The function of centromeric DNA in the yeast Saccharomyces cerevisiae has been studied in detail. Twelve of the sixteen S. cerevisiae centromeres have been sequenced to date, and a consensus sequence has been identified. This sequence consists of a central region, conserved centromere DNA element II (CDE II), which is 78-86 bp in length, greater than 87% A+T-rich, and tends to be arranged in runs of As and runs of Ts. The central region is flanked on one side by a highly conserved 8 bp sequence (CDE I) and on the other side by a highly conserved 25 bp sequence (CDE III) which contains partial inverted dyad symmetry around a central C/G base pair. Mutational analyses have been used to determine the importance of the consensus sequences to centromere function. A protein which binds to the CDE I sequence and at least one protein that binds to the CDE III sequence have been identified. The roles of these proteins in centromere function in mitosis and meiosis are currently under investigation. In this study, totally synthetic DNA was used to create centromere mutants that could not have been made easily by any other method. Functional analysis of these mutants have confirmed and extended the findings of other workers. First, the results provide supporting evidence for the idea that the centromere consensus sequence is sufficient to confer wild-type mitotic and meiotic function to a replicated plasmid or chromosome. Second, they support the idea that the highly conserved base pairs in CDE III are important for maintaining the symmetry and functionality of CDE III. Third, the results suggest that a protein important for proper mitotic centromere function binds to CDE III and interacts directionally with something located at or near CDE II. Fourth, they confirm the importance of the A+T-richness of CDE II for proper mitotic and meiotic centromere function, and they provide evidence that the ability of CDE II to form a bend may also be important for both mitotic and meiotic centromere function. Finally, one mutant functioned better on plasmids than on chromosomes, a finding that has interesting implications for chromosome structure and function.

The U14 RNA of Saccharomyces cerevisiae is an essential small nuclear RNA (snRNA) associated with the nucleolus. Repression of U14 RNA synthesis was shown to result in impaired production of 18S rRNA, manifest as a dramatic decrease in the ratio of mature 18S and 25S RNAs. This effect was evident within one generation after the onset of U14 gene repression and correlated well with depletion of U14 snRNA. Results from pulse-chase assays revealed the basis of the imbalance to be underaccumulation of 18S RNA and its 20S precursor. This effect appears to result from: (1) impairment of processing of the 35S rRNA transcript at sites that define the 20S species and (2) rapid turnover of an unusual 18S-containing intermediate. These results constitute the first demonstrated involvement of an essential snRNA in rRNA maturation. Functional mapping was directed at assessing the importance of sequence elements that are: (1) conserved among the yeast and smaller mouse U14 RNAs and (2) unique to the yeast species. First, the functional equivalency of the yeast and mouse U14 RNAs was examined in a test strain, in which wild-type U14 synthesis is induced by galactose and repressed by glucose. The experimental RNAs included mouse U14 and several yeast:mouse bi- and tri-partite hybrid RNAs, all transcribed from yeast U14 gene signals in a plasmid. Next, plasmid-encoded yeast U14 DNA was subjected to deletion and base substitution mutagenesis. Functional characterization showed that phylogenetically conserved sequences in the yeast and mouse U14 RNAs are interchangeable, provided that a terminal stem composed of 5$\sp\prime$ and 3$\sp\prime$ segments is preserved. This result argues that the yeast and mouse U14 RNAs are functional homologs and that the U14 RNAs are universally important. The result also identified two internal yeast specific elements which are required for function of U14 in yeast. An effort was also made to develop a genetic system for analyzing structural features of rDNA involved in 18S rRNA processing, with a view to establishing U14-dependent pre-rRNA maturation assays. To this end, a plasmid-borne rDNA operon was constructed, in which the 18S RNA coding sequence was "tagged" by an oligonucleotide insertion. The hybridization tag was shown to behave as a silent mutation with no apparent interference with rRNA processing, ribosome assembly or incorporation of tagged ribosomes into polysomes. The analysis of deletions constructed with the tagged operon revealed that 18S RNA production in Saccharomyces cerevisiae requires colinear expression of intact 25S RNA.

