WO2000022169A1 - DETECTION USING Snz AND Sno GENES AND PROTEINS - Google Patents
DETECTION USING Snz AND Sno GENES AND PROTEINS Download PDFInfo
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- WO2000022169A1 WO2000022169A1 PCT/US1999/023596 US9923596W WO0022169A1 WO 2000022169 A1 WO2000022169 A1 WO 2000022169A1 US 9923596 W US9923596 W US 9923596W WO 0022169 A1 WO0022169 A1 WO 0022169A1
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Definitions
- the present invention relates to probes and assays using the family of SNZ and SNO genes and proteins to detect particular microorganisms in a sample, and the use of the same as antibiotic targets for eradicating those microbes.
- the primary regulators of cellular growth and proliferation in multicellular organisms are growth factors and hormones. Similar regulation of growth and proliferation exists in unicellular organisms, such as Saccharomyces cerevisiae, but nutrient availability is typically the most important extracellular cue. Identification of the factors involved in nutrient limitation can lead to elucidation of many important regulatory factors.
- Snzlp Snooze 1 protein
- Eucarya phylogenetic domains
- Bacteria, and Archea phylogenetic domains
- its mRNA accumulates specifically in response to nutrient-limited growth arrest
- Basun, E. L. Fuge, E. K., Padilla, P. A. and Werner-Washburne, M. (1996)
- a Stationary-phase gene in Saccharomyces cerevisiae is a member of a novel, highly conserved gene family J. Bad. 178: 6865-6872; Padilla, P.A., Fuge, E.K., Crawford, M.E., Errett, A. and Werner-Washburne, M.
- Snzlp is the most highly conserved protein present in all three phylogenetic domains, sharing 60% identity with Snz homologues in bacteria and archaea
- SOR1 Functional characterization of SOR1, a gene required for resistance to photosensitizing toxin in the fungus Cercospora nicotianae, Current Genetics 34: 478-485; Ehrenshaft, M., Jenns, A. E., Chung, K. R. and Daub, M. E. (1998) SOR1, a gene required for photosensitizer and singlet oxygen resistance in Cercospora fungi, is highly conserved in divergent organisms, Mol Cell i : 603-609; Padilla, P.A., et al. (1998)). S.
- SNZ1 is situated proximal to the centromere on the right arm of chromosome XIII.
- SNZ2 and SNZ3 are located in the telomeric regions on the left arms of chromosomes XIV and VI, respectively, within a 7-kb region that is nearly identical between these two chromosomes. This duplicated region is not observed in the telomeric regions of other yeast chromosomes. All three SNZ genes are adjacent to another conserved gene family, SNO (Snooze proximal Open reading frame).
- Sno proteins are less highly conserved (approximately 40% identity between yeast, B. subtilis, and M. jannaschii) than Snz proteins, suggesting that there are fewer constraints on the structure and sequence of Sno proteins. Although Sno proteins, like Snz proteins, have an unknown function, they show some sequence similarity to glutamine amidotransferases.
- SNZ and SNO genes are not present in all organisms nor are they present in all species of related lineages.
- SNZ and SNO are present in H. influenzae but not in the related E. coli.
- M. thermoautotrophicum and M. jannaschii the location of the two genes adjacent to each other is conserved.
- SNZ and SNO genes have not been identified in either vertebrates or invertebrates. However, they have been identified in medically significant microorganisms including: M. tuberculosis, M. leprae, H. influenzae, S. pneumoniae, F. tularensis, and F. neoformans. Therefore, characterization of the processes in which Snz and Sno proteins participate in some but not other organisms may have major implications for management of disease and drug design.
- HisF and HisH interact to produce aminoimidazole carboxamide ribonucleotide in E.coli (Winkler).
- His7p a multifunctional protein, catalyzes the same reaction in yeast.
- Snolp is the glutamine amidotransferase and Snz1 p the acceptor domain for transfer of an amido group to an as yet unknown substrate.
