A Kluyveromyces lactis mutant defective in lac9 cannot induce ,-galactosidase or galactokinase activity and is unable to grow on lactose or galactose. When this strain was transformed with the GAL4 positive regulatory gene of Saccharomyces cerevisiae it was able to grow on lactose or galactose as the sole carbon source. Transformants bearing GAL4 exhibited a 4.5-h generation time on galactose or lactose, versus 24 h for the nontransformed lac9 strain. A K. lactis lac9 strain bearing two integrated copies of GAL4 showed 3.5-fold induction of ,3-galactosidase activity and 1.8-fold induction of galactokinase activity compared with 15.6-fold and 4.4-fold induction, respectively, for the LAC9 wild-type strain. In transformants bearing 10 integrated copies of GAL4, the induced level of I-galactosidase was nearly as high as in the LAC9 wild-type strain. In addition to restoring lactose and galactose gene expression, GAL4 in K. lactis lac9 mutant cells conferred a new phenotype, severe glucose repression of lactose and galactose-inducible enzymes. Glucose repressed 11-galactosidase activity 35-to 74-fold and galactokinase activity 14-to 31-fold in GAL4 transformants, compared with the 2-fold glucose repression exhibited in the LAC9 wild-type strain. The S. cerevisiae MEL] gene was repressed fourfold by glucose in LAC9 cells. In contrast, the MEL] gene in a GAL4 lac9 strain was repressed 20-fold by glucose. These results indicate that the GAL4 and LAC9 proteins activate transcription in a similar manner. However, either the LAC9 or GAL4 gene or a product of these genes responds differently to glucose in K. lactis.The galactose-melibiose regulon of Saccharomyces cerevisiae and the galactose-lactose regulon of Kluyveromyces lactis have several common features. Both have a galactose gene cluster with the order GAL7-GALIO-GAL1 (2, 24). These clusters code for galactose-1-phosphate (gal-1-P) uridyl transferase (GAL7), UDP-galactose-4-epimerase (GALIO), and galactokinase (GALl) (10,24 These phenomenological and organizational similarities suggest functional and mechanistic similarities between the two regulons. Recently it has been shown that the K. lactis GALl-GAL10-GAL7 cluster is inducible by galactose in S. cerevisiae (Webster and Dickson, unpublished results), indicating GAL4 and GAL80 regulation of the K. lactis cluster. These data directly imply that GAL4 and LAC9 activate transcription in a similar manner. No direct implication between the similarity of GAL80 and LACIO can be drawn from these data. Since GAL4 protein must bind to upstream activator sequences (UASGAL) (3,14) in order to activate transcription, one would expect the K. lactis GAL genes to * Corresponding author.have sequences related to UASGAL, and they do (4; S. Bhairi, Ph.D. thesis, University of Kentucky, Lexington, 1984; Webster and Dickson, unpublished results).Despite the phenomenological and organizational similarities the regulons are strikingly different in their response to glucose. The galactose-melibiose regulon is severely repressed by gluco...
Mutants of Kluyveromyces lactis defective in lactose transport were identified among lactose-resistant revertants of lactose-sensitive strains. The mutations are closely linked to the beta-galactosidase gene, LAC4, and they are located in a previously identified gene, LAC12, which has been shown to code for a lactose permease. Our data establish that LAC12 is the only lactose permease gene in K. lactis. The lactose permease also transports galactose. LAC12 is transcribed in a direction opposite to that of LAC4, there being about 2.5 kb between their transcription start sites. Transcription of LAC12 is inducible as is that of all other structural genes in the lactose-galactose regulon of K. lactis.
Mast cells are central in the development of several allergic diseases and contain a number of pre-formed mediators. b-tryptase, the most abundant mast cell product, is increasingly recognized as a key inflammatory mediator, as it causes the release of cytokines, particularly the chemokine IL-8, from both inflammatory and structural cells. The molecular mechanisms, however, remain largely unknown. In this study we sought to investigate whether b-tryptase could induce IL-8 expression in human airway smooth muscle (ASM) cells and to explore the molecular mechanisms involved. We found that purified human b-tryptase stimulated IL-8 production in a timeand concentration-dependent manner, which was inhibited by protease inhibitors and mimicked by recombinant human b-tryptase, but not by the protease-activated receptor-2 (PAR-2) agonist SLIGKV-NH 2 , consistent with the low-level expression of PAR-2 protein in these cells. b-tryptase also up-regulated IL-8 mRNA expression, as analyzed by RT-PCR and real-time PCR, which was abolished by the transcription inhibitor actinomycin D. Reporter gene assay showed that b-tryptase-induced IL-8 transcription was mediated by the transcription factors activator protein-1, CCAAT/enhancer binding protein, and NF-kB, and chromatin immunoprecipitation assay demonstrated that b-tryptase induced in vivo binding of these transcription factors to the IL-8 gene promoter. Furthermore, b-tryptase stabilized IL-8 mRNA, suggesting additional post-transcriptional regulation. Collectively these findings show that b-tryptase upregulates IL-8 expression in ASM cells through a PAR-2-independent proteolytic mechanism and coordinated transcriptional and posttranscriptional regulation, which may be of particular importance in understanding the role and the mechanisms of action of b-tryptase in regulating chemokine expression in mast cell-related disorders.
Hsc70's expected binding site on helix II of the J domain of T antigens appears to be blocked in its structure bound to tumor suppressor pRb. We used NMR to map where mammalian Hsc70 binds the J domain of murine polyomavirus T antigens (PyJ). The ATPase domain of Hsc70 unexpectedly has its biggest effects on the NMR peak positions of the C-terminal end of helix III of PyJ. The Hsc70 ATPase domain protects the C-terminal end of helix III of PyJ from an uncharged paramagnetic probe of chelated Gd(III), clearly suggesting the interface. Effects on the conserved HPD loop and helix II of PyJ are smaller. The NMR results are supported by a novel assay of Hsc70's ATP hydrolysis showing that mutations of surface residues in PyJ helix III impair PyJ-dependent stimulation of Hsc70 activity. Evolutionary trace analysis of J domains suggests that helix III usually may join helix II in contributing specificities for cognate hsp70s. Our novel evidence implicating helix III differs from evidence that Escherichia coli DnaK primarily affects helix II and the HPD loop of DnaJ. We find the pRb-binding fragment of E2F1 to be intrinsically unfolded and a good substrate for Hsc70 in vitro. This suggests that E2F1 could be a substrate for Hsc70 recruited by T antigen to an Rb family member. Importantly, our results strengthen the chaperone hypothesis for E2F release from an Rb family member by Hsc70 recruited by large T antigen. That is, it now appears that Hsc70 can freely access helix III and the HPD motif of large T antigen bound to an Rb family member.
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