WO2000022169A1 - DETECTION USING Snz AND Sno GENES AND PROTEINS - Google Patents

Abstract

A probe, kit and method of using Snz and Sno genes and proteins for detection of foreign organisms in a sample. The sample can include cell culture, DNA samples, or patient specimens. The probe is utilized in a variety of assays, including Northern blots, Southern blots, Western blots, ELISAs and other detection assays.

Description

DETECTION USING Snz AND Sno GENES AND PROTEINS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing of U.S. Provisional Patent Application Serial No. 60/103,599, entitled SNZ1 PROTEIN/SNZ1 GENE-POTENTIAL ANTIBIOTIC TARGET, filed on October 9, 1998, and the specification thereof is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention (Technical Fieldl:

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.

Background Art:

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.

It is established that Snzlp (Snooze 1 protein) is present in all three phylogenetic domains (i.e. Eucarya, Bacteria, and Archea) and that its mRNA accumulates specifically in response to nutrient-limited growth arrest (Braun, 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. (1998) The highly conserved, coregulated SNO and SNZ gene families in Saccharomyces cerevisiae respond to nutrient limitation, J. Bacter. 180: 5718-5726). SNZ1 homologues in β. subtilis and H. brasiliensis also respond to potentially nutrient-limiting signals (Mitchell, C, Morris, P. W. and Vary, J. C. (1992) Amino acid sequences of several Bacillus subtilis proteins modified by apparent guanylylation, Molecular Microbiology 6: 157 9-1581 ; Sivasubramaniam, S., Vanniasingham, V. M., Tan, C. T. and Chua, N.H. (1995), Characterization of HEVER, a novel stress-induced gene from Hevea-brasiliensis, Plant Molec Biol 29: 173-178). This led to the deduction that Snz protein functions as part of an ancient response to nutrient limitation.

Subsequent work confirmed that Snzlp is the most highly conserved protein present in all three phylogenetic domains, sharing 60% identity with Snz homologues in bacteria and archaea (Braun, E. L., et al. (1996); Das, S., Yu, L., Gaitatzes, C, Rogers, R., Freeman, J., Bienkowska, J., Adams, R. M., Smith, T. F. and Lindelien, J. (1997) Biology's new rosetta stone, Nature 385: 29-30; Galperin, M. Y. and Koonin, E. V. (1997) Sequence analysis of an exceptionally conserved operon suggests enzymes for a new link between histidine and purine biosynthesis, Molec Microbiol 24: 443- 445; Ehrenshaft, M., Bilski, P., Li, M., Chignell, C. F. and Daub, M. E. (1999) A highly conserved sequence is a novel gene involved in de novo vitamin B6 biosynthesis, Proceedings of the National Academy of Sciences USA: PNAS 96: 9374-9378; Ehrenshaft, M., Chung, K. R., Jenns, A. E. and Daub, M. D. (1999) 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. cerevisiae has two additional SNZ genes, SNZ2 and SNZ3 that are approximately 99% identical to each other and 80% identical to Snzlp. 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.

Although present in all three phylogenetic domains, the SNZ and SNO genes are not present in all organisms nor are they present in all species of related lineages. For example, SNZ and SNO are present in H. influenzae but not in the related E. coli. However, it is probably significant that SNZ and SNO genes are adjacent to each other because all organisms that carry SNZ genes also carry SNO genes. And, with only two exceptions, 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.

The functions of Snzlp and Snolp have not been determined. Disruption of the SNZ1 gene or all three pairs of SNZ-SNO genes in yeast is not lethal (Braun, E. L., et al. (1996); Padilla, P. A., et al. (1998)). However, because their sequences and proximity are so highly conserved, it is likely that these proteins share an important fundamental physiological function, including distant relationships with proteins involved in amino acid, vitamin, and nucleic acid biosynthesis.

Protein sequence comparisons suggest that Snz proteins bear limited resemblance to metabolic enzymes ThiG, TrpC, and HisF in E.coli and that Sno proteins bear resemblance to glutamine amidotransferases such as HisH and GuaA (Zalkin, H. (1993) The amidotransferases, Advances in Enzvmoloαv and Related Areas of Molecular Biology. 203-209; Galperin, M. Y. and Koonin, E. V. (1997) Sequence analysis of an exceptionally conserved operon suggests enzymes for a new link between histidine and purine biosynthesis, Molec Microbiol 24: 443-445). HisF and HisH interact to produce aminoimidazole carboxamide ribonucleotide in E.coli (Winkler). His7p, a multifunctional protein, catalyzes the same reaction in yeast. Thus it has been proposed that Snolp is the glutamine amidotransferase and Snz1 p the acceptor domain for transfer of an amido group to an as yet unknown substrate. However, 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. The failure to readily associate Snz and Sno proteins with already identified control networks or metabolic pathways has led to suggestions that they function in a novel salvage pathway that interconverts amino acids or nucleotides (Galperin, M. Y., et al. (1997); Padilla, P.A., et al. (1998)).