- the reported Sno1 and Snzl p alignments do not constitute strong evidence that they are orthologues of HisF and it is unlikely because his7SNZ1SN01 mutants require histidine. Nonetheless, physical interaction and involvement has been shown in the same process.
- a preferred embodiment of the present invention comprises a probe comprising at least one sequence from the group of SNZ1, SNZ2, SNZ3, SN01, SN02, SN03, Snzl p, Snz2p, Snz3p, Snol p Sno2p, Sno3p, and fragments thereof, for detecting and determining origin of a foreign organism in a sample.
- the preferred embodiment also comprises a kit for detecting and determining origin of a foreign organism in a sample comprising a Snz/Sno probe, a marker, and a detection method for detecting said probe.
- the kit preferably comprises a probe comprising at least one sequence selected from the group consisting of SNZ1, SNZ2, SNZ3, SN01, SN02, SN03, Snzlp, Snz2p, Snz3p, Snol p Sno2p, Sno3p, and fragments thereof, and additionally preferably comprises a means for detecting and determining origin of a foreign organism in a sample comprising at least one source selected from the group consisting of cell culture, DNA sample, and patient specimen.
- the marker comprises at least one marker selected from the group consisting of fluorescent, radioactive, magnetic, and other markers, and preferably the detection method comprises at least one method selected from the group consisting of Northern blotting, Western blotting, Southern blotting, ELISA, in situ hybridization, and other assays.
- the present invention also comprises an assay for detecting and determining origin of a foreign organism in a sample comprising the steps. of probing the sample with a Snz/Sno probe having a marker; and detecting the marker.
- Probing the sample with a Snz/Sno probe having a marker preferably comprises probing the sample with at least one sequence selected from the group consisting ol SNZI, SNZ2, SNZ3, SN01, SN02, SN03, Snzl p, Snz2p, Snz3p, Snol p Sno2p, Sno3p, and fragments thereof; and preferably probing with at least one marker selected from the group consisting of fluorescent, radioactive, and other markers; and optionally detecting utilizing at least one method selected from the group consisting of Northern blotting, Western blotting, Southern blotting, ELISA, in situ hybridization, and other assays; and preferably probing at least one source selected from the group consisting of cell culture, DNA
- the present invention additionally comprises a method for treating a disease with antibiotics comprising the steps oftargeting a Snz/Sno target for receiving the antibiotic; and administering the antibiotic.
- the step of targeting the Snz/Sno target comprises targeting at least one sequence selected from the group consisting of SNZ1, SNZ2, SNZ3, SN01, SN02, SN03, Snzlp, Snz2p, Snz3p, Snol p, Sno2p, Sno3p, and fragments thereof; and preferably wherein the step of targeting the Snz Sno target comprises targeting the metabolic pathway of the Snz/Sno target.
- administering the antibiotic comprises administering an antibiotic which impedes the metabolic pathway of the target.
- a primary object of the present invention is the development of a probe useful for determining origin of foreign proteins/genes.
- Another object of the present invention is the use of Snz/Sno proteins/genes to probe for origin of foreign proteins/genes.
- a further object of the present invention is the use of Snz/Sno genes/fragments or protein products to serve as targets for antibiotic therapy.
- Yet another object of the present invention is the use of Snz/Sno transcriptional elements to serve as targets for Snz/Sno gene/protein regulation.
- a primary advantage of the present invention is the ability to eliminate certain organisms as potential hosts of foreign DNA/proteins.
- Another advantage of the present invention is the ability to quickly screen for origin of foreign genes/proteins.
- a further advantage of the present invention is the ability to screen and target for treatment using the same gene/protein.
- Yet another advantage of the present invention is the ability to use novel pathways to target destruction of foreign organisms.
