The carboxyl-terminal region of human interferon {gamma} is important for biological activity: mutagenic and NMR analysis

Deletion of nine amino acids from the carboxyl terminus of humanIFN (residues 138–146; LFRGRRASQ) resulted in a 7-foldincrease in specific antiviral activity. Similar increases inreceptor binding affinity were seen. Deletion of residues 136and 137 (QM) had little additional effect, but removal of Ser135resulted in a sharp drop in antiviral activity. Further removalof residues 133 and 134 (KR) lowered antiviral activity to 1%of the peak value. Comparison of the proton NMR spectra of selecteddeletions down to residue 132 showed that there was no significantchange in the core protein structure. Deletions down to residue125 had the same antiviral activity as those to 132, but changescould now be seen in the aromatic proton NMR spectrum of thisshorter derivative. Substitution of the homologous murine sequencebetween residues 124 and 130 (human SPAAKTG; murine LPESSLR)resulted in only a small decrease in antiviral activity, furthersuggesting that the precise sequence in this region was notcritical for activity. Ser135 was substituted with a numberof other amino acids with little or no change in activity. Theimportance of the residues between 131 and 134 for biologicalactivity was corroborated by mutagenesis, although some substitutionsin this region were tolerated.     

Expression of functional papain precursor in Saccharomyces cerevisiae: rapid screening of mutants

A microbial expression system for the study of the cysteineprotease papain has been developed as a more useful alternativeto the insect cell/baculovirus expression system we have previouslyused. A synthetic papain precursor (propapain) gene was expressedin the yeast Saccharomyces cerevisiae under the control of the-factor promoter. Efficient expression required fusion of thepropapain sequence with the yeast -factor prepro region anda yeast host cell defective in the synthesis of vacuolar proteases.Surprisingly, the glycosylated form of the inactive papain precursoris not secreted, but accumulates within the yeast cell. Completeconversion of the intracellular zymogen into active mature papaincould be achieved in vitro. Purified recombinant papain producedby the yeast system has kinetic characteristics similar to thoseof the natural enzyme. An advantage of the yeast expressionsystem over the baculovirus/insect cell system is that we canperform mutagenesis and screening of papain mutants very efficiently.We have set up a ‘one-tube’ screening procedurefor the simultaneous characterization of numerous mutants ofthe papain precursor. Yeast cells are grown and lysed in microtiterplate wells and the released papain precursor is then activatedto mature papain. This assay allows easy discrimination betweenproteins with close to wild type properties and proteins thatare not functional. We have applied this assay to investigatethe spectrum of amino acids which are tolerated at Asnl75 ofpapain using two independently derived libraries of mutantsat this position. Many amino acid substitutions at this positionare not accepted; only the reintroduction of Asn restored normalfunction.     

Assembly of phosphofructokinase-1 from Saccharomyces cerevisiae in extracts of single-deletion mutants

Phosphofructokinase-1 from Saccharomyces cerevisiae is an octameric enzyme comprising two non-identical subunits, α and β, which are encoded by the unlinked genes PFK1 and PFK2. In this paper, assembly and reactivation of the enzyme have been studied in cell-free extracts of single-deletion mutants. In contrast to the previously described lack of phosphofructokinase-1 activity in cell-free extracts of these mutants, we could measure a temporary enzyme activity immediately after lysis of protoplasts. This result supports the assumption that each of the subunits forms an enzyme structure which is active in vivo but not stable after cell disruption. Upon mixing of separately prepared cell-free extracts of both deletion mutants very low activity could be measured. About 40% of the wild-type activity was regained when both mutants were mixed prior to disruption. The reactivation rate could be slightly increased by addition of ATP and fructose 6-phosphate and was found to be a function of the growth state, particularly of the β-subunit-carrying cells. The individual subunits did not interact with Cibacron Blue F3G-A, a biomimetic ligand of phosphofructokinase-1. After reassembly of both subunits in vitro a strong affinity of the reconstituted phosphofructokinase-1 to the dye-ligand was observed. The inability of the subunits to reconstitute under certain conditions seems to result from alterations of the intracellular environment following disruption. These changes give rise to induce an unproductive side reaction like self-aggregation of the subunits. Because reconstitution of phosphofructokinase-1 from S. cerevisiae behaves in a similar way to that of hemoglobin and luciferase, we would speculate a general mechanism for assembly of oligomeric proteins in vivo. © 1998 John Wiley & Sons, Ltd.     