To gain a better understanding of the architecture of the ribosome in general, and the structure of the ribosome within the tRNA binding sites specifically, photoreactive tRNA derivatives were cross-linked to the Escherichia coli ribosome. Protein and RNA components in the immediate vicinity of the tRNA were identified by cross-linking yeast tRNA$\sp{\rm Phe}$ molecules, substituted with the photoreactive nucleoside 2-azidoadenosine at positions 37, 73 or 76, to the ribosomal tRNA binding sites. These tRNA derivatives were good substrates for aminoacyl-tRNA synthetase and were specifically bound and cross-linked to the A (aminoacyl), P (peptidyl) and E (exit) sites on the ribosome. Yeast tRNA$\sp{\rm Phe}$, containing 2-azidoadenosine at position 37, was used to probe the decoding domain on the 30S subunit since the photoreactive base was immediately adjacent to the 3$\sp\prime$ end of the anticodon. From the A, P and E sites, cross-linking was exclusively to the 30S subunit and both proteins and 16S rRNA were labeled. While protein S7 was cross-linked in the A and P sites, protein S11 was labeled in the E site. The 16S rRNA nucleotides C1317 and C1359 were labeled in the P site, and U1135, C1226, C1228, C1237, C1249 and C1284 in the A site. In addition, the E site-bound tRNA cross-linked nucleotide(s) within the 3$\sp\prime$ terminal 29 bases of the 16S rRNA. While the tRNA-16S rRNA cross-links in the A and P sites are generally consistent with the current three-dimensional models of the 3$\sp\prime$ major domain, they suggest that this region of the 16S rRNA should be positioned closer to nucleotide C1400, which was previously cross-linked to the 5$\sp\prime$ anticodon base of P site-bound tRNA$\sb1\sp{\rm Val}$. Since several immune electron microscopy studies place C1400 deep in the cleft of the 30S subunit, both the 16S rRNA nucleotides and protein S7, cross-linked in this study, must also be associated with this unique topographical feature. In the E site, the anticodon loop is proposed to reside on the platform of the 30S subunit, since both the 3$\sp\prime$ end of 16S rRNA and protein S11 have been located in this region by immune electron microscopy. When tRNA$\sp{\rm Phe}$ derivatives containing 2-azidoadenosine at either position 73 or 76 were bound and cross-linked to the A site, protein L27 was the main target of labeling. The cross-linking of protein L27 by azidoadenosines at or near the aminoacyl-end of tRNA in the A site adds to a mounting body of evidence suggesting that this protein is a central component of the peptidyl transferase center. While no ribosomal RNA was labeled by the tRNA probe substituted at position 73, significant labeling of 23S rRNA occurred when the derivative substituted at position 76 was cross-linked to the A site.

In this dissertation, I report on the first in-depth study of RNA recombination in any virus system. In turnip plants infected with turnip crinkle virus (TCV) genomic RNA, satellite RNA D (sat-RNA D) and certain altered transcripts of sat-RNA C, sat-RNA D/C recombinants are found to accumulate. This exchange of RNA can be classified as targeted, aberrant homologous recombination as varying lengths of sat-RNA D are found joined to one of 5 consecutive bases within sat-RNA C. These 5 nucleotides are contained within a larger sequence, Motif I, which is one of three sequences we propose act as replicase recognition signals. We propose that recombination between these RNAs occurs utilizing a replication dependent mechanism in which the replicase, after synthesizing a full length or nearly full length sat-RNA D molecule, reinitiates polymerization at Motif I within sat-RNA C to generate a chimeric D/C species. Detailed analysis of the role of Motif I in this exchange of RNAs was carried out. The sequence of the signal is not required for infectivity of sat-RNA C transcripts but is required for RNA recombination. Thirteen different point mutations were introduced into and around Motif I; all alterations allowed infectivity of the transcripts yet several of these same base changes abolish RNA recombination. Small insertions and deletions generated at three unique restriction sites in sat-RNA C had no effect upon transcript infectivity, however, some of these alterations were found to affect recombination. The addition of residues at the predicted 5$\sp\prime$ border of Motif I (a Bam HI site) or 32 residues upstream (a Mlu I site) allowed recombination to occur; a deletion of 4 bases 13 bases downstream (an Apa I site) did not allow for RNA exchange. Secondary structure modelling of a 78 base region encompassing Motif I indicated that a dual-stem loop structure could be formed. Analysis of the sequences of the loops suggests that the two loops may interact. Two base alterations which appeared to disrupt the formation of the larger stem were selected for further analysis. Compensatory mutations were generated to repair the nucleotide mismatches introduced by these two alterations; repairing of the base pairing (replacement of the original A:U pair with a G:C pair) restored recombination activity.