- Fig. 1 A is a diagram of a SNZ2/3 disruption mutation
- Fig. 1B is a diagram of a SNZ2 3 SN02/3 deletion mutation
- Fig. 1C is a diagram of a SNZ1 deletion mutation
- Fig. 1D is a diagram of a SNZ1 SN01 deletion mutation
- Fig. 1E is a diagram of a SN01 disruption mutation
- Fig. 2A is a Northern blot probed with SNZ3, SN03, and SNZ1 to show relative timing of expression;
- Fig. 2B is a Northern blot probed with SN01, SNZ1, and BCY1 to show timing of SN01 expression
- Fig. 3 is a Northern analysis of SNZ1 expression in an snz2 snz3 mutant during growth to stationary phase;
- Fig. 4 is a Western analysis of Snz proteins
- Fig. 5 is a Northern analysis of SNZ and SNO mRNA accumulation in nitrogen-starved cells, blotted with SNZ3 and SNZ1 (5A), and SN03 and SN01;
- Fig. 6 is a Northern analysis of SNZ1 expression in the presence or absence of auxotrophic requirements
- Fig. 7A is a photograph of growth of snz and sno mutants on minimal medium without uracil, with or without 6-AU;
- Fig. 7B is a photograph of growth of snz and sno mutants on YPD medium supplemented with methylene blue, in the presence or absence of light;
- Fig. 8 shows results of expression of deletion plasmids
- Fig. 9 shows expression of SNZ1 in relation to presence of GCN4 boxes
- Fig. 10A is the sequence listing for the SNZ1 ORF
- Fig. 10B is the sequence listing for the SNZ1 protein
- Fig. 10C is the sequence listing for the SNZ2/3 ORF
- Fig. 10D is the sequence listing for the SNZ2/3 protein
- Fig. 10E is the sequence listing for the SN01 ORF
- Fig. 10F is the sequence listing for the SN01 protein
- Fig. 10G is the sequence listing for the SN02/3 ORF
- Fig. 10H is the sequence listing for the SN02/3 protein
- Fig. 101 is the sequence listing for the SNZ1-SN01 promoter.
- Fig. 10J is the sequence listing for the SNZ2/3-SN02/3 promoter. DESCRIPTION OF THE PREFERRED EMBODIMENTS (BEST MODES FOR CARRYING OUT THE INVENTION) The conditions that induce expression of SNZ1 and SN01 orthologues clearly demonstrate that these genes respond globally to stress. In yeast, SNZ1 is induced during stationary phase
- SNZ1 orthologues in other organisms also respond to stress conditions including ethyolene and salicylic acid in the rubber-tree plant Hevea brasiliensis (Sivasubramaniam, S., et al. (1995)), oxidative stress in the bacterium ⁇ . subtilis (Antlemann, H., Bernhardt, J., Schmid, R., Mach, H., Voelker, U. and Hecker, M. (1997) First steps from a two-dimensional protein index towards a response-regulation map for Bacillus subtilis, Electrophoresis 18: 1451-1463) and the fungus C. nicotianae (Ehrenshaft, M., et al. (1998)).
- Wild-type SNZ1 exhibits a complex pattern of gene expression when auxotrophic strains were starved for the required supplements (although snzl deletion mutants themselves were not auxotrophic)(Padilla, P.A., (1998)).
- SNZ1 mRNA accumulates in trp-1-1, Ieu2, ura3-52, or ade2-1 single mutants when cultures are deprived of tryptophan, leucine, uracil, or adenine but notably not to histidine (in a his3-1 mutant).
- SNZ1 mRNA is completely repressed when tryptophan or leucine is added to the medium but not after uracil or adenine is added.
- the results with uracil and adenine are surprising because exogenously added uracil or adenine generally repressess genes involved in the respective de novo or salvage biosynthetic pathways (Lacroute, F. (1968) Regulation of Pyrimidine biosynthesis in
- SNZ1 may be induced in response to conditions that alter the balance of intracellular nucleotides.
- SNZ1 is induced in the presence of 6-azauracil (6-AU), an inhibitor of purine and pyrimidine biosynthetic pathways, resulting in altered GTP and UTP pools (Hampsey, M. (1997) A review of phenotypes in Saccharomyces cerevisiae, Yeast 13: 1099-1133).
- SNZ1 is highly induced (65-fold) in the presence of DNA-damaging agents (Jelinsky, S., et al. (1999)).
