Peroxisomal beta-oxidation--a metabolic pathway with multiple functions.

Fatty acid degradation in most organisms occurs primarily via the beta-oxidation cycle. In mammals, beta-oxidation occurs in both mitochondria and peroxisomes, whereas plants and most fungi harbor the beta-oxidation cycle only in the peroxisomes. Although several of the enzymes participating in this pathway in both organelles are similar, some distinct physiological roles have been uncovered. Recent advances in the structural elucidation of numerous mammalian and yeast enzymes involved in beta-oxidation have shed light on the basis of the substrate specificity for several of them. Of particular interest is the structural organization and function of the type 1 and 2 multifunctional enzyme (MFE-1 and MFE-2), two enzymes evolutionarily distant yet catalyzing the same overall enzymatic reactions but via opposite stereochemistry. New data on the physiological roles of the various enzymes participating in beta-oxidation have been gathered through the analysis of knockout mutants in plants, yeast and animals, as well as by the use of polyhydroxyalkanoate synthesis from beta-oxidation intermediates as a tool to study carbon flux through the pathway. In plants, both forward and reverse genetics performed on the model plant Arabidopsis thaliana have revealed novel roles for beta-oxidation in the germination process that is independent of the generation of carbohydrates for growth, as well as in embryo and flower development, and the generation of the phytohormone indole-3-acetic acid and the signal molecule jasmonic acid.

PHYTOCHROME KINASE SUBSTRATE4 modulates phytochrome-mediated control of hypocotyl growth orientation

Gravity and light are major factors shaping plant growth. Light perceived by phytochromes leads to seedling deetiolation, which includes the deviation from vertical hypocotyl growth and promotes hypocotyl phototropism. These light responses enhance survival of young seedlings during their emergence from the soil. The PHYTOCHROME KINASE SUBSTRATE (PKS) family is composed of four members in Arabidopsis (Arabidopsis thaliana): PKS1 to PKS4. Here we show that PKS4 is a negative regulator of both phytochrome A- and B-mediated inhibition of hypocotyl growth and promotion of cotyledon unfolding. Most prominently, pks4 mutants show abnormal phytochrome-modulated hypocotyl growth orientation. In dark-grown seedlings hypocotyls change from the original orientation defined by seed position to the upright orientation defined by gravity and light reduces the magnitude of this shift. In older seedlings with the hypocotyls already oriented by gravity, light promotes the deviation from vertical orientation. Based on the characterization of pks4 mutants we propose that PKS4 inhibits changes in growth orientation under red or far-red light. Our data suggest that in these light conditions PKS4 acts as an inhibitor of asymmetric growth. This hypothesis is supported by the phenotype of PKS4 overexpressers. Together with previous findings, these results indicate that the PKS family plays important functions during light-regulated tropic growth responses

PHYTOCHROME KINASE SUBSTRATE1 regulates root phototropism and gravitropism.

Light promotes the expression of PHYTOCHROME KINASE SUBSTRATE1 (PKS1) in the root of Arabidopsis thaliana, but the function of PKS1 in this organ is unknown. Unilateral blue light induced a negative root phototropic response mediated by phototropin 1 in wild-type seedlings. This response was absent in pks1 mutants. In the wild type, unilateral blue light enhanced PKS1 expression in the subapical region of the root several hours before bending was detectable. The negative phototropism and the enhanced PKS1 expression in response to blue light required phytochrome A (phyA). In addition, the pks1 mutation enhanced the root gravitropic response when vertically oriented seedlings were placed horizontally. The negative regulation of gravitropism by PKS1 occurred even in dark-grown seedlings and did not require phyA. Blue light also failed to induce negative phototropism in pks1 under reduced gravitational stimulation, indicating that the effect of pks1 on phototropism is not simply the consequence of the counteracting effect of enhanced gravitropism. We propose a model where the background level of PKS1 reduces gravitropism. After a phyA-dependent increase in its expression, PKS1 positively affects root phototropism and both effects contribute to negative curvature in response to unilateral blue light.