U14 and U3 are phylogenetically conserved small nucleolar RNAs (snoRNAs). Both RNAs are required for 18S ribosomal RNA synthesis in the yeast Saccharomyces cerevisiae and both have been reported to be associated with rRNA precursors. Loss of either snoRNA disrupts nucleolytic processing of precursor rRNA, suggesting that each has a role in this process. This study addresses three issues related to U14 and U3 biochemistry. The objectives include: (1) development of detailed functional maps of the conserved and essential box C and box D sequence elements in U14; (2) postulation of a secondary folding model for yeast U14 RNA; and, (3) identification of the RNA polymerase responsible for U14 gene expression in S. cerevisiae. The sequence requirements for box C and box D function in U14 have been determined by site-directed mutagenesis. Functional effects were evaluated in a test strain dependent on galactose for expression of wild-type U14; activity of mutant U14 RNAs was assessed in glucose medium. The results show that the first GA bases of the box C sequence UGAUGA and the final GA bases of the box D sequence GUCUGA are essential. Mutations at these positions abolish or severely reduce U14 accumulation. Similar effects were observed for box C mutants transcribed from the heterologous GAL1 promoter. This latter result suggests that box C is not required for expression, but influences turnover. Mutagenesis of the first GA doublet of box C in U3A (CGAUGA) showed that only a G $\to$ C mutation is lethal. The occurrence of another candidate box C element in U3A is discussed. A hypothetical secondary structure model is proposed for U14 based on biochemical and genetic data. In the model the functionally important domains Y2 (specific to yeast), box C element and 18S-A and 18S-B (both complementary to 18S rRNA) are exposed in single-stranded regions. The essential elements box D and Y1 participate in double-stranded regions. Expression of U14 and other small nuclear RNAs has been examined in yeast strains in which RNA polymerase II or III is conditionally defective. The relative abundances of U14, snR190 and snR10 remain surprisingly constant for several generation-equivalents, following a shift to the non-permissive conditions. The significance of these results are discussed in the context of polymerase structure and function and snoRNA turnover rates.

OBJECTIVE: Patients of corneal opacity are increasing year by year. The treatment for this disease is relatively straightforward: corneal transplant from a human donor. However, there is a global shortage of donor corneas, leading to many patients not being able to receive the treatment even though the procedure itself is well developed. Scientists have continuously tried to find alternative methods of human corneal transplant. Recently, the porcine cornea has become a research focus as a transplantation alternative because of mechanical and morphological similarities to the human cornea. This study focused on analyzing the porcine corneal endothelial cell (PCEC), specifically its density-dependent characteristics in vitro and its proliferation capabilities. The hypothesis was tested that PCECs could proliferate most efficiently under a high-density seeding environment and could maintain cell efficacy for 5 passages. METHODS: PCECs were isolated from three fresh porcine corneas and seeded into 24-well petri dishes at 10,000 cells/cm2 (high density) and 6,700 cells/cm2 (low density). Cells were cultured for 96 hours before subculturing with the same conditions onto the next passage. Samples from doubling times of each passage were collected and analyzed, and tight junction markers were stained with proteins ZO-1 (zonula occludens-1) and N-cadherin to test for tight junction functionality. RESULTS: The high-density group averaged a faster doubling time at 38.3 hours (n = 8) compared with the doubling time of the low-density group at 43.2 hours (n = 8). PCECs were able to maintain proliferation and matured for 8 passages until losing cell morphology (more drastic for the high-density group) and cell tight junction (both groups) at the ninth passage. Doubling times were also increased drastically for both density groups at the ninth passage. CONCLUSION: Examination of PCEC growing conditions led to the discovery that growth density is crucial to the overall quality of the corneal endothelial cells. From these data and past research, high-density growth conditions of ≥10,000 cells/cm2 were more beneficial than a low-density environment of 6,700 cells/cm2. PCECs were able to maintain tight junction functionality and cell morphology for 8 passages when grown to subconfluency. Based on the analysis of these results, the PCEC appears to be a more accessible corneal study target because of the many characteristics similar to the human corneal endothelial cell (HCEC), making it a possible alternative in medical transplant research in the future. This potential of the PCEC, if successfully developed, will increase the arsenal of choices for doctors and researchers around the world in the combat against corneal opacity.