- snz mutants are not sensitive to the DNA-damaging agent EMS, suggesting that SNZ1 does not encode a DNA repair enzyme.
- SNZ and SNO genes are not essential. However, snzl and snol mutants grew poorly in three different conditions. These phenotypes were (1) sensitivity to 6-azauracil, (2) sensitivity to conditions that generate single oxygen, and (3) slow growth on synthetic medium lacking pyridoxine.
- PPR1 pyrimidine pathway regulator
- SNZ1 does not contain a PPR1 recognition element making it unlikely that SNZ1 induction or snzl mutant sensitivity is due directly to Ppr1.
- SNZ1 contain any sequence motifs suggesting that it is a transcription factor.
- uracil suppresses both snzl and pprl
- S/VZ1 has a different function in pyrimidine metabolism.
- the snzl sensitivity to 6-AU leads to the conclusion that Snz proteins may be responding directly or indirectly to imbalances in nucleotide pools but not directly through PPR1 or PPR2. Additionally, the lack or motifs of DNA-binding protein leads to the conclusion that PPR1 and PPR2 and SNZ proteins do not have the same function.
- Methylene blue is a vital dye that generates singlet oxygen in the presence of light. Singlet oxygen is highly reactive and causes intracellular damage to proteins, DNA, membranes, and other macromolecules and structures.
- SOR1 and SNZL The association between snz mutants and singlet oxygen sensitivity was made after the discovery of the link of SOR1 and SNZL It seems possible that methylene blue causes a turnover in RNA or an increase in DNA repair, which results in an alteration of intracellular nucleotide concentrations, in turn resulting in an induction of SNZ1 and SN01.
- the Snz1 p-Sno1 p complex which may have a glutamine amidotransferase activity, plays a role in nucleotide metabolism and is induced in response to stresses that cause an imbalance, maintenance of or decrease in the concentrations of nucleotides.
- Pyridoxine is an essential vitamin that is synthesized by a wide variety of organisms, including yeast. In yeast, only two genes, PDX2 and PDX3, have been reported to be involved in pyridoxine biosynthesis. PDX3 encodes pyridoxamine phosphate oxidase, and PDX2 describes a mutant locus on the right arm of chromosome XIII that leads to pridoxine auxotrophy (Hawthorne, D.C. and Mortimer, R.K.
- SNZ and SNO genes are found in all three phylogenetic domains, these genes are not present in all organisms.
- Examples of organisms lacking SNZ and SNO orthologues include E. coli, Borrelia burgdorferi, Synechocystis spp., Helicobacter pylori, Mycoplasma pneumoniae, and Mycoplasma genitalium.
- the majority of the organisms that lack SNZ1 and SN01 orthologues reside in nutrient-rich environments within the host, e.g., in the stomach or intestine, or adjacent to damaged cells.
- SNZ and SNO are important for survival in nutrient-poor conditions and organisms that reside in relatively nutrient-rich conditions may have no selective pressure to maintain these genes.
- the presence of SNZ and SNO genes in plants may be indicative of the frequency of nutrient limitation experienced by plants due either to poor soils or drought.
- Snz and Sno proteins have not been identified in animals, which are organisms that are unable to maintain viability under frequent severe starvation conditions. Because Snz and Sno proteins are not present in all organisms and because they may function in different and novel pathways, these proteins and genes can be used as markers to distinguish different organisms. These include standard Western, Northern, and Southern blots, as well as other assays, including in situ hybridization assays, PCR and ELISA tests. The proteins or pathways can also be targets for useful antibiotic therapeutics.
- SNZ1 transcription increases as yeast cells center stationary phase (quiescence) (Braun et al. 1996; Padilla et al. 1998). Stationary-phase transcription promoter elements and proteins have future broad applications in the controlled expression of heterologous genes in quiescent cells. These results suggest the existence of both positive and negative regulatory elements in the SNZ1 promoter.
- the positive regulatory element may involve any or all of four putative GCN4 recognition elements (i.e. "boxes") that are located in the intergenic region (MW1457). The use of these elements to regulate SNZ1 expression has not been previously demonstrated in any aspect.