The Arabidopsis PHYTOCHROME KINASE SUBSTRATE2 protein is a phototropin signaling element that regulates leaf flattening and leaf positioning.

In Arabidopsis (Arabidopsis thaliana), the blue light photoreceptor phototropins (phot1 and phot2) fine-tune the photosynthetic status of the plant by controlling several important adaptive processes in response to environmental light variations. These processes include stem and petiole phototropism (leaf positioning), leaf flattening, stomatal opening, and chloroplast movements. The PHYTOCHROME KINASE SUBSTRATE (PKS) protein family comprises four members in Arabidopsis (PKS1-PKS4). PKS1 is a novel phot1 signaling element during phototropism, as it interacts with phot1 and the important signaling element NONPHOTOTROPIC HYPOCOTYL3 (NPH3) and is required for normal phot1-mediated phototropism. In this study, we have analyzed more globally the role of three PKS members (PKS1, PKS2, and PKS4). Systematic analysis of mutants reveals that PKS2 (and to a lesser extent PKS1) act in the same subset of phototropin-controlled responses as NPH3, namely leaf flattening and positioning. PKS1, PKS2, and NPH3 coimmunoprecipitate with both phot1-green fluorescent protein and phot2-green fluorescent protein in leaf extracts. Genetic experiments position PKS2 within phot1 and phot2 pathways controlling leaf positioning and leaf flattening, respectively. NPH3 can act in both phot1 and phot2 pathways, and synergistic interactions observed between pks2 and nph3 mutants suggest complementary roles of PKS2 and NPH3 during phototropin signaling. Finally, several observations further suggest that PKS2 may regulate leaf flattening and positioning by controlling auxin homeostasis. Together with previous findings, our results indicate that the PKS proteins represent an important family of phototropin signaling proteins.

Investigation of single tryptophan proteins encapsulated in TEOS-derived sol-gel matrices by fluorescence spectroscopy /

In the work reported here, optically clear, ultrathin TEOS derived sol-gel slides which were suitable for studies of tryptophan (Trp) fluorescence from entrapped proteins were prepared by the sol-gel technique and characterized. The monitoring of intrinsic protein fluorescence provided information about the structure and environment of the entrapped protein, and about the kinetics of the interaction between the entrapped protein and extemal reagents. Initial studies concentrated on the single Trp protein monellin which was entrapped into the sol-gel matrices. Two types of sol-gel slides, termed "wet aged", in which the gels were aged in buffer and "dry-aged", in which the gels were aged in air , were studied in order to compare the effect of the sol-gel matrix on the structure of the protein at different aging stages. Fluorescence results suggested that the mobility of solvent inside the slides was substantially reduced. The interaction of the entrapped protein with both neutral and charged species was examined and indicated response times on the order of minutes. In the case of the neutral species the kinetics were diffusion limited in solution, but were best described by a sum of first order rate constants when the reactions occurred in the glass matrix. For charged species, interactions between the analytes and the negatively charged glass matrix caused the reaction kinetics to become complex, with the overall reaction rate depending on both the type of aging and the charge on the analyte. The stability and conformational flexibility of the entrapped monellin were also studied. These studies indicated that the encapsulation of monellin into dry-aged monoliths caused the thermal unfolding transition to broaden and shift upward by 14°C, and causedthe long-term stability to improve by 12-fold (compared to solution). Chemical stability studies also showed a broader transition for the unfolding of the protein in dry-aged monoliths, and suggested that the protein was present in a distribution of environments. Results indicated that the entrapped proteins had a smaller range of conformational motions compared to proteins in solution, and that entrapped proteins were not able to unfold completely. The restriction of conformational motion, along with the increased structural order of the internal environment of the gels, likely resulted in the improvements in themial and long-term stability that were observed. A second protein which was also studied in this work is the metal binding protein rat oncomodulin. Initially, the unfolding behavior of this protein in aqueous solution was examined. Several single tryptophan mutants of the metal-binding protein rat oncomodulin (OM) were examined; F102W, Y57W, Y65W and the engineered protein CDOM33 which had all 12 residues of the CD loop replaced with a higher affinity binding loop. Both the thermal and the chemical stability were improved upon binding of metal ions with the order apo < Ca^^ < Tb^"^. During thermal denaturation, the transition midpoints (Tun) of Y65W appeared to be the lowest, followed by Y57W and F102W. The placement of the Trp residue in the F-helix in F102W apparently made the protein slightly more thermostable, although the fluorescence response was readily affected by chemical denaturants, which probably acted through the disruption of hydrogen bonds at the Cterminal end of the F-helix. Under both thermal and chemical denaturation, the engineered protein showed the highest stability. This indicated that increasing the number of metal ligating oxygens in the binding site, either by using a metal ion with a higher coordinatenumber (i.e. Tb^*) which binds more carboxylate ligands, or by providing more ligating groups, as in the CDOM33 replacement, produces notable improvements in protein stability. Y57W and CE)OM33 OM were chosen for further studies when encapsulated into sol-gel derived matrices. The kinetics of interaction of terbium with the entrapped proteins, the ability of the entrapped protein to binding terbium, as well as thermal stability of these two entrapped protein were compared with different levels of Ca^"*^ present in the matrix and in solution. Results suggested that for both of the proteins, the response time and the ability to bind terbium could be adjusted by adding excess calcium to the matrix before gelation. However, the less stable protein Y57W only retained at most 45% of its binding ability in solution while the more stable protein CDOM33 was able to retain 100% binding ability. Themially induced denaturation also suggested that CDOM33 showed similar stability to the protein in solution while Y57W was destabilized. All these results suggested that "hard" proteins (i.e. very stable) can easily survive the sol-gel encapsulation process, but "soft" proteins with lower thermodynamic stability may not be able to withstand the sol-gel process. However, it is possible to control many parameters in order to successfully entrap biological molecules into the sol-gel matrices with maxunum retention of activity.

