The DNA sequence of the Tyl activating region from the CYC7-H2 mutant of Saccharomyces cerevisiae is presented. Analysis of the data revealed the presence of four simian virus 40-type enhancer core sequences. Two of the Tyl enhancer cores are contiguous with sequences also homologous to the diploid control site at MATa. We postulate that these two Tyl regions of :30 base pairs are regulatory blocks, and we have analyzed deletions to ascertain whether they are necessary for effects of Tyl on adjacent gene expression. We found that activation is lost when a restriction fragment encompassing both postulated regulatory blocks is deleted. Deletion of restriction fragments that remove only one of the two regulatory blocks has little or no effect on Tyl activating ability in haploid yeast cells or on repression of this function in diploid yeast cells. Because the most significant internal homologies in the restriction fragments analyzed are the putative regulatory blocks, these observations suggest that enhancer-like sequences are involved in cell-type control of Tyl effects on gene expression.The CYC7-H2 mutation in Saccharomyces cerevisiae causes overproduction of the respiratory protein, iso-2-cytochrome c. This regulatory mutation was caused by insertion of a 5.5-kilobase (kb) sequence in the 5' noncoding region of the CYC7 structural locus (1). The inserted sequence is a member of the Tyl family of dispersed repetitive elements found in yeast (2).Tyl insertion places CYC7-H2 expression under control of yeast cell type, which can be a, a, or a/a (see ref. 3 for review). In either a or a haploid cells of yeast, the CYC7-H2 mutation causes a 20-fold overproduction of iso-2-cytochrome c. In a/a diploid cells, CYC7-H2 expression is repressed -10-fold. The a and a cell types are established by the presence of either a or a alleles, respectively, at the mating type locus (MAT). MATa Cell-type dependent regulation of the CYC7-H2 mutation is shared by certain other Tyl insertion mutations in yeast (see ref. 6 for review). Among these are the CYP34, cargA+Oh, and ADH2C mutations in which Tyl insertions were shown to be within a region between 125 and 600 bp upstream from the various coding sequences. In all of these insertion mutants, the Ty element is oriented so that its direction of transcription is opposite that of the affected gene. Also, it has been found that the steady-state mRNA corresponding to each of these genes is increased or made constitutively, but size and 5' map position of the transcript are not affected. These observations show that the Tyl element is not providing a new transcription initiation site but is regulating transcription from various upstream positions. This effect is similar in certain respects to the action of enhancer sequences, such as that of the simian virus 40 (SV40) 72-bp repeat (see ref. 7 for review). However, unlike the SV40 enhancer, the orientation of Ty sequences inserted at the 5' end of the affected genes may be important. A similar orientation effect is associated wi...
PI? ilcidelp h iu , Pen n sy 1 L W n ici I 9 I 04 SummaryThe stabilities and optima with respect to temperature and pH of the P-glucosidase, Avicelase, and carboxymethylcellulase (CMCase) activities of Thermomonospora sp., in the culture filtrate, culture whole broth, and filtrate after sonication of culture solids, are reported. The P-glucosidase is cell associated and has an optimal activity at about pH 6.5 and 55°C. In the whole culture broth, it has a half-life of about 8 hr at 55°C and less than 1 hr at 60°C. while the half-life of the activity in the sonicated, cell-free filtrate is less than 1 hr at 55°C. The Avicelase and CMCase activities occur in the extracellular culture fluid and have optima at about pH 7.0 and 5.9, and 65 and 7O"C, respectively. The CMCase activity is stable over 24 hr at 60"C, but declines by 50% in the same period at 65°C. The Avicelase activity declines by 15% over 24 hr at 55°C. and by 50% at 60°C. The highest pH studied (pH 7.3) was the most destabilizing for all three activities. The thermostable characteristics of the cellulases from 7herniomonosporcr appear to make them suitable for commercial saccharification processes operating at elevated temperatures.
The saccharification of cellulosic substrates by cellulase from Thermomonospora fusca, strain YX, was influenced by the presence of various substances. Cellobiose was strongly inhibitory, reducing the activity against swollen cellulose to 25% at 5% concentration. Glucose had much less effect, reducing activity to 40% at 20% concentration. Ethanol was found to be only slightly inhibitory to the cellulase, reducing activity by about 15% at 6% concentration, but much more inhibitory to the cell-bound beta-glucosidase activity. Of the numerous metal ions examined, Ca(2+) and Co(2+) at 0.1mM-1.0mM concentration were found to be slightly activating under assay conditions, while 1.0mM Pb(2+) and Hg(2+) were the most inhibitory ions. The implications of these results for the design of commercial processes for ethanol production from cellulose are discussed.
The carboxymethyl-cellulase and Avicelase activities of Thermoactinomyces sp., strain YX, were produced simultaneously with cell growth. Throughout the growth phase these activities were primarily extracellular, with up to 50% adsorbed to residual cellulosic substrate at any one time. On the other hand, the
The cell-associated beta-glucosidase activity of Thermomonospora fusca, strain YX, showed both PNPGase and cellobiase activities. The cellobiase activity was found by HPLC assay to have very low product inhibition, whereas the PNPGase activity was more significantly inhibited. Of the various sugars and sugar analogs tested for inhibition of the PNPGase activity, gluconolactone had the greatest effect. The low product inhibition of the cellobiase activity was further demonstrated by the production of glucose syrups to 20% concentration from both cellobiose and swollen cellulose (Avicel). This characteristic is of practical importance in the development of a commercial process for the production of glucose syrups from cellulose. Growth experiments gave further evidence for the probability of separate enzymes for the PNPGase and cellobiase activities.
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