Inflammation is important in mediating host defense. However, chronic inflammation can lead to the development of numerous lifelong disorders. Specialized pro-resolving lipid mediators (SPMs) are lipid mediators that actively inhibit inflammation and enhance resolution. SPMs are generated through the actions of various lipid mediator biosynthetic enzymes such as 5-lipoxygenase (5LO or ALOX5). While there has been a lot of progress elucidating lipoxygenase- mediated biosynthetic pathways leading to SPM production, the upstream cues governing activation and regulation of ALOX5 are still not fully understood. Our laboratory discovered that engagement of the NOD2 pathway led to the production of not only pro-inflammatory lipid mediators, but also SPMs. In order to understand how NOD2 was mediating these effects, we investigated the role of NOD2 in influencing the post-translational modification, localization, and stabilization of ALOX5. We found that while the presence of NOD2 promoted the activating phosphorylation of ALOX5, it also decreased the levels of ALOX5 in a dose-dependent manner. Further pulse-chase analysis indicated that although NOD2 initially decreased ALOX5 protein levels, it ultimately prolonged the half-life of total and phosphorylated ALOX5. It was also observed that certain disease associated NOD2 variants caused dysregulated phosphorylation of ALOX5 or dysregulated localization of activated ALOX5. Lastly, mass spectrometry analysis of ALOX5 in M1 or M2 polarized THP-1 macrophages stimulated with the NOD2 agonist MDP indicated differential proline hydroxylation of ALOX5 under these conditions. Understanding how NOD2 influences ALOX5 may shed light on the mechanisms which are dysregulated under pathological conditions and could inform the potential utility of various inhibitors of lipid mediator production or of NOD2/RIP2 signaling for treatment of specific inflammatory diseases.

BACKGROUND: Myelodysplastic syndromes (MDS) are a group of blood disorders associated with defective blood cell production and differentiation. In approximately 30 percent of cases, MDS has a chance of progressing into acute myeloid leukemia, which is a type of blood cancer. Important hallmarks of MDS are abnormal DNA promoter methylation and aberrant mRNA alternative splicing. Both activities could lead to the silencing of important tumor suppressor genes which accelerates the disease progression. OBJECTIVE: The study focused on investigating the possible relationship between DNA methylation level and alternative splicing activity in the hope of targeting the two critical factors of MDS simultaneously. There is currently no cure for MDS, and the result could be useful in developing a novel therapy. METHODS: The myeloid cell line K562 was either treated with 1)demethylating agent 5’-Azacytidine (AZA) or 2) splicing inhibiting agent Pladienolide B (PB) and their effect on alternative splicing and DNA methylation level were explored respectively. The DNA methylation level and the alternative splicing activity of the drug treated cells were assessed through DNA methylation dot blot assay and the fluorescent microscope images respectively. RESULTS: The inhibition of DNA methylation could lead to a decrease in splicing activity and the inhibition of splicing might also lead to a decrease in DNA methylation suggesting a positive correlation between the two activities. CONCLUSIONS: The study was successful in discovering a possible presence of interconnection between the two critical activities related to the pathogenesis of MDS. Further studies are still needed to examine the exact mechanism of how one activity could impact the other. / 2026-02-14T00:00:00Z