- the sequence immediately upstream of the GCN4 boxes may have a deleterious effect or counteract Gcn4 protein activity, hence contributing to negative SNZ1 regulation, because SNZ1 expression is greatly increased when these sequences are deleted (MW1454 and MW1460).
- Additional results suggest that SNZ1 regulation is affected by c/s-acting elements (other than the GCN4 boxes) because SNZ1 expression through the culture cycle remains unchanged in a gcn4 deletion mutant (Fig. 9). These results indicate that SNZ1 regulation occurs at the level of transcription and that increased transcription during the post diauxic phase can be attained from a plasmid-based construct. Location and identification of these elements provide yet another potential target for antibiotic action.
- the usefulness of this invention is derived from the observation that SNZ and SNO genes are not present in all organisms. Hence, using standard techniques the sequences can be used as probes to identify pathogenic and non-pathogenic organisms that harbor these sequences.
- the SNZ gene has not yet been identified in humans or vertebrates. However, the human pathogen
- Cryptococcus neoformans does carry a SNZ gene.
- One specific application of this invention would be to distinguish or follow the presence of S/VZ-bearing infectious microorganisms in humans.
- the presence of even minute amounts of Cryptococcus can be ascertained in a potentially infected individual by using PCR (Polymerase Chain Reaction) and primers specific to the SNZ gene.
- SNZ and SNO genes may be involved in novel metabolic pathways, the products of these genes can be used as targets to combat infections such as that for Cryptococcus as mentioned. Since these genes are involved in pyridoxine (vitamin B6) metabolism, agents can be identified that specifically inhibit Snz and Sno protein function thereby interfering with essential metabolic processes and causing cell death of the microrganism.
- pyridoxine vitamin B6
- yeast 1% yeast extract, 2% peptone, 2% glucose
- synthetic complete (SC) medium 0.67% Bacto-yeast nitrogen base without amino acids [Difco], 2% glucose; supplemented with auxotrophic requirements but lacking the amino acids for which one is selecting, unless indicated otherwise
- nonsupplemented YNB 2% glucose, 0.17% Bacto-yeast nitrogen base without amino acids and ammonium sulfate [Difco], 16.7% succinate buffer.
- YNB media supplied YNB
- adenine 0.06 mg/ml
- uracil uracil
- tryptophan and histidine
- leucine 0.03 mg/ml
- Solid media contained 2% agar.
- yeast cells were shaken at 250 rpm in 100 ml of medium at 30°C for the time indicated.
- strains produced for this study were derived from the common laboratory strains W303-1A (MW644) and W303-1 B (MW647). All yeast transformations were performed by the lithium acetate protocol or the quick-colony method. Transformants were selected on SC media lacking only the auxotrophic requirement used in the selection.
- PCR fragments containing the appropriate wild-type genes were obtained from common laboratory strain S288C (MW481) and introduced by linear transformation into a W303-1 strain.
- W303-1 strains were transformed with the YCplac22 CEN plasmid.
- Escherichia co/ XL2-Blue cells were used for propagation of all plasmids and were cultured in Luria broth with ampicillin according to the manufacturer's recommendations (Stratagene).
- the snz2-1 and snz3-1 disruption alleles were constructed by inserting a 1.8-kb Kpnl LEU2 fragment, generated by PCR from YCplad 81 , into SNZ2 and a 1.4-kb Kpnl TRP1 fragment, generated by PCR from YCplac22, into the Kpnl site of SNZ3 (Fig. 1 A).
- Chromosome blot analysis and Southern blot analysis were used to confirm the disruption of SNZ2 and SNZ3.
- the sno2 ⁇ 3snz2 ⁇ 3 sno3 ⁇ 3snz3 ⁇ 3 strain was constructed by inserting a 1.8-kb Sail LEU2 fragment generated by PCR from YCplad 81 into the Sail sites in SN03 and SNZ3, respectively. This resulted in the deletion of the SNZ2 and -3 and SN02 and -3 promoter region as well as 178 bp of the SN02 and -3 coding region and 575 bp of the SNZ2 and -3 coding region (Fig. 1B). The deletions were confirmed by Southern analysis.
