Browsing by Subject "Gene expression regulation"
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Item Identification and characterization of cystatin-related epididymal and spermatogenic (CRES) expression in anterior pituitary gonadotropes(Texas Tech University, 2001-08) Sutton-Walsh, Heather GayleMale factor infertility contributes to half of all infertility cases, yet current knowledge remains unable to diagnose all but gross abnormalities in male reproductive physiology. In an effort to identify proteins involved in initiating epididymal sperm maturation, our lab identified the cystatin-related epididymal spermatogenic (CRES) gene based on its preferential expression in the initial epididymal segment. Cres represents a unique subgroup of the cystatin family of cysteine protease inhibitors in that its expression is restricted to reproductive tissues and its sequence predicts specificity distinct from classic cystatins. CRES protein is localized to the caput epididymal lumen and the sperm acrosome. Both compartments contain active proteases responsible for protein processing events that are required for sperm function; however, the regulatory mechanisms controlling these processing events are poorly understood. These observations led to the proposal that CRES protein regulates protein processing in these tissues. We hypothesized that CRES might also regulate proteolysis in the pituitary gland for two reasons: (1) the pituitary gland expresses a number of proteases involved in preprotein processing; and (2) the pituitary gland is functionally connected with the gonad via the hypothalamic-pituitary-gonadal axis. Thus, the goal of these studies was to determine whether CRES is expressed in the pituitary gland and if so, to examine its regulation by hypothalamic and/or gonadal factors. These studies demonstrate that CRES is expressed in the anterior pituitary gland and is co-localized intracellularly with leutinizing hormone (LH) in gonadotrope secretory granules. To examine the regulation of Cres mRNA, we developed a semi-quantitative RT-PCR assay and showed that, unlike steroid hormones, GnRH is the primary physiologic regulator of Cres gene expression. Specifically, GnRH administration causes a rapid decrease in Cres mRNA levels, an effect opposite from its potent stimulation of LHp mRNA expression. In contrast, intracellular CRES and LHP protein levels were regulated similarly by steroid hormones, consistent with their being packaged within the same subset of secretory granules. Taken together, these studies indicate that CRES function is influenced by subtle alterations in the hypothalamic-pituitary-gonadal axis, suggesting it may regulate processing events important at specific times for gonadotrope secretory functions.Item Prevalent and differential herpesviral gene regulation mediated by 3'-untranslated regions(2012-08) McClure, Lydia Virginia; Sullivan, Christopher S.Herpesviral infections are currently incurable and are associated with severe human diseases, such as cancer. Kaposi’s Sarcoma-associated Herpesvirus (KSHV), like all herpesviruses, undergoes a long-term, latent infection where few viral products are made as a mechanism to evade the host immune system. Recently, the KSHV latent genome was shown to have bivalent histone marks thought to keep the virus poised for replication. However, it is unclear how the virus prevents spurious leaky transcription from this primed state. The 3' untranslated region (3'-UTR) of transcripts is a common site of gene expression regulation, however less than half of the KSHV 3'-UTRs have been mapped and few studies have interrogated their role during infection. The work presented here is the first large-scale map and analysis of the KSHV 3'-UTRs. Four methods were used to identify the 3'-UTRs expressed by the ~85 KSHV genes, including prediction algorithms, 3'-RACE, DNA tiling array, and next generation deep sequencing analysis. The role of each KSHV 3'-UTR in gene expression was then examined using luciferase reporter assays and showed a surprising prevalence of negative regulation conveyed during latent infection. Sequential deletions across numerous 3'-UTRs indicated RNA structure is likely involved in this regulation. In addition, several KSHV 3'-UTRs conveyed an increase in translation during lytic infection through enhanced recognition by the cap-dependent translation initiation machinery activated via the MNK1 kinase. A second mechanism of KSHV gene regulation was identified through motifs encoded in the K7 3'-UTR. This work indicated that a previously characterized RNA element and a novel putative hairpin are both partially responsible for negative regulation conveyed by the K7 3'-UTR. We hypothesize that these structural motifs control expression of the K7 transcript by altering its sub-cellular location and/or via RNA stability. This work represents a broad 3'-UTR study that mapped the KSHV 3'-UTRs and is the first large-scale functional analysis of 3'-UTRs from a large genome virus. We have implicated post-transcriptional mechanisms, along with known transcriptional regulation, in viral evasion of the immune response during latency and the escape of viral-mediated host shutoff. These results identify new potential targets for therapeutic intervention of KSHV-associated disease.Item The contribution of quorum sensing to the pathogenesis of pseudomonas aeruginosa in burn wound infections(Texas Tech University, 2001-05) Rumbaugh, Kendra P.Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes severe infections in burned patients. In this work, we examined the contribution ofthe cell-to-cell communication systems or quorum sensing (QS) systems to the pathogenesis of P aeruginosa infection of burn wounds. For these studies, we used the thermally-injured mouse model and specific mutants that carry deletions in genes encoding specific components of the P aeruginosa QS systems {lasR, rhlR, lasl and rhll). In comparison with their parent strain (PAOl), all mutants displayed decreased lethality. The mutants were defective in their ability to spread systemically throughout the body of the mice. In addition, the lasR (PAO-Rl) and the lasl/rhll (PAO-JP2) mutants were defective in their ability to spread locally within the burned skin at 8 and 16 hours post-bum/infection. The defects in the PAO-JP2 strain were complemented upon the introduction of a plasmid carrying intact lasl and rhll genes. To determine if the defect in PAO-JP2 is due to the loss of one or more of the QScontrolled virulence factors, isogenic mutants that carry deletions in lasA, lasB, lasAAasB, toxA, lasB/toxA or rpoS were examined. With the exception of the rpoS mutant, all mutants were defective in their in vivo virulence. However, none was as severely defective as PAO-JP2. Our attempt to ascertain the role of autoinducers as virulence factors was confounded by the influence of the solvent used to purify them. Host production of cytokines in response to P aeruginosa infection in bum wounds was examined using the Multi-Probe Template/RNase protection assay. The expression of several proinflammatory and hematopoietic cytokines was up-regulated in burned mice infected with PAOl at 40 hours post-bum/infection. In contrast, the expression of most of these cytokines was not enhanced in burned mice infected with PAO-JP2. These results suggest that: (1) the QS systems play an important role in the pathogenesis of P aeruginosa in bum wound infections; (2) their effects may be contributed to by autoinducers or other, yet undefined, QS-controlled factors; and (3) QS may play a role in modulating the host immune system in response to P. aeruginosa.Item The regulation of Vibrio cholerae MDR efflux Pump in a TolC mutant of Escherichia coli(Texas Tech University, 2004-05) Ramasubramian, BhagavathiThe emergence of antibiotic-resistance among bacterial pathogens is becoming a serious threat to human health and welfare. One mechanism by which bacteria become resistant to antibiotics is to remove them via a multiple drug resistant (MDR) efflux pump. The first multidrug resistance pump was described in tumor cells that were resistant to chemotherapeutic agents. More recently, MDRs have been characterized in both Gram-negative and Gram-positive bacteria. Vibrio cholerae, a Gram-negative non-invasive enteric pathogen, is the causative agent of the severe diarrheal disease cholera. The first multi-drug efflux pump of V. cholerae, VceAB, was isolated and characterized by Colmer et al. In that study the authors cloned a 6.6 kb SalI-Hind III fragment of V cholerae into pBR322, pVC2 and demonstrated that this plasmid was capable of providing both V. cholerae and an Escherichia coli to/C mutant with resistance to a variety of toxic compounds and antibiotics. The authors also demonstrated that this 6.6 kb DNA fragment contained two genes, vceA and vceB, which encoded proteins whose amino acid sequences shared a high degree of similarity with the EmrA and EmrB proteins of E.coli, respectively. Since those studies were published, our laboratory has identified the outer membrane component of this efflux pump, OepX, whose gene resides in an operon with vceA and vceB and a regulatory gene vceR, whose product is a member of the TetR/AcrR transcriptional regulator family and which negatively regulates the oepX-vceA-vceB operon. During that study, the authors constructed an in-frame vceA::phoA gene fusion in pVC2 (pi328). In this construct the phoA gene is fused in-frame to the vceA gene at position 753 I have employed this reporter fusion to examine the regulation of the oepX-vceA- vceB (i.e., VceAB MDR) operon. The activity of this gene fusion, measured by alkaline phosphatase assay was found to be up regulated in the tolC mutant strain. The purpose of this study was to decipher the cause of this regulation which seems to be a tolC effect.Item The RING finger binding protein is a nuclear membrane protein that interacts with RUSH transcription factors(Texas Tech University, 2001-05) Mansharamani, Malini; Chilton, Beverly S.; Lee, Vaughan H.; Pfarr, Curt M.; Pressley, Thomas A.; Webster, Daniel R.; Whelley, Sandra M.Molecular regulation of Uteroglobin gene expression by progesterone and prolactin is mediated by RUSH transcription factors. The RUSH family of proteins, which includes rabbit RUSH-la and P and the human, mouse and plant homologs of RUSH lα, are SWI/SNF related chromatin remodeling proteins. These proteins have a novel C3HC4 RING finger, at their -COOH terminus that has been implicated in mediating protein-protein interactions. When this motif was identified in RUSH proteins, it was used to screen an expression library to isolate cDNAs for proteins that complex with it. A single phage clone (~1.6kb insert) was identified. Sequence analysis of this RING Finger Binding Protein (RFBP) clone, revealed a partial cDNA that lacked an initiator codon but contained a stop codon. RACE PCR was then used to extend the 5' and 3' ends of the cDNA. The predicted amino acid sequence from the composite cDNA