Synechococcus sp. PCC 7002 CpcB lyase null mutations alter phycocyanin chromophore function and, consequently, affect the redistribution of excitation energy via the light state transition /

Phycobilisomes are the major light harvesting complexes for cyanobacteria and phycocyanin is the primary phycobiliprotein of the phycobilisome rod. The phycocyanobilin lyases responsible for chromophorylating the phycocyanin p subunit (CpcB) have been recently identified in the cyanobacterium Synechococcus sp. PCC 7002. Surprisingly, mutants missing the CpcB lyases were nevertheless capable of producing pigmented phycocyanin. 10K absorbance measurements revealed that the energy states of the p phycocyanin chromophores were only subtly shifted; however, 77K steady state fluorescence emission spectroscopy showed excitation energy transfer involving the targeted chromophores to be highly disrupted. Such evidence suggests that phycobilin orientation within the binding domain is specifically modified. We hypothesized that alternate, less specific lyases are able to act on the p binding sites. A phycocyanin linker-polypeptide deficient mutant was similarly characterized. The light state transition, a short term adaptation of the photosynthetic light harvesting apparatus resulting in the redistribution of excitation energy among the photo systems, was shown to be dominated by the reallocation of phycocyanin-absorbed excitation energy. Treatment with a high M phosphate buffer effectively prevented the redistribution of both chlorophyll a- and phycobilisome- absorbed excitation energy, suggesting that the two effects are not strictly independent. The mutant strains required a larger redistribution of excitation energy between light states, perhaps to compensate for their loss in phycobilisome antenna function.

Metabolic, biochemical and molecular profiling of Catharanthus roseus flower cultivars and transformed hairy roots for monoterpenoid indole alkaloid accumulation /