- the snz1 ⁇ 3 sno1 ⁇ 3 strain was produced by outward-directed PCR of the snzl ⁇ l construct from the pWFY12 plasmid. This modification resulted in the deletion of 751 bp of the SNZ1 coding region and 159 bp of the SN01 coding region as well as the 450 bp between the two genes (Fig. 1 D). These strains were mated with sno2 ⁇ 3snz2 ⁇ 3 sno3 ⁇ 3snz3 ⁇ 3 strain, and hapioid segregants from tetrads were isolated to obtain the snz, sno sextuple mutant (MW980). The construction of the snzl ⁇ 2 strain (Fig. 1C) has been previously described.
- the heterozygous diploid sno1-1/SN01 strain, carrying snol disrupted by URA3 at amino acid 139 (of 224) (Fig. 1 E), and a control ssa4/SSA4 strain (MW1434) were obtained from Mike Snyder. Conventional dissection techniques were used to obtain the hapioid mutant strains.
- Example 3 We assessed the numbers of SNZ genes in several laboratory yeast strains by Southern hybridization of chromosome blots. All of the strains we examined carried a single copy of a gene closely related to SNZ1 but carried variable numbers of SNZ2 and -3 genes. S288C, W303, and YPH contain a single SNZ1 homologue and two genes more closely related to SNZ2 and -3 (data not shown). At least one strain, ⁇ 1278, which grows pseudohyphally, contains a single copy of SNZ1 and does not contain genes related to SNZ2 or SNZ3. Finally, DS10, which is derived from S288C, contains a fourth gene related to SNZ2 and -3 on chromosome II. Although we do not know the chromosomal position of this fourth SNZ gene, its similarity to SNZ2 and SNZ3 suggests that it may have arisen from gene duplication in the telomeric region.
- SNZ and SNO are separated by a single gene, whose product has some homology to thioesterases.
- P. horikoshii and A.fulgidus SNZ and SNO are also in an apparent operon.
- Example 4 Northern analysis of total RNA was used to assay SNZ2 and SNZ3 mRNA accumulation during growth to stationary phase (Fig. 2A).
- Total RNA was prepared with the Purescript RNA isolation kit (Gentra Systems Inc.) except that glass beads were used to lyse the cells. Briefly, the cells were vortexed with the glass beads for 30 s and put on ice for an additional 30 s, a cycle which was repeated three times. This produced a better yield of total RNA, especially for stationary-phase cells. Electrophoresis, hybridizations, and digitizing of autoradiograms were performed as previously described. We refer to these mRNAs as SNZ2/3 because the close identity between SNZ2 and SNZ3 does not allow distinction between their mRNAs.
- SNZ and SNO genes are conserved during evolution and because their relative orientations in yeast suggested that they share common promoter elements, we wanted to determine whether they were coregulated.
- Northern analysis of total RNAs revealed that SN01 and SNZ1 mRNAs do exhibit the same pattern of expression in cells grown to stationary phase (Fig. 2B).
- SN02/3 mRNAs which are also indistinguishable from each other by Northern analysis, accumulate at the same time as SNZ2/3 mRNAs (Fig. 2A).
- rRNAs are used as a control for loading because most mRNAs, e.g., that for actin, decrease in abundance in stationary phase and we currently have no RNA that remains constant in both exponential and stationary phases and that therefore could be used as an internal reference.
- SNZ1 and BCY1 as controls in some blots to allow us to demonstrate the intactness of mRNAs in cells grown to stationary phase (Fig. 2), because the mRNA levels of both SNZ1 and BCY1 in cells grown to stationary phase are known.
- Fig. 2 we concluded from this that the SNZ-SNO gene parts are coregulated and that the ability to coregulate these genes might have been an important factor in maintaining their proximity and orientations during evolution.
- Snz and Sno proteins interact.
- Snz proteins are part of a complex, in stationary-phase cells, with an apparent molecular mass of approximately 230 kDa (Fig. 4).