Genetic transformation of auxotrophic mutants of the filamentous yeast Trichosporon cutaneum using homologous and heterologous marker genes

A transformation system for the filamentous yeast Trichosporon cutaneum based on auxotrophic markers is presented and techniques for the induction, isolation and characterization of mutants are described. A number of auxotrophic mutants were isolated and characterized by using biosynthetic precursors and/or inhibitors. A mutant unable to grow in the presence of ornithine could be complemented successfully by spheroplast transformation experiments using the cloned Aspergillus nidulans ornithine transcarbamoylase gene (argB gene) as selection marker with an efficiency of 5-100 transformants per microgram of DNA. In these transformants the heterologous argB gene was present in multiple tandem copies and the transforming DNA was found to remain stable after more than 50 generations in non-selective media. The same mutant could be complemented by a T. cutaneum cosmid gene library and a complementary cosmid was subsequently isolated from this library by a sib-selection strategy. This cosmid transformed T. cutaneum spheroblasts with an efficiency of 50-200 colonies per microgram of DNA. Southern blot analyses were consistent with the view that the transforming sequences became stably integrated into the host genome at the homologous site.     

A truncated antenna mutant of Chlamydomonas reinhardtii can produce more hydrogen than the parental strain

Photoproduction of H₂ gas was examined in the Chlamydomonas reinhardtii tla1 strain, CC-4169, containing a truncated light-harvesting antenna, along with its parental CC-425 strain. Although enhanced photosynthetic performance of truncated antenna algae has been demonstrated previously (Polle et al. Planta 2003; 217:49-59), improved H₂ photoproduction has yet to be reported. Preliminary experiments showed that sulfur-deprived, suspension cultures of the tla1 mutant could not establish anaerobiosis in a photobioreactor, and thus, could not photoproduce H₂ gas under conditions typical for the sulfur-deprived wild-type cells (Kosourov et al. Biotech Bioeng 2002; 78:731-40). However, they did produce H₂ gas when deprived of sulfur and phosphorus after immobilization within thin (∼300 μm) alginate films. These films were monitored for long-term H₂ photoproduction activity under light intensities ranging from 19 to 350 μE m⁻² s⁻¹ PAR. Both the tla1 mutant and the CC-425 parental strain produced H₂ gas for over 250 h under all light conditions tested. Relative to the parental strain, the CC-4169 mutant had lower maximum specific rates of H₂ production at low and medium light intensities (19 and 184 μE m⁻² s⁻¹), but it exhibited a 4-times higher maximum specific rate at 285 μE m⁻² s⁻¹ and an 8.5-times higher rate at 350 μE m⁻² s⁻¹ when immobilized at approximately the same cell density as the parental strain. As a result, the CC-4169 strain accumulated almost 4-times more H₂ than CC-425 at 285 μE m⁻² s⁻¹ and over 6-times more at 350 μE m⁻² s⁻¹ during 250-h experiments. These results are the first demonstration that truncating light-harvesting antennae in algal cells can increase the efficiency of H₂ photoproduction in mass culture at high light intensity.     