Catharanthlls rosellS (L.) G Don is a commercially significant flower species and in addition is the only source of the monoterpenoid indole alkaloids (MIA) vinblastine and vincristine, which are key pharmaceutical compounds that are used to combat a number of different cancers. Therefore, procurement of the antineoplastic agents is difficult but essential procedure. Alternatively, CatharanthllS tissue cultures have been investigated as a source of these agents; however they do not produce vindoline, which is an obligate precursor to vinblastine and vincristine. The interest in developing high MIA cultivars of Catharantlws rosellS has prompted metabolic profiling studies to determine the variability of MIA accumulation of existing flowering cultivars, with particular focus on the vindoline component ofthe pathway. Metabolic profiling studies that used high performance liquid chromatography of MIAs from seedlings and young leaf extracts from 50 different flowering cultivars showed that, except for a single low vindoline cultivar (Vinca Mediterranean DP Orchid), they all accumulate similar levels of MIAs. Further enzymatic studies with extracts from young leaves and from developing seedlings showed that the low vindoline cultivar has a IO-fold lower tabersonine-16-hydroxylase activity than those of CatharanthllS rosellS cv Little Delicata. Additionally, studies aimed at metabolic engineering ofvindoline bios}l1thesis in Catharanthus rosellS hairy root cultures have been performed by expressing the last step in vindoline biosynthesis [Dcacetylvindoline-4-0- acetyltransferase (DAT)]. Enzymatic profiling studies with transformed hairy roots have confirmed that over-expressing DAT leads to lines with high levels of O-acetyltransferase activity when compared to non-expressing hairy roots. One particular DA T over111 expressing hairy root culture (line 7) contained 200 times the OAT activity than leaves of control lines. Additional MIA analyses revealed that DAT over-expressing hairy roots have an altered alkaloid profile with significant variation in the accumulation of h6rhammericine. Further analysis of transformed hairy root line 7 suggests a correlation between the expression of OAT activity and h6rhammericine accumulation with root maturation. These studies show that metabolic and selective enzymatic profiling can enhance our ability to search for relevant MIA pathway mutants and that genetic engineering with appropriate pathway genes shows promise as a tool to modify the MIA profile of Catharanthus roseus.

Primary characterization of genes involved in the colony development of the insect pathogenic fungus Metarhizium anisopliae /

Strain improvement of the insect pathogenic fungus Metarhizium anisopUae is necessary to increase its virulence towards agricultural pests and thus improve its commercial efficacy. Nevertheless, the release of genetically modified conidia in crop fields may negatively affect the ecosystem. Controlling conidiation is a potential means of limiting the release of engineered strains since conidia are the infective propagules and the means of dispersal. The purpose of this study was to research the colony development of M. anisopUae to identify potential targets for genetic manipulation to control conidiation. Following Agrobacterium tumefaciem insertional mutagenesis, phenotypic mutants were characterized using Y-shaped adaptor dependent extension PCR. Four of 1 8 colony development recombinants had T-DNA flanking sequences with high homology to genes encoding known signaling pathway proteins that regulate pathogenesis and/or asexual development in filamentous fungi. Conidial density counts and insect bioassays suggested that a Serine/Threonine protein kinase COTl homolog is not essential for conidiation or virulence. Furthermore, a choline kinase homolog is important for conidiation, but not virulence. Finally, the regulator of G protein signaling CAG8 and a NADPH oxidase NoxA homolog are necessary for conidiation and virulence. These genes are candidates for further investigation into the regulatory pathways controlling conidiation to yield insight into promising gene targets for biocontrol strain improvement.

The Catharanthus roseus 16-hydroxytabersonine O-methyltransferase involved in vindoline biosynthesis /