- the antibody to the common N-terminal peptide that was produced is capable of recognizing all three Snz proteins in yeast (data not shown).
- the complex is only present when Snzl p is present (Fig. 4).
- Snzl p is part of a protein complex in stationary-phase cells.
- yeast cells were transformed with the GAL4-bd-SNZ1 vector and a yeast GAL4-ad library. Plasmids from cells that grew on medium lacking histidine were isolated for further study. The results of this screen yielded two plasmids with activating genes SNZ2 and SN01.
- the plasmid containing SN01 includes all of the ORF except for the first 40 nucleotides.
- the plasmid containing SNZ2 includes more than three-fourths of the coding region.
- auxotrophic cells that have all wild-type SNZ and SNO genes were transferred from rich, glucose-based medium to nonsupplemented, nitrogen-limiting medium (YNB medium).
- YNB medium nonsupplemented, nitrogen-limiting medium
- Northern analysis revealed that SNZ1 and SN01 mRNAs accumulate in cells transferred to nonsupplemented YNB medium whereas SNZ2/3 and SN02/3 mRNAs did not (Fig. 5).
- SNZ1 mRNA accumulation in W303-derived strains that are auxotrophic for a single nutrient SNZ1 mRNA accumulated in the tr ⁇ 1-1 (MW1128), ade2-1 (MW1203), and ura3-1 (MW1207) mutants in YNB medium but did not accumulate in the his3-11 (MW1201) or the prototrophic (MW1199) strains incubated in YNB medium (Fig. 6). SNZ1 mRNA did not accumulate when tryptophan was added to YNB medium in which the trp1-1 mutant (MW1128) was incubated.
- SNZ1 mRNA accumulation in Ieu2-3, 112 trp1-1 ura3-1 ade2-1 his3-11, 15 can1-100 (MW644) cells incubated in YNB medium with supplements was suppressed with the addition of uracil and adenine.
- the SNZ1 mRNA accumulation in ade2-1 (MW1203) and ura3-1 (MW1207) mutants incubated in YNB medium with supplements was not suppressed (Fig. 6).
- a Northern blot of total RNA isolated from cells grown overnight in YPD medium to an OD 60 o of 2.0 to 3.0 and from cells transferred from YPD to YNB medium for 90 min is shown.
- the blot was probed with SNZL Auxotrophic strains were incubated with (+) or without (-) their specific auxotrophic requirements. Genotypes of the strains are as follows: Ieu2-3, 112 trp1-1 ura3-1 ade2-1 his3-11, 15 can1-100 (MW644); trp1-1 (MW1128); his3-11, 15 (MW1201); ade2-1 (MW1203); ura3-1 (MW1207); and canl- 100 (mW1199). Ethidium bromide-stained rRNAs are shown to indicate loading. The Northern blot was probed with ACT1 as a control. The autoradiograph was exposed for 1 day.
- SNZ1 induction in response to limitation of specific nutrients is a function of the auxotrophies of a given strain and is not a function of strain background.
- 6-AU sensitivity was evaluated on SC medium lacking uracil and supplemented with 6-AU to a final concentration of 30.0 ⁇ g/ml.
- Mycophenolic acid (MPA) sensitivity was scored on SC medium lacking adenine, guanine, and uracil and supplemented with MPA at a final concentration of 30.0 ⁇ g/ml.
- uracil When indicated uracil was added to the SC medium to a final concentration of 30.0 ⁇ g/ml. Strains were incubated at 30°C for 2 days. 6-AU inhibits IMP dehydrogenase, encoded by PUR5, and OMP decarboxylase, encoded by URA3. Strains carrying any of the snzl mutant alleles (Fig. 1), including snz1 ⁇ 2 and snzl ⁇ 2 snol ⁇ 3, and strains carrying snol mutations are extremely sensitive to 6-AU (Fig. 7A). Growth of snz and sno mutants and control strains on minimal medium without uracil, with or without 6-AU.
- a control is shown for each mutant strain; the control has the same auxotrophic markers as the mutant strain.