Increase in gene expression by respiratory-deficient mutation

Respiratory-deficient mutants (rho- cells) of Saccharomyces cerevisiae produced about 10 times as much human (h-) lysozyme as did wild-type strains (rho+ cells) when the GAL10 promoter was used in an expression plasmid with the h-lysozyme gene. Introduction of intact mitochondria into the rho- cells resulted in a significant decrease in the production of h-lysozyme, indicating that the rho- mutation increased the expression of the h-lysozyme gene. The copy number of the expression plasmid was not responsible for the increased expression. The level of h-lysozyme mRNA in the rho- cells was also much higher than that in the rho+ cells especially at the stationary phase. The increased expression of the h-lysozyme gene was also observed when a glyceraldehyde-3-phosphate dehydrogenase gene promoter and the PHO5 promoter were used in the expression plasmid. The rho- mutation also increased the expression of the PHO5 gene under the control of the HIS5 promoter in a plasmid and the ACT1 gene in the yeast chromosome, but did not increase the expression of the ribosomal RNA gene. In contrast to the rho- mutants, pet mutants did not show higher gene expression compared with wild-type strains.     

高产柠檬酸的SHAM特性黑曲霉突变株的选育

研究了SHAM (水杨酸氧肟酸 )对产酸的影响。 5mg/ 6 0mL的SHAM基本抑制柠檬酸的产生。当W 3菌株在加有 10mg/ 10mLSHAM(L1)的平板上生长时 ,只能着生 10 %的孢子 ,在加有 2 0mg/ 10mLSHAM (L2 )的平板上生长时不能着生孢子。据此选育出不能够在L1平板上着生孢子的SHAM敏感株和在L2平板上能够着生孢子的SHAM抗性株。通过发酵试验 ,SHAMS- 2菌株产柠檬酸比出发菌株W 3高 4 0 % ,转化率达到 91 7%     

A Conditional Sterol Esterification Defect in Yeast having either a sec1 or sec5 Mutation in the Secretory Pathway

Two temperature-conditional secretory mutations, sec1 and sec5, cause the accumulation of post-Golgi vesicles when strains containing these mutations are grown at 37°C. In addition to accumulating vesicles, the mutants do not esterify free sterol on rich media at the restrictive temperature. It is the high level of inositol in the media that causes this condition in the yeast Saccharomyces cerevisiae, not a defective steryl ester synthase or lack of substrates. When strains containing the sec1 or sec5 mutation were transformed separately with a plasmid carrying SEC1 and SEC5, the esterification and secretory defects were alleviated. Double mutants containing sec6, sec14 or sec18 with either a sec1 or sec5 mutation have normal esterification levels. Strains with suppressor mutations were isolated that grew at 37°C, esterified sterols and had diminished accumulation of vesicles, when grown at the restrictive temperature on defined media with additional inositol. Electron microscopy was used to examine vesicle accumulation, the number of lipid droplets, and to further characterize the esterification defect. When grown at 37°C on defined medium, the strains with sec5 or sec1 accumulated the usual secretory vesicles, but when grown under similar conditions with elevated levels of inositol, accumulated an additional vesicular-like body. © 1997 John Wiley & Sons, Ltd.     

Classical transketolase functions as the formaldehyde-assimilating enzyme during growth of a dihydroxyacetone synthase-negative mutant of the methylotrophic yeast Hansenula polymorpha on mixtures of xylose and methanol in continuous cultures

Contrary to expectation, a mutant of Hansenula polymorpha blocked in dihydroxyacetone (DHA) synthase was able to assimilate methanol-carbon when grown in chemostat culture on mixtures of xylose and methanol. Incubation of a DHA synthase- and DHA kinase-negative double mutant resulted in DHA accumulation, indicating that a DHA synthase-type of reaction was involved. Low residual DHA synthase activity subsequently was shown to be present when using an assay with improved sensitivity. This activity was not associated with the (mutated) DHA synthase protein, which was still present in the peroxisomes, but with the enzyme transketolase. Transketolase from methanol grown cells was purified (525-fold) to homogeneity in 9% yield. The native enzyme was dimeric, as has been reported fro other transketolases, with a subunit molecular weight of 74000. The affinity of the purified enzyme for formaldehyde was low (K_m = 5 mM ), but high for xylulose-5-phosphate (ca. 10 μM ). The in vivo functioning of transketolase in formaldehyde assimilation, and the influence of the hydration state of formaldehyde is discussed.