Madagascar periwinkle (Catharanthus roseus) produces the well known and remarkably complex dimeric anticancer alkaloids vinblastine and vincristine that are derived by coupling vindoline and catharanthine monomers. This thesis describes the novel application of carborundum abrasion (CA) technique as a tool for large scale isolation of leaf epidermis enriched proteins. This technique was used to facilitate the purification to apparent homogeneity of 16-hydroxytabersonine-16-0-methyltransferse (l60MT) that catalyses the second step in the 6 step pathway that converts tabersonine into vindoline. This versatile tool was also used to harvest leaf epidermis enriched mRNAs that facilitated the molecular cloning of the 160MT. Functional expression and biochemical characterization of recombinant 160MT enzyme showed that it had a very narrow substrate specificity and high affinity for 16-hydroxytabersonine, since other closely related monoterpene indole alkaloids (MIAs) did not act as substrates. In addition to allowing the cloning of this gene, CA technique clearly showed that 160MT is predominantly expressed in Catharanthus leaf epidermis, in contrast to several other OMTs that appear to be expressed in other Catharanthus tissues. The results provide compelling evidence that most of the pathway for vindoline biosynthesis including the 0- methylation of 16-hydroxytabersonine occurs exclusively in leaf epidermis, with subsequent steps occurring in other leaf cell types. Small molecule O-methyltransferases (OMTs) (E.C. 2.1.1.6.x) catalyze the transfer of the reactive methyl group of S-adenosyl-L-methionine (SAM) to free hydroxyl groups of acceptor molecules. Plant OMTs, unlike their monomeric mammalian homologues, exist as functional homodimers. While the biological advantages for dimer fonnation with plant OMTs remain to be established, studies with OMTs from the benzylisoquinoline producing plant, Thalictrum tuberosum, showed that co-expression of 2 recombinant OMTs produced novel substrate specificities not found when each rOMT was expressed individually (Frick, Kutchan, 1999) . These results suggest that OMTs can fonn heterodimers that confer novel substrate specificities not possible with the homodimer alone. The present study describes a 160MT model based strategy attempting to modify the substrate specificity by site-specific mutagenesis. Our failure to generate altered substrate acceptance profiles in our 160MT mutants has lead us to study the biochemical properties ofhomodimers and heterodimers. Experimental evidence is provided to show that active sites found on OMT dimers function independently and that bifunctional heterodimeric OMTs may be fonned in vivo to produce a broader and more diverse range of natural products in plants.

A comparative study of protoheme and heme d catalases : role of the heme and the heme pocket in catalysis and ligand binding

Catalase dismutes H20 2 to O2 and H20. In successive twoelectron reactions H20 2 induces both oxidation and reduction at the heme group. In the first step the protoheme prosthetic group of beef liver catalase forms compound I, in which the heme has been oxidized from Fe3+ to Fe4+=0 and a porphyrin radical has been created. Compound II is formed by the oneelectron reduction of comp I. It retains Fe4+=0 but lacks the porphyrin radical and is catalytically inert. Molecular structures are available for Escherichia coli Hydroperoxidase II, Micrococcus Iysodeiktus, Penicillium vitale and beef liver enzymes, which contain different hemes and heme pockets. In the present work, the pockets and substrate access channels of protoheme (beef liver & Micrococcus) and heme d (HPII of E. coli and Penicillium) catalases have been analysed using Quanta™ and CharmMTM molecular modeling packages on the Silicon Graphics Iris Indigo 2 computer. Experimental studies have been carried out with two catalases, HPII (and its mutants) and beef liver. Fluoride and formate' are inhibitors of both enzymes, and their binding is modulated by the heme and by distal residues N201 & H128. Both HPII and beef liver enzymes form compound I with H202 or peracetate. The reduction of beef liver enzyme compound I to II and the decay of compound II are accelerated by fluoride. The decay of compound II is also accelerated by formate, and this reagent acts as a 2-electron donor towards compound I of both enzymes. It is concluded that heme d enzymes (Penicillium and HPII of E. coli) are formed by autocatalytic transformation of protoheme in a modified pocket which contains a characteristic serine residue as well as a partially occluded heme channel. They are less active than protoheme enzymes but also do not form the inactive compound II species. Binding of peroxide as well as fluoride and formate is prevented by mutation of H128 and modulated by mutation of N201.