- Strains are as follows: control A, MW740; snz1 ⁇ 2 strain, MW926; snz1 ⁇ 3sno1 ⁇ 3 strain, MW908; snz1 ⁇ 2 YC- plac19 strain, MW1435; control B MW1071 : sextuple snz1,2,3 ⁇ 3 sno1,2,3 ⁇ 3 mutant strain, MW980; control C, MW1283; snz2,3 ⁇ 3 sno2,3 ⁇ 3 strain, MW1286; control D, MW1434; heterozygous snol- 1/SN01 strain, MW1359; sno1-1 strain, MW1427.
- the growth inhibition is specific to strains carrying snzl or snol mutations, and is not observed with SNZ1 snz2,3 sno2,3 mutants or wild-type controls (Fig. 7A).
- the 6-AU sensitivity cosegregates with snzl mutation through numerous crosses in both S288C and W303 strain backgrounds.
- snzl ⁇ 2 mutants were not complemented by mating to a SNZ1 ura3 strain or by transformation with a CEN plasmid carrying the SNZ1 structural gene and 953 bp of the region upstream of the start codon. This data suggests that the snzl ⁇ 2 mutant allele may cause a dominant-negative effect or an imbalance between Snzlp and Snol p resulting in a mutant phenotype under these conditions or that the snz1 ⁇ 2 mutant allele results in an alteration of SN01 expression.
- the 6-AU sensitivity of the snol mutant is complemented in diploids that are heterozygous for SN01, heterozygous for URA3 at the SN01 locus, and homozygous at the ura3-52 locus, i.e., they contain only the URA3 gene used to disrupt SNOL Additionally, snol mutants have a slower growth rate on SC-uracil media than the ssa4::URA3 mutant control and the sno1-1 heterozygous diploid (Fig. 7A). We conclude from these results that loss of either Snzl or Snol protein function is responsible for the 6-AU sensitivity observed in these mutants.
- the snz1,2,3 ⁇ 3 sno1,2,3 ⁇ 3, the snz2,3 ⁇ 3 sno2,3 ⁇ 3 are not sensitive to growth on methylene blue (Fig. 7B).
- sensitivity to methylene blue is only exhibited when Snzlp or Snol p is absent.
- Snol p an imbalance between Snzl p and Snol p, i.e., the presence of either Snzl p or Snol p and the absence of the other protein, results in the sensitivity to methylene blue.
- the sno1-1 mutant sensitivity to methylene blue is complemented in a sno1-1/SN01 heterozygous mutant (Fig. 7B).
- FIG. 8 details the results of our initial experiments.
- MW1272 which contains the entire SN01-SNZ1 intergenic region, exhibits essentially the same expression profile through the culture cycle as the genomic SNZ.
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AU11079/00A AU1107900A (en) | 1998-10-09 | 1999-10-08 | Detection using (snz) and (sno) genes and proteins |
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Non-Patent Citations (2)
Title |
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OKRESZ L. ET AL.: "T-DNA Trapping of a cryptic promoter identifies an ortholog of highly conserved SNZ growth arrest response genes in Arabidopsis", PLANT SCIENCE, vol. 138, 23 November 1998 (1998-11-23), pages 217 - 228, XP002922972 * |
PADILLA P.A. ET AL.: "The Highly Conserved, Coregulated SNO and SNZ Gene Families in Saccharomyces cerevisiae Respond to Nutrient Limitation", JOURNAL OF BACTERIOLOGY, vol. 180, no. 21, November 1998 (1998-11-01), pages 5718 - 5726, XP002922971 * |
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WO2006013010A3 (en) * | 2004-07-31 | 2006-06-08 | Metanomics Gmbh | Preparation of organisms with faster growth and/or higher yield |
JP2008507960A (en) * | 2004-07-31 | 2008-03-21 | メタノミクス ゲーエムベーハー | Production of organisms with faster growth and / or higher yield |
US20100050296A1 (en) * | 2004-07-31 | 2010-02-25 | Metanomics Gmbh | Preparation of organisms with faster growth and/or higher yield |
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