To study peptide absorption in chickens, an intestinal peptide transporter cDNA (cPepT1) was isolated from a chicken cDNA library. The cDNA was 2,914-bp and encoded a protein of 714 amino acid residues. Twenty-three di-, tri-, and tetra-peptides were used for functional analysis of cPepT1 in Xenopus oocytes and Chinese hamster ovary (CHO) cells. For most di- and tripeptides tested, the Kt was in the micromolar range, except Lys-Lys and Lys-Trp-Lys. Northern analysis demonstrated that cPepT1 is expressed strongly in the small intestine, and at lower levels in kidney and cecum. These results demonstrated the presence and functions of a peptide transporter in chickens. cPepT1 mRNA abundance was evaluated in response to developmental and dietary regulations. In Experiment 1, eggs at incubation day 18 (E18) and Cobb chicks after hatch (d 0) were sampled before treatments. Three groups of chicks were fed diets containing 12, 18, or 24% crude protein (CP). Feed intake of chicks fed the 18 or 24% CP diets was restricted to that of chicks fed the 12% CP diet. In Experiment 2, a fourth group with free access to the 24% CP diet was added. cPepT1 mRNA abundance was quantified from northern blots. By d 0, there was a 50-fold increase in cPepT1 mRNA abundance compared with E 18. In chicks fed the 12% CP diet, cPepT1 mRNA abundance decreased throughout the 35 d. Chicks fed 18 or 24% CP diets showed an increase in cPepT1 mRNA abundance with time. In chicks with free access to the 24% CP diet, cPepT1 mRNA decreased until d 14 but returned to an intermediate level at d 35. Our results indicate that cPepT1 mRNA is regulated by both dietary protein and developmental stage. To investigate the kinetics of an ovine peptide transporter (oPepT1), CHO cells were transfected with oPepT1 cDNA. Uptake of Gly-Sar by transfected cells was pH-dependent, concentration-dependent, and saturable. Competition studies showed that all di-, tri-, and tetra-peptides inhibited uptake of Gly-Sar. Pretreatment of the cells with staurosporine resulted in an increase in peptide transport. This increase was blocked by pretreatment with PMA. The results indicate that protein kinase plays a role in oPepT1 function. / Ph. D.

Black bears are omnivorous, and tend to be opportunistic feeders, in that they will eat what is readily abundant or available. The end-products of intestinal digestion are absorbed by the body through the action of transporter proteins expressed on the brushborder membrane of small intestinal epithelial cells. The goal of this study was to increase the understanding of the physiological processes associated with nutrient assimilation by black bears. Distribution and relative abundance of mRNA of a peptide transporter (PepT1), a glucose transporter (SGLT1), two AA transporters (NBAT, bo,+AT), and a digestive enzyme, aminopeptidase N (APN), in the intestinal tract of black bears were investigated. Ten bears were used for this study. For tissue collection, the intestine was removed from the animal and divided into five sections. Each collected section was opened longitudinally, rinsed in ice-cold PBS, and the mucosal scrapings were stored at -80&#61616;C. Total RNA was extracted and quantified by spectrophotometry. Abundance of PepT1, SGLT1, NBAT, bo,+AT, and APN mRNA was determined by performing Northern blots, using bear cDNA probes. Northern blot data were quantified by densitometric analysis, with the abundance of each gene expressed relative to GAPDH. Abundance of PepT1 (P < 0.05), APN (P < 0.05), and SGLT1 (P < 0.0001) changed quadratically from the proximal to the distal intestine with abundance being greatest in the midregion. Abundance of bo,+AT mRNA increased linearly (P < 0.05) from the proximal to distal intestine. Abundance of NBAT mRNA did not change among intestinal segments.The absolute number of molecules of mRNA/ng of total RNA for each gene was determined using Real-Time PCR. Similar to the Northern results, abundance of PepT1 (P < 0.0003), SGLT1 (P < 0.0003), and APN (P < 0.02) changed quadratically from the proximal to distal intestine with abundance being greatest in the mid-region, and bo,+AT mRNA increased linearly (P < 0.0001) from the proximal to distal intestine. NBAT mRNA abundance also increased linearly (P < 0.0001) from proximal to distal intestine. PepT1 mRNA was present at tenfold or greater levels than AA transporter mRNA in all segments of the intestine, suggesting that di- and tripeptides constitute the major form in which AAs are absorbed. NBAT and bo,+AT mRNA abundance was greater towards the distal portion of the intestine, suggesting their importance in salvaging remaining unabsorbed AAs.These results indicate that the mRNA of nutrient transporters examined and APN are differentially expressed throughout the gastrointestinal tract of black bears, suggesting their involvement in nutrient assimilation. / Master of Science