Aspects of the hemes and modulation of hydrogen donors in catalases from bovine liver, yeast, and escherichia coli

Catalase is the enzyme which decomposes hydrogen peroxide to water and oxygen. Escherichia coli contains two catalases. Hydroperoxidase I (HPI) is a bifunctional catalase-peroxidase. Hydroperoxidase II (HPII) is only catalytically active toward H202. Expression of the genes encoding these proteins is controlled by different regimes. HPJI is thought to be a hexamer, having one heme d cis group per enzymatic subunit. HPII wild type protein and heme containing mutant proteins were obtained from the laboratory of P. Loewen (Univ. of Manitoba). Mutants constructed by oligonucleotidedirected mutagenesis were targeted for replacement of either the His128 residue or the Asn201 residue in the vicinity of the HPII heme crevice. His128 is the residue thought to be analogous to the His74 distal axial ligand of the heme in the bovine liver enzyme, and Asn201 is believed to be a residue critical to the function of the enzyme because of its role in orienting and interacting with the substrate molecule. Investigation of the nature of the hemes via absorption spectroscopy of the unmodified catalase proteins and their derived pyridine hemochromes showed that while the bovine and Saccharomyces cerevisiae catalase enzymes are protoheme-containing, the HPII wild type protein contains heme d, and the mutant proteins contain either solely protoheme, or heme d-protoheme mixtures. Cyanide binding studies supported this, as ligand binding was monophasic for the bovine, Saccharomyces cerevisiae, and wild type HPII enzymes, but biphasic for several of the HPII mutant proteins. Several mammalian catalases, and at least two prokaryotic catalases, are known to be NADPH binding. The function of this cofactor appears to be the prevention of inactivation of the enzyme, which occurs via formation of the inactive secondary catalase peroxide compound (compound II). No physiologically plausible scheme has yet been proposed for the NADPH mediation of catalase activity. This study has shown, via fluorescence and affinity chromatography techniques, that NADPH binds to the T (Typical) and A (Atypical) catalases of Saccharomyces cerevisiae, and that wild type HPII apparently does not bind NADPH. This study has also shown that NADPH is unlike any other hydrogen donor to catalase, and addresses its features as a unique donor by proposing a mechanism whereby NADPH is oxidized and catalase is protected from inactivation via the formation of protein radical species. Migration of this radical to a position close to the NADPH is also proposed as an adjunct hypothesis, based on similar electron migrations that are known to occur within metmyoglobin and cytochrome c peroxidase when reacted with H202. Validation of these hypotheses may be obtained in appropriate future experiments.

Development of packaging cell lines for rescuing BAV2 viral vectors

The construction of adenovirus vectors for cloning and foreign gene expression requires packaging cell lines that can complement missing viral functions caused by sequence deletions and/or replacement with foreign DNA sequences. In this study, packaging cell lines were designed to provide in trans the missing bovine adenovirus functions, so that recombinant viruses could be generated. Fetal bovine kidney and lUng cells, acquired at the trimester term from a pregnant cow, were tranfected with both digested wild type BAV2 genomic DNA and pCMV-EI. The plasmid pCMV-EI was specifically constructed to express El of BAV2 under the control of the cytomegalovirus enhancer/promoter (CMV). Selection for "true" transformants by continuous passaging showed no success in isolating immortalised cells, since the cells underwent crisis resulting in complete cell death. Moreover, selection for G418 resistance, using the same cells, also did not result in the isolation of an immortalised cell line and the same culture-collapse event was observed. The lack of success in establishing an immortalised cell line from fetal tissue prompted us to transfect a pre-established cell line. We began by transfecting MDBK (Mardin-Dardy bovine kidney) cells with pCMV-El-neo, which contain the bacterial selectable marker neo gene. A series of MDBK-derived cell lines, that constitutively express bovine adenoviral (BAV) early region 1 (El), were then isolated. Cells selected for resistance to the drug G418 were isolated collectively for full characterisation to assess their suitability as packaging cell lines. Individual colonies were isolated by limiting dilution and further tested for El expression and efficiency of DNA uptake. Two cell lines, L-23 and L-24, out of 48 generated foci tested positive for £1 expression using Northern Blot analysis. DNA uptake studies, using both lipofectamine and calcium phosphate methods, were performed to compare these cells, their parental MDBK cells, 8 and the unrelated human 293 cells as a benchmark. The results revealed that the new MDBKderived clones were no more efficient than MDBK cells in the transient expression of transfected DNA and that they were inferior to 293 cells, when using lacZ as the reporter gene. In view of the inherently poor transfection efficiency of MDBK cells and their derivatives, a number of other bovine cells were investigated for their potential as packaging cells. The cell line CCL40 was chosen for its high efficiency in DNA uptake and subsequently transfected with the plasmid vector pCMV El-neo. By selection with the drug G418, two cell lines were isolated, ProCell 1 and ProCell 2. These cell lines were tested for El expression, permissivity to BAV2 and DNA uptake efficiency, revealing a DNA uptake efficiency of 37 % , comparable to that of CCL40. Attempts to rescue BAV2 mutants carrying the lacZ gene in place of £1 or £3 were carried out by co-transfecting wild type viral DNA with either the plasmid pdlElE-Z (which contains BAV2 sequences from 0% to 40.4% with the lacZ gene in place of the £1 region from 1.1% to 8.25%) or with the plasmid pdlE3-5-Z (which contains BAV2 sequences from 64.8% to 100% with the lacZ gene in place of the E3 region from 75.8% to 81.4%). These cotransfections did not result in the generation of a viral mutant. The lack of mutant generation was thought to be caused by the relative inefficiency ofDNA uptake. Consequently, cosBAV2, a cosmid vector carrying the BAV2 genome, was modified to carry the neo reporter gene in place of the £3 region from 75.8% to 81.4%. The use of a single cosmid vector earring the whole genome would eliminate the need for homologous recombination in order to generate a viral vector. Unfortunately, the transfection of cosBAV2- neo also did not result in the generation of a viral mutant. This may have been caused by the size of the £3 deletion, where excess sequences that are essential to the virus' survival might have been deleted. As an extension to this study, the spontaneous E3 deletion, accidently discovered in our viral stock, could be used as site of foreign gene insertion.

