This study focuses on the gene (glnA) encoding glutamine synthetase from the cyanobacterium Synechococcus sp. strain PCC 7942. A molecular analysis of the gene was initiated to test the hypothesis that expression of glnA is regulated at the level of transcription and that this regulation is reflective of global control of nitrogen metabolism in Synechococcus 7942. The method used for testing the hypothesis involved first isolating and sequencing the glnA gene and its flanking regions, and second, analyzing the RNA produced by the glnA gene by transcript mapping. Sequence analysis of the glnA gene revealed high homology within the open reading frame at the nucleotide level when compared with glnA from the cyanobacterium Anabaena 7120, and lower homology with glnA from other bacteria. Comparisons of the deduced amino acid sequences showed a similar pattern of highest homology between the cyanobacterial glutamine synthetases, with lower homologies in comparison with other bacteria. Northern analysis using the Synechococcus glnA gene as a hybridizing probe revealed a transcript of 1.6 kb, verifying expression of glnA. It was upon these three criteria--heterologous hybridization with the glnA gene of Anabaena 7120, sequence comparisons with several other bacterial glnA genes, and identification of a 1.6 kb transcript--that the open reading frame was identified as the structural gene (glnA) of glutamine synthetase. The results of transcript mapping indicate that transcription begins at a start site 142 nucleotides upstream of the translational start when cells are grown under either nitrogen sufficient or nitrogen deficient (N for 10 hours) conditions. When cells are grown in a medium lacking nitrogen for only 4.5 hours, the transcriptional start site maps to a position 139 nucleotides upstream of the translational start. The identification of two transcriptional start sites, 3 base pairs apart, might be an artifact inherent in the experimental strategy, or it may imply transcriptional control. A sequence consisting of TAGGAT is present 14 or 17 bases upstream of the two transcriptional starts, respectively, and is similar to other Synechococcus promoters (TAGAAT in psbA1 and TATTAT in psbA2) as well as the $-$10 promoter, (TATAAT), found in enteric bacterial $\sigma\sp{70}$ promoters. Two overlapping sequences, GTTACA and CAAAAG, are positioned in the $-$35 promoter region. The first of these resembles the $-$35 region for three light responsive genes (TTTACA for psbA1 and psbA2, and TTCACA for psbA3) from Synechococcus. It also resembles, TTGACA, found at $-$35 in unregulated promoters of enteric bacteria. The other sequence, CAAAAG, resembles two Anabaena 7120 $-$35 promoter regions, CAAAAC in glnA, and CATAAC in nifH, which are nitrogen regulated. Taken together, the presence of two potential $-$35 promoter regions and two transcriptional start sites, indicates transcriptional regulation of glnA in Synechococcus 7942.

MRP13 was defined by biochemical criteria as a 35-kilodalton small subunit protein of the yeast mitochondrial ribosome. The MRP13 gene was identified by immunological screening of a yeast genomic library in $\lambda$gt11 and a functional copy of the gene has been cloned on a 2.2-kilobase BglII fragment. Sequencing of this fragment showed that the MRP13 coding region specifies as 324-amino acid basic protein with a calculated M$\sb{\rm r}$ of 37,366. Computer searches failed to reveal any significant sequence similarity to previously identified ribosomal proteins or to the sequences in the current National Biomedical Research Foundation data base. Cells carrying disrupted copies of MRP13 lacked the MRP13 protein but were not impaired for growth on nonfermentable carbon sources. However, in comparison to the wild type, mrp13-$\Delta$2::TRP1 mutant cells had a lower rate of whole cell respiration, an unusual profile on in vivo labeled mitochondrial translation products and an abnormal profile of ribosomal subunits in sucrose gradient centrifugation. Mutants lacking MRP13 were also impaired in their ability to undergo the transition from growth on high concentrations of glucose to growth on nonrepressing carbon sources. This mutant phenotype suggests an important role for the MRP13 protein under conditions where cells are actively increasing their capacity for the synthesis of mitochondrial encoded proteins. Analysis of the sequence in the MRP13 5$\sp\prime$-flanking region revealed the closely linked gene for the cytoplasmic ribosomal protein RP39A. The RP39A coding region begins at nucleotide $-$846 and ends at $-$325 with respect to the MRP13 translational start. The steady-state levels of the MRP13 mRNA were determined in response to carbon catabolite repression, variation in the mitochondrial genetic background, and increased gene dosage of MRP13. In $\rho\sp+$ cells, transcript levels were repressed severalfold by growth in glucose as compared with growth in either galactose or nonfermentable carbon sources. In respiratory-deficient strains ($\rho\sp{\rm o,}$ mit$\sp-$), however, transcription appeared to be largely derepressed even in the presence of high concentrations of glucose. Thus, the MRP13 mRNA is a member of a class of yeast nucleus-encoded RNAs whose transcription responds to changes in the mitochondrial genetic background. Despite high levels of the MRP13 transcripts in $\rho\sp{\rm o}$ cells, the MRP13 protein did not accumulate, suggesting that the protein is relatively unstable in the absence of ribosome assembly. Cells carrying the MRP13 gene on a multiple-copy plasmid overproduced the mRNA in rough proportion to the gene dosage and the protein accumulated to a significant but lesser extent. The results indicate that MRP13 expression is regulated predominantly at the transcriptional level in response to catabolite repression and the cellular capacity for respiration. In addition, the levels of the MRP13 protein appear to be modulated posttranscriptionally by degradation of excess, unassembled polypeptides.

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