and saliva. The effects of exogenous microbial DNA on human DNA profiles were analyzed by spiking human DNA with differing amounts of microbial DNA using the Promega PowerPlex® 16 HS kit. Although there were no significant effects to human DNA quantitation, two microbial species, B. subtilis and M. smegmatis, amplified an allelic artifact that mimics a true allele (‘5’) at the TPOX locus in all samples tested, interfering with the interpretation of the human profile. Lastly, the number of contributors of theoretically generated 2-, 3-, 4-, 5-, and 6-person mixtures were evaluated via allele counting with the Promega PowerPlex® Fusion 6C system, an amplification kit with the newly expanded core STR loci. Maximum allele count in the number of contributors for 2- and 3-person mixtures was correct in 99.99% of mixtures. It was less accurate in the 4-, 5-, and 6-person mixtures at approximately 90%, 57%, and 8%, respectively. This work provides guidance in addressing some of the limitations of current DNA technologies.
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    Alternative Assembly Pathways of the 20S Proteasome and Non-canonical Complexes
    (2018-12) Panfair, Dilrajkaur; Balakrishnan, Lata; Kusmierczyk, Andrew; Randall, Stephen; Rubenstein, Eric; Anderson, Gregory
    The 20S proteasome, a multi-subunit protease complex, present in all domains of life and some orders of bacteria, is involved in degradation of the majority of cellular proteins. Structurally, it is made of α and β subunits arranged in four heptameric rings, with inner two β-rings sandwiched between outer two α-rings. The 20S proteasome in prokaryotes usually has one type of α and one type of β subunits, whereas eukaryotes have seven distinct types of α and seven distinct types of β subunits. Unlike the highly conserved structure of proteasome, its assembly pathway is different across the domains. In archaea and eukaryotes, proteasome assembly begins with α subunit interactions leading to the α-ring formation. By contrast, bacterial proteasome assembly pathway bypasses the α-ring formation step by initiating assembly through an α and β subunit interaction first. These early interactions are not well understood due to their highly rapid and dynamic nature. This dissertation focused on understanding the early events in proteasome assembly and contributed three significant findings. First, the archaeal proteasome assembly can also begin without formation of α-rings, demonstrating the coexistence of a bacterial-like assembly pathway. Second, a novel assembly intermediate was identified in yeast, and its composition argues for the presence of a similar α-ring independent assembly pathway. Third, the assembly chaperone Pba3-Pba4 prevents the formation of high molecular weight complexes arising from spontaneous and non-productive interactions among the α subunits. These findings provide a broader understanding of proteasome biogenesis and suggest considering proteasome assembly event as a network of interactions rather than a linear pathway. The results also shed light on assembly chaperone’s contribution in increasing the efficiency of proteasome assembly by streamlining the productive interactions.
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    Cold response biomarker identification in strawberry
    (2018-07-17) Deitch, Zachary M.; Randall, Stephen
    Strawberry (Fragaria spp.) is an agricultural crop grown often in temperate regions that has high variability in its susceptibility to freezing injury. To breed cultivars for frost and freezing tolerance, identification of molecular markers associated with low temperature tolerance is advantageous. In this work, I investigated a high-throughput method for protein assays and western blotting. Success in streamlining these processes saves an immense amount of time and allows for the processing of more samples and obtaining larger datasets. Thirty-three octoploid varieties were tested for their accumulation of five different potential biomarkers in response to cold exposure. It was found that total dehydrin content, has the strongest potential to be reliable biomarkers for breeding programs. Previous work identified seven putative dehydrins in Fragaria, where two were purified and positively identified by mass spectrometry and determined to be COR47-like (SKn) and XERO2-like (YnSKn). This work demonstrated that cold tolerance positively correlated with dehydrin protein expression levels. To understand the cold-regulated expression of dehydrins as a function of cold exposure time, the levels of transcripts and corresponding proteins were examined in strongly cold tolerant (Alta) and lesser cold tolerant (FDP817, NCGR1363) Fragaria diploid genotypes. The COR47-like (SKn) and XERO2-like (YnSKn) dehydrins both had higher transcript accumulation and protein levels in the more cold tolerant line in comparison to the two less cold tolerant lines. Lack of correlation between transcript and resulting COR47 protein level in Alta were observed at several different timepoints, where protein accumulation preceded an increase in RNA. This trend was not seen with XERO2. This initiated an investigation to discover at what level COR47 is being regulated. First, the COR47 coding region was sequenced for all the genotypes to confirm against the predicted sequence. In addition, since two isoforms of the COR47 gene exist, and could possibly explain the discrepancy in transcript counts, primers were designed for both isoforms and RT-qPCR was performed to examine the transcripts of COR47 more closely. Through examination of the non-congruence of COR47 transcripts and protein, it was found that transcriptional mechanisms of regulation are not involved, and that post transcriptional and post-RNA splicing mechanisms are likely to be responsible for the observed trend in Alta. Conclusions from this work demonstrate that dehydrin transcripts and dehydrin protein accumulations are strong potential biomarkers for identifying low temperature tolerance in diploid strawberry.