Glucosamine resistance in the yeast, Saccharomyces cerivisae [sic]

By using glucosamine resistant mutants of Saccharomyces ceriv~sa~ an attempt was made to discover the mechanisms which cause glucose repression and/or the Crabtree effect. The strains used are 4B2, GR6, lOP3r, GR8l and GRI08. 4B2 is a wild type yeast while the others are its mutants. To characterize the biochemical reactions which made these mutants resistant to glucosamine poisoning the following experiments were done~ 1. growth and respiration; 2. transport of sugars; 3. effect of inorganic phosphate (Pi): 4. Hexokinase; 5. In yivo phosphorylation. From the above experiments the following conclusions may be drawn: (i) GR6 and lOP3r have normal respiratory and fermentative pathways. These mutants are resistant to glucosamine poisoning due to a slow rate of sugar transport which is due to change in the cell membrane. (ii) GR8l has a normal respiratory pathway. The slow growth on fermentable carbon sourCEE indicates that in GR8l the lesion is in or associated with the glycolytic pathway. The lower rate of sugar transport may be due to a change in energy metabolism. The invivo phosphorylation rate indicates that in GR81 facilitated diffusion is the dominant transport mechanism. (iii) GR108 msa normal glycolytic pathway but the respiratory pathway is abnormal. The slow rate of sugar transport is due to a change in energy metabolism. The lower percentage of in vivo phosphorylation is probably due to a lowered availability of ATP because of the mitochondrial lesion. In all mutants resistance to glucosamine poisoning is due to a lower rate of utilization of ATP. which is caused by various mechanisms (see above), making less ADP available for phosphorylation via ATP synthase which utilizes inorganic phosphate. Because of the lower utilization of Pi, the concentration of intra-mitochondrial Pi does not go down thus protecting mutants from glucosamine poisoning.