Interpreting DNA profiles manually can potentially call into question subjectivity between analysts who may interpret specific results differently. There are multiple features of a DNA profile that can complicate interpretation, which include allelic dropout and drop-in, allele sharing, and polymerase chain reaction (PCR) artifacts, which can all confound manual interpretation of DNA profiles. Difficulties in interpretation of DNA profile evidence can also be caused by degradation of the DNA itself, which can be caused by various environmental factors. Over the last 15 years, developments in DNA profile interpretation using probabilistic genotyping software (PGS) have been made in order to assist in the complicated task of interpreting and deconvoluting a challenging mixture. Among these PGSs is DNA-View® Mixture Solution™, a continuous-model program that is based on stochastic modeling. In this research, Mixture Solution was used to provide statistical analyses on DNA mixtures that were subject to various levels of degradation, through the assignment of a likelihood ratio (LR) to the mixture profile. The LR would either support the hypothesis that the person of interest (POI) contributed to the mixture, or support the contrary hypothesis, that the POI was not one of the contributors. Mixtures were prepared at four different contributor ratios with varying combinations of three levels of degradation: no degradation, partial degradation, and full degradation, using controlled heating to systematically degrade the DNA template prior to amplification. Using two hypothesis tests, Mixture Solution was used to compute LRs for each of the mixtures with a variety of defined POIs. Results showed that Mixture Solution successfully generated appropriate LRs for all 20 mixtures in this research, with no Type I errors that falsely excluded a known contributor from a mixture via an LR less than one. Even with the greatest level of degradation and at the most disproportionate ratio of contributors, Mixture Solution was able to assign an LR to each contributor that confirmed their presence in the mixture. When the DNA of a POI was subjected to degradation, decreases in the LR values were observed when compared to the values computed for undegraded DNA from the same POI. However, in all mixtures, Mixture Solution was able to assign an LR with “moderate support” or higher to each of the POIs in both tests, regardless of the level of degradation.

Ribozymes are small RNA molecules that cleave specific RNA molecules enzymatically. Ribozymes can be designed to target specific RNA molecules, thereby modulating gene expression. A hammerhead ribozyme was designed to cleave inhibin $\alpha$ subunit mRNA. This ribozyme was synthesized as double-stranded oligodeoxynucleotides and cloned into plasmid pGEM-4Z. In vitro transcripts of this cloned ribozyme and a cloned rat inhibin $\alpha$ subunit cDNA were incubated together both at 50$\sp\circ$C and at 37$\sp\circ$C. Northern blot analysis revealed two extra bands of the expected size representing the cleavage products. The cleavage was specific. No cleavage was observed when in vitro antisense transcripts of rat inhibin $\alpha$ subunit cDNA were incubated with the ribozyme. Since our goal is to down-regulate inhibin production using ribozyme, a DNA construct carrying the ribozyme was constructed to generate transgenic mice. The ribozyme was connected to the 3$\sp\prime$ end of the E. coli lacZ gene, and was placed under the control of the H-2K$\sp{\rm b}$ gene promoter. Two transgenic mouse lines were established from one transgenic founder, because the injected DNA integrated at two sites, which segregated independently from each other. In one line lacZ expression was detected in tissues including the cornea, the snout, the tongue, the palm of paws, the ventral midline, the pituitary, the dorsum near the spinal cord, on day 13 embryos. Northern blot analysis indicated that ribozyme expression was detected in adult spleen. These results demonstrated that (1) this engineered ribozyme cleaved inhibin $\alpha$ subunit transcripts in vitro, suggesting that there were no secondary structures which interfere with the cleavage activity of the ribozyme; (2) two transgenic mouse lines carrying the ribozyme were established, and expression of the ribozyme was determined.