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    Glycine max and Glycine soja are capable of cold acclimation
    (Wiley, 2017-12) Robison, Jennifer; Arora, Nigam; Yamasaki, Yuji; Saito, M.; Boone, J.; Blacklock, Brenda; Randall, Stephen; Biology, School of Science
    Soybean has been considered a cold intolerant species; based largely upon seed germination and soil emergent evaluations. This study reports a distinct acquisition of cold tolerance, in seedlings, following short acclimation periods. Diversity in cold responses was assessed in eight cultivars of Glycine max and six accessions of G. soja. All varieties of soybean significantly increased in freezing tolerance following acclimation. This study indicates soybean seedlings are indeed capable of sensing cold and acquiring cold tolerance. Germination rates after cold imbibition were negatively correlated with maturity group, but positively correlated with cold acclimation potential in G. soja. Seed fatty acid composition was varied between the species, with Glycine soja accessions containing about 2-times more linolenic acid (18:3) than G. max. Furthermore, high levels of linoleic acid (18:2) in seeds were positively correlated with germination rates following cold imbibition in G. soja only. We suggest that domestication has not impacted the overall ability of soybean to cold acclimate at the seedling stage and that there is little variation within the domesticated species for ability to cold acclimate. Thus, this brief comparative study reduces the enthusiasm for the “wild” species as an additional source of genetic diversity for cold tolerance.
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    Recent Activities in the Center for Membrane Biosciences
    (Office of the Vice Chancellor for Research, 2011-04-08) Blazer-Yost, Bonnie L.; Randall, Stephen; Minto, Robert; Birch, Garrison; Haydon, Julie; Bacallao, Robert; Gattone, Vincent; Blacklock, Brenda
    The Center for Membrane Biosciences (CMB) is active in facilitating collaborative research among center members and other IUPUI community members. A number of seed grants have been made and the results from two will be presented. Recent major funding from the NSF supports a CMB-centered program that promotes intensive undergraduate research opportunities. Project 1: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the slow growth of fluid-filled cysts in the kidney tubules and liver bile ducts. We identified LPA (lysophosphatic acid) as a component of cyst fluid that stimulates secretory Cl- and compensatory water flux into cysts through binding of receptors on the basolateral membrane of renal cells. LPA concentrations measured in ADPKD cyst fluid and in normal serum are sufficient to maximally stimulate ion transport. Thus, cyst fluid seepage and/or leakage of vascular LPA into the interstitial space are capable of stimulating secretion from epithelial cells resulting in cyst enlargement. Project 2: Upon the recent acquisition of Center-supported high-resolution mass spectrometers at IUPUI, methods for the analysis of lipid and protein samples to support nascent research endeavors within the CMB are being developed. Identification and quantification of sphingolipids in biological samples as well as other lipidomic experiments will be presented. Project 3: The IUPUI URM Immersion in Interdisciplinary Research in Biological Signaling program targets underrepresented minorities in the biological sciences, and through early and sustained undergraduate research experiences that are intensely mentored at multiple levels, aims to increase the number of underrepresented minorities achieving graduate degrees in the Biological Sciences. The first cohort will begin research in the program during the summer of 2011 and are currently in the selection process.
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    Sex Chromosome Evolution in Blow Flies
    (2020-08) Andere, Anne Amarila; Picard, Christine; Hahn, Matthew; Berbari, Nicolas; Randall, Stephen; Liu, Yunlong
    Chromosomal mechanisms of sex determination vary greatly in phylogenetically closely related species, indicative of rapid evolutionary rates. Sex chromosome karyotypes are generally conserved within families; however, many species have derived sex chromosome configurations. Insects display a plethora of sex chromosome systems due to rapid diversification caused by changes in evolutionary processes within and between species. A good example of such a system are insects in the blow fly family Calliphoridae. While cytogenetic studies observe that the karyotype in blow flies is highly conserved (five pairs of autosomal chromosomes and one pair sex chromosome), there is variation in sex determining mechanisms and sex chromosome structure within closely related species in blow flies. The evolutionary history of sex chromosomes in blow fly species have not been fully explored. Therefore, the objective of this research was to characterize the sex chromosome structures in four species of blow flies and investigate the selective forces which have played a role in shaping the diverse sex chromosome system observed in blow flies. The blow fly species used in this study are Phormia regina, Lucilia cuprina, Chrysomya rufifacies and Chrysomya albiceps. Phormia regina,and Lucilia cuprina have a heteromorphic sex chromosome system and are amphogenic (females produce both male and female offspring in equal ratio). In contrast, Chrysomya rufifacies and Chrysomya albiceps, have a homomorphic sex chromosome system, are monogenic (females produce unisexual progeny), have two types of females (arrhenogenic females – male producers and thelygenic females – female producers), and sex of the offspring is determined by the maternal genotype. To accomplish these tasks, a total of nine male and female individual draft genomes for each of the four species (including three individual draft genomes of Chrysomya rufifacies – male, and the two females) were sequenced and assembled providing genomic data to explore sex chromosome evolution in blow flies. Whole genome analysis was utilized to characterize and identify putative sex chromosomal sequences of the four blow fly species. Genomic evidence confirmed the presence of genetically differentiated sex chromosomes in P. regina and L. cuprina; and genetically undifferentiated sex chromosomes in C. rufifacies and C. albiceps. Furthermore, comparative analysis of the ancestral Dipteran sex chromosome (Muller element F in Drosophila) was determined to be X-linked in P. regina and L. cuprina contributing to sex chromosome differentiation but not sex-linked in C. rufifacies and C. albiceps. Evolutionary pressures are often quantified by the ratio of substitution rates at non-synonymous (dN) and synonymous (dS) sites. Substitution rate ratio analysis (dN/dS) of homologous genes indicated a weaker purifying selection may have contributed to the loss of sex-linked genes in Muller element F genes of the undifferentiated sex chromosome as compared to the differentiated sex chromosome system. Overall, the results presented herein greatly expands our knowledge in sex chromosome evolution within blow flies and will reinforce the study of sex chromosome evolution in other species with diverse sex chromosome systems.