No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

Using two related dominant negative screens, galactose-negative cDNAs were isolated that contained C. albicans TUB2 (beta-tubulin), NHP6 (non-histone protein 6), CIT2 (citrate synthase) and MRPS9 (mitochondrial ribosomal protein S9) sequences. CaTUB2 encodes beta-tubulin, a component of the microtubule system of the cytoskeleton. Overexpression of this sequence in S. cerevisiae resulted in lethality associated with cell shape changes characteristics of an arrest in the G2/M phase of the cell division cycle. However, overexpression of an ADH1-CaTUB2 fusion in C. albicans did not affect cell shape. CaNHP6 encodes a protein that shows a high level of sequence identity to ScNhp6A and ScNhp6B, and is closely related to High Mobility Group proteins from other eukaryotes. CaNHP6 overexpression in S. cerevisiae cells caused cell cycle arrest. Expression of an ADH1-CaNHP6 fusion in C. albicans did not affect growth rate, but affected both of cell and colony morphogenesis. These effects were not dependent upon the PKC pathway. CaMRPS9 encodes a protein of the small mitochondrial ribosome subunit, and overexpression of this protein caused the slow development of galactose lethality in S. cerevisiae. The development of lethality correlated with the emergence of petite mutants, indicating that the overexpression of CaMRPS9 interferes with S. cerevisiae mitochondrial function, thereby preventing growth on galactose. Similarly, CaCIT2 expression was presumed to block growth of S. cerevisiae on galactose by interfering with respiratory metabolism. Separate approaches were taken to isolate CaGCN4. This gene was of interest because (a) hypha-specific promoters contain GCRE-like sequences [Gcn4 Response Element], (b) amino acid starvation promotes yeast-to-hypha morphogenesis, and (c) GCN-like responses are thought to occur in C. albicans. The 5'-truncated C. albicans GCN4 cDNA was isolated by functional complementation of a S. cerevisiae Delta gcn4 mutation. The 5'-region of the CaGCN4 ORF, including 624 bp of 5'-untranslated region, was isolated by PCR. The sequences of the overlapping 5' and 3'fragments revealed a major ORF capable of encoding a 323 aa protein with significant homology in its C-terminal bZIP domain to ScGcn4 and other fungal Gcn4-like proteins. The existence of this ORF in the C. albicans genome was confirmed by PCR. The CaGCN4 cDNA contained two upstream ORFs and was encoded by a single exon. The contribution of C. albicans Gcn4 to yeast-to-hypa morphogenesis remains to be verified.

Major histocompatibility complex (MHC) class II associated invariant chain is a chaperone of the mammalian MHC class II antigen presentation pathway. It is responsible for targeting the MHC class II dimer to the endocytic pathway, which allows for the loading of exogenous antigens onto the MHC class II receptor. Two genes showing significant sequence similarity to the mammalian invariant chain gene have been identified in rainbow trout. S25-7 and INVX are thought to play a role within the teleost MH class II antigen presentation pathway similar to that performed by the mammalian invariant chain. In vivo and in vitro methods have been used to investigate the regulation of these genes upon immune system activation. To induce an immune response both rainbow trout and the macrophage-like cell line RTS-11 were treated with phorbol myristate acetate (PMA). PMA is a stimulator of protein kinase C, which is involved in the activation of both B and T cells. After PMA treatment, individuals were sacrificed at several time points and tissues were collected for analysis. Up-regulation of IL-1β transcript, as detected by RT-PCR analysis, demonstrated the successful induction of an immune response in vivo. S25-7 transcript levels remained unchanged in gill, spleen, peripheral blood leukocytes and liver. However, the transcript was found to be significantly decreased in head kidney beginning 24 hours post-stimulation. INVX transcript levels remained unchanged in all tissues analyzed. In addition, the level of S25-7 transcript is higher in all tissues than INVX transcript. Western blot analysis detected both S25-7 and INVX proteins within gill and spleen, indicating the presence of cell populations capable of presenting extracellular antigen within the MH class II dimer. Similar to transcript analysis, the level of invariant chain in these tissues remained unchanged during immune system activation. Neither INVX nor S25-7 was detected in head kidney, indicating this tissue may not function as a secondary immune tissue, as previously thought. Finally, western blot analysis failed to detect either INVX or S25-7 protein within liver, as expected in a non-immune tissue. RTS11 analysis demonstrated a similar pattern of invariant chain expression in vitro. S25-7 transcript levels remained stable throughout the PMA stimulation time course, while INVX transcript was down-regulated at 48 and 96 hours post-stimulation, with a subsequent increase at 168 hours. However, both S25-7 and INVX protein levels remained unchanged during immune stimulation. These results demonstrate a pattern of invariant chain tissue expression, providing information about the type of immune functions carried out at these sites. In addition, the results indicate that neither INVX nor S25-7 is up-regulated, at either the transcript or protein level upon immune system activation. This is in contrast to the mammalian invariant chain, which is up-regulated at both the transcript and protein level during an immune response, indicating that teleosts have evolved a unique system of immune regulation.

Major histocompatibility complex (MHC) class II associated invariant chain is a chaperone of the mammalian MHC class II antigen presentation pathway. It is responsible for targeting the MHC class II dimer to the endocytic pathway, which allows for the loading of exogenous antigens onto the MHC class II receptor. Two genes showing significant sequence similarity to the mammalian invariant chain gene have been identified in rainbow trout. S25-7 and INVX are thought to play a role within the teleost MH class II antigen presentation pathway similar to that performed by the mammalian invariant chain. In vivo and in vitro methods have been used to investigate the regulation of these genes upon immune system activation. To induce an immune response both rainbow trout and the macrophage-like cell line RTS-11 were treated with phorbol myristate acetate (PMA). PMA is a stimulator of protein kinase C, which is involved in the activation of both B and T cells. After PMA treatment, individuals were sacrificed at several time points and tissues were collected for analysis. Up-regulation of IL-1β transcript, as detected by RT-PCR analysis, demonstrated the successful induction of an immune response in vivo. S25-7 transcript levels remained unchanged in gill, spleen, peripheral blood leukocytes and liver. However, the transcript was found to be significantly decreased in head kidney beginning 24 hours post-stimulation. INVX transcript levels remained unchanged in all tissues analyzed. In addition, the level of S25-7 transcript is higher in all tissues than INVX transcript. Western blot analysis detected both S25-7 and INVX proteins within gill and spleen, indicating the presence of cell populations capable of presenting extracellular antigen within the MH class II dimer. Similar to transcript analysis, the level of invariant chain in these tissues remained unchanged during immune system activation. Neither INVX nor S25-7 was detected in head kidney, indicating this tissue may not function as a secondary immune tissue, as previously thought. Finally, western blot analysis failed to detect either INVX or S25-7 protein within liver, as expected in a non-immune tissue. RTS11 analysis demonstrated a similar pattern of invariant chain expression in vitro. S25-7 transcript levels remained stable throughout the PMA stimulation time course, while INVX transcript was down-regulated at 48 and 96 hours post-stimulation, with a subsequent increase at 168 hours. However, both S25-7 and INVX protein levels remained unchanged during immune stimulation. These results demonstrate a pattern of invariant chain tissue expression, providing information about the type of immune functions carried out at these sites. In addition, the results indicate that neither INVX nor S25-7 is up-regulated, at either the transcript or protein level upon immune system activation. This is in contrast to the mammalian invariant chain, which is up-regulated at both the transcript and protein level during an immune response, indicating that teleosts have evolved a unique system of immune regulation.

No description available.