Partial characterization of the cloned dihydrofolate reductase gene of Saccharomyces cerevisiae

The cloned dihydrofolate reductase gene of Saccharomyces cerevisiae (DFR 1) is expressed in Escherichia coli. Bacterial strain JF1754 transformed with plasmids containing DFR 1 is at least 5X more resistant to inhibition by the folate antagonist trimethoprim. Expression of yeast DFR 1 in E. coli suggests it is likely that the gene lacks intervening sequences. The 1.8 kbp DNA fragment encoding yeast dhfr activity probably has its own promotor, as the gene is expressed in both orientations in E. coli. Expression of the yeast dhfr gene cloned into M13 viral vectors allowed positive selection of DFR 1 - M13 bacterial transfectants in medium supplemented with trimethoprim. A series of nested deletions generated by nuclease Bal 31 digestion and by restriction endonuclease cleavage of plasmids containing DFR 1 physically mapped the gene to a 930 bp region between the Pst 1 and Sal 1 cut sites. This is consistent with the 21,000 molecular weight attributed to yeast dhfr in previous reports. From preliminary DNA sequence analysis of the dhfr DNA fragment the 3' terminus of DFR 1 was assigned to a position 27 nucleotides from the Eco Rl cut site on the Bam Hi - Eco Rl DNA segment. Several putative yeast transcription termination consensus sequences were identified 3' to the opal stop codon. DFR 1 is expressed in yeast and it confers resistance to the antifolate methotrexate when the gene is present in 2 - 10 copies per cell. Plasmid-dependent resistance to methotrexate is also observed in a rad 6 background although the effect is somewhat less than that conferred to wild-type or rad 18 cells. Integration of DFR 1 into the yeast genome showed an intermediate sensitivity to folate antagonists. This may suggest a gene dosage effect. No change in petite induction in these yeast strains was observed in transformed cells containing yeast dhfr plasmids. The sensitivity of rad 6 , rad 18 and wild-type cell populations to trimethoprim were unaffected by the presence of DFR 1 in transformants. Moreover, trimethoprim did not induce petites in any strain tested, which normally results if dhfr is inhibited by other antifolates such as methotrexate. This may suggest that the dhfr enzyme is not the only possible target of trimethoprim in yeast. rad 6 mutants showed a very low level of spontaneous petite formation. Methotrexate failed to induce respiratory deficient mutants in this strain which suggested that rad 6 might be an obligate grande. However, ethidium bromide induced petites to a level approximately 50% of that exhibited by wild-type and rad 18 strains.

The genetics of glucosamine in yeast : cytoplasmic determinants conferring resistance

Two cytoplasmic, glucosamine resistant mutants of Saccharomyces cerevisiae, GR6 and GR10, were examined to determine whether or not the lesions involved were located on mitochondrial DNA. Detailed investigation of crosses of GR6 and GR10 or their derivatives to strains bearing known mitochondrial markers demonstrated that: 1. the frequency of glucos~~ine resistance in diploids was independent of factors influencing mitochondrial marker output. 2. upon tetrad analysis a variety of tetrad ratios was observed for glucosamine resistance whereas mitochondrial markers segregated 4:0 or 0:4 (resistant:sensitive). 3. glucosamine resistance and mitochondrial markers segregated differentially with time. 4. glucosamine resistance persisted following treatment of a GRIO derivative with ethidium bromide at concentrations high enough to eliminate all mitochondrial DNA. 5. haploid spore clones displayed two degrees of glucosamine resistance, weak and strong, while growth due to mitochondrial mutations was generally thick and confluent. 6. a number of glucosamine resistant diploids and haploids, which also possessed a mithchondrial resistance mutation, were unable to grow on medium containing both glucosamine and the particular drug involved. 3 These observations 1~ 6 provided strong evidence that the cytoplasmic glucosamine resistant mutations present in GR6 and GRiO were not situated on mitochondrial DNA. Comparison of the glucosamine resistance mutations to some other known cytoplasmic determinants revealed that: 7. glucosamine resistance and the expression of the killer phenotype were separate phenomena. 8. unlike yeast carrying resistance conferring episomes GR6 and GR10 were not resistant to venturicidin or oligomycin and the GR factor exhibited genetic behaviour different from that of the episomal determinants. These results 7--+8 suggested that glucosamine resistance was not associated with the killer determinant nor with alleged yeast episomes. It is therefore proposed that a yeast plasmid(s), previously undescribed, is responsible for glucosamine resistance. The evidence to date is compatible with the hypothesis that GR6 and GR10 carry allelic mutations of the same plasmid which is tentatively designated (GGM).