sequence (4286-bp) is that of a putative Type IV P-type ATPase. P-type ATPases are membrane transporters that use the energy of ATP hydrolysis to transport substrate across the membrane. Genomic cloning and ClustalW alignment indicate that RFBP is an atypical P-type ATPase that has only seven of eight core regions and nine of ten transmembrane domains typical of this family of proteins. Core region D that contains transmembrane domain four is absent from this protein. Western blot analysis, coupled with immunoelectron microscopy data, indicates that RFBP is present in the inner nuclear membrane. Coimmunoprecipitation and GST pulldown experiments showed a direct interaction between RUSH and RFBP. The RUSH binding site lies within aa 612-804 of the RFBP protein. Competitive quantitative RT-PCR indicates that RFBP is ubiquitous in its expression, with the expression pattern correlating with that of RUSH in these same tissues. In addition, expression of RFBP is hormonally regulated in the endometrium, suggesting that RFBP function and expression may be closely linked with the function of the RUSH proteins in regulating gene expression in the reproductive system. Current studies provide important information about RFBP as a RUSH binding interaction between the proteins to shed light on the mechanism of hormone regulation of uteroglobin gene expression.Item Transcriptional regulation of the cystatin-related epididymal spermatogenic (Cres) gene in the reproductive tract and the identification of novel Cres-like genes(Texas Tech University, 2002-08) Hsia, NelsonAn essential event in male reproduction is formation of functionally mature spermatozoa. Testicular spermatozoa are non-functional and require critical post-testicular modifications in the long convoluted tubule known as the epididymis. Our laboratory previously identified a gene potentially involved in sperm maturation we called cystatin-related epididymal spermatogenic {Cres) which was restricted in its expression to the initial segment epididymidis. Subsequently, it was determined that CRES protein is secreted into the initial segment epididymal lumen and is present in sperm acrosomes and anterior pituitary gonadotrophs. CRES and recently identified CRES-Iike proteins appear to represent a unique subgroup in the cystatin superfamily of cysteine protease inhibitors based on the highly reproductive-specific expression and their divergence in key sequences found in classical cystatins. Recent studies in our laboratory suggest that CRES acts as a cross-class inhibitor of the serine protease prohormone convertase 2 (PC2), a protease involved in prohormone processing. Therefore, CRES may regulate specific proteolytic processing in reproductive tissues. One aspect of understanding sperm maturation is to identify the molecular mechanisms which control the reproductive-specific expression of genes important in reproductive processes. The goal of these transcriptional regulation studies was to identify transcription factors and their cognate motifs that are critical for the mRNA expression of the Cres gene. Analysis of DNA-protein interactions demonstrates that CC/V\T/enhancer binding protein p transcription factor is required for basal levels of Cres gene expression in the epididymis and the anterior pituitary gonadotrophs. To begin to identify the Cres 5'-flanking sequences necessary for Crestspecific expression, we generated transgenic mice that contained approximately 1.6 kb of C/-es5'-flanking sequence driving the chloramphenicol acetyltransferase (CAT) gene. These studies demonstrated that 1.6 kb of Cres 5'-flanking sequence specifically drives CAT expression to the round spermatids of the testis but not in any other tissues including the epididymis and pituitary. Therefore, this promoter sequence can recapitulate the pattern of Cres-specific expression in the testis. The reproductive-specific expression of Cres gene and two other genes similar to Cres, cystatin T and testatin, suggests there may be a group of Creslike genes that function primarily in the reproductive tract, much like the growing number of ADAM (A Disintegrin And Metalloprotease) proteins in the reproductive tract. By screening nucleotide databases, we identified two new members of the Cres subgroup of family 2 cystatins, called Cres2 and Cres3 and demonstrated they exhibit reproductive-specific expression. Cres2 mRNA expression is epididymal-specific, whereas the Cres3 mRNA is present in the testis, epididymis, and ovary. Taken together, these studies demonstrate that epididymal and pituitary transcription of the Cres gene requires the C/EBPp transcription factor for basal levels of expression; however, testicular expression is not dependent on C/EBPs. Interestingly, Cres mRNA expression can be recapitulated only in the testis with 1.6 kb of the Cres promoter, suggesting the elements that control epididymal and pituitary expression of the Cres gene are either not contained in the 1.6 kb 5'- flanking region or perhaps repressors elements within this fragment suppress expression. In addition, we have identified two new members of the Cres subgroup of family 2 cystatins that show restricted expression to reproductive tissues, suggesting these genes have diverged not only in function but also in their transcriptional regulation as compared to the ubiquitously expressed cystatins. Therefore, multiple Cres-\\ke genes may have evolved to have specific and overlapping roles in proprotein processing imperative for reproductive processes.