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Les microARN (miARN) sont des petits ARN non-codants pour des protéines qui permettent d’inhiber la traduction d’ARN messagers. Pour obtenir un miARN, un gène de miARN passe à travers une voie de maturation dans laquelle il sera coupé à deux reprises par les enzymes Drosha et Dicer. Pour interagir avec les enzymes, les gènes de miARN possèdent une structure générale en tige-boucle. Cependant, les détails de cette structure sont encore peu connus. L’objectif principal de ce projet était d’établir s’il y a une relation entre l’efficacité de maturation et la dynamique de la structure. Pour cela, l’efficacité de maturation de plusieurs variants de miARN a été évaluée par Northern Blot. La dynamique de la structure a été mesurée par un programme informatique à partir de l’information de la séquence. Une corrélation de 0,74 avec une valeur p de 0,02206 a été obtenue entre les dynamiques et les ratios d’efficacités de maturation de miARN. Cette corrélation est supérieure à celle obtenue basée sur l’énergie libre des structures prédites les plus stables qui n’atteignent pas 0,6. Les mutants de miR128-1 et miR188 ont été découverts comme diminuant la maturation. De plus, les mutants de miR125a, miR188 et miR330 affectent le site de clivage de Drosha. Une meilleure connaissance de la dynamique de l’ARN impliquée dans la maturation permettrait de définir l’impact des mutations dans les séquences de miARN ou encore de prédire les séquences pouvant générer des miARN. / MicroRNAs (miRNAs) are small non-coding RNAs, which can inhibit target messenger RNAs translation. In order to obtain a miRNA, two enzymes, Drosha and Dicer cut the gene of miRNA. The RNA interacts with the proteins by its general hairpin structure. However, the details of the structure are still missing. The objective of this project is to establish if there is a relation between the efficiency of maturation and the RNA’s structural dynamics. In order to do this, the maturation efficiency of miRNA variants is measured by Northern Blot. The structural dynamics is measured by a program assessing the information of the sequence. The correlation between the dynamics and the maturation efficiency of the miRNA is 0.74 with a p-value of 0.02206. This correlation is superior to those based on free energy, which does not reach 0.6. The tested mutants of miR128-1 and miR188 have inhibited maturation; also, those of miR125a, miR188 and miR330 have modified the cleavage site of Drosha. A better knowledge of the dynamic structure involved in maturation would help define the impact of miRNA mutation or to predict sequences that are able to generate miRNAs.

Streptomyces are medically important soil-living bacteria that undergo morphological changes from spores to aerial hyphae and are important producers of bioactive compounds including antibiotics. Their gene expression is tightly regulated at the early level of transcription and translation. In the transcriptional control, sigma factors play a central role; the model organism Streptomyces coelicolor possesses astonishing 65 sigma factors. The expression of sigma factors themselves is controlled on the post-transcriptional level through the action of sRNAs that modify their mRNA level. However, only several sigma factors in Streptomyces have known regulons and also their sRNAs-mediated regulation has not been studied so far. According to previously measured gene expression data, we selected several highly expressed sigma factors. Using mutant strains with HA-tagged sigma factors, regulons of two important sigma factors, SigQ and HrdB, were analyzed by ChIP-seq procedure. Other sigma factors were further studied to see if they possess asRNAs, using 5' and 3' RACE method and northern blotting. Our data confirm the essentiality of HrdB sigma factor during the vegetative phase of growth. The other sigma factor, SigQ, has been revealed to be an important regulator of nitrogen metabolism and osmotic...

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No description available.