SUMMARY OF THE INVENTION (DISCLOSURE OF THE INVENTION)

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 sample, and patient specimen.

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. Preferably, 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. Preferably, 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.

Other objects, advantages and novel features, and further scope of applicability of the present invention will be set forth in part in the detailed description to follow, taken in conjunction with the accompanying drawings, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated into and form a part of the specification, illustrate several embodiments of the present invention and, together with the description, serve to explain the principles of the invention. The drawings are only for the purpose of illustrating a preferred embodiment of the invention and are not to be construed as limiting the invention. In the drawings:

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; and

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

(Braun, E.L, et al. (1996); Padilla, P.A., et al. (1998)), nutrient starvation (Padilla, P.A., et al. (1998)), oxidative stress (Padilla, P.A., et al., (1998)), and DNA-damaging conditions (Jelinsky, S. and Samson, L. (1999) Global response of Saccharomyces cerevisiae to an alkylating agent, Proceedings of the National Academy of Sciences USA 96: 1486-1491). 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 β. 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

Saccharomyces cerevisiae, Journal of Bacteriology 95: 824-8732; Daignan-Fomier, B. and Fink, G. (1992) Coregulation of purine and histidine biosynthesis by the transcriptional activators Bas1 and Bas2, Proc Natl Acad Sci 89: 6746-6750; Seron, K., Blondel, M.O., Haguenauer-Tsapis, R. and Voland, C. (1999) Uracil-induced down-regulation of the yeast uracil permease, Journal of Bacteriology 181: 1793-1800). This led to the suggestion that the condition that induces SNZ1 in the absence of uracil and adenine is not relieved as rapidly upon supplementation as that for amino acid starvation.

In addition, two observations led to conclusion that SNZ1 may be induced in response to conditions that alter the balance of intracellular nucleotides. First, 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). Second, SNZ1 is highly induced (65-fold) in the presence of DNA-damaging agents (Jelinsky, S., et al. (1999)). However, snz mutants are not sensitive to the DNA-damaging agent EMS, suggesting that SNZ1 does not encode a DNA repair enzyme.

The 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.

To address the first of these, we examined the factors that affect pyrimidine metabolism. Mutations in two other genes, PPR1 and PPR2, are also sensitive to 6-AU. PPR1 (pyrimidine pathway regulator) encodes a transcriptional regulator of several pyrimidine pathway genes. SNZ1 does not contain a PPR1 recognition element making it unlikely that SNZ1 induction or snzl mutant sensitivity is due directly to Ppr1. Nor does SNZ1 contain any sequence motifs suggesting that it is a transcription factor. Thus, although 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.

To address the second of the conditions, we looked at the effects of methylene blue upon snz. Methylene blue (MB) 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. 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 sensitivity of either snzl or snol mutants, but not the double mutant, to methylene blue, provides evidence that the balance and physical interaction of these proteins may be needed to maintain metabolites that are targets for the methylene blue. Based on these results, 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.

To address the third of these conditions, we looked at the biosynthesis of pyridoxine. 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. (1960) Chromosome mapping in Saccharomyces cerevisiae: centromere-linked genes, Genetics 45: 1085-1110-60; Mortimer, R.K., Schild, D., Contopoulou, C.R. and Kans, J.A. (1991) Genetic and physical maps of Saccharomyces cerevisiae, Methods in Enzymology ΛW. 827- 863). Neither the sequence nor the function of PDX2 are known. However, based on linkage mapping data, PDX2 is linked to the SNZ1 gene and may be allelic to SNZ1 or SNOL

To examine the relationship of the 6-AU^s and Pdx^" phenotypes, mutants lacking functional ORFs were spotted onto media containing either 6-AU or pyridoxine. Three different snz and sno mutants were examined to validate the phenotypes. The results showed that all three mutants were inhibited by 6-AU and required pyridoxine; all three mutants responded similarly to any given treatment; pyridoxine did not suppress the 6-AU inhibition even at concentrations considerably greater than was necessary for growth on medium lacking pyridoxine; the mutants were consistently more sensitive on medium containing 6-AU and lacking pyridoxine than for either condition individually; the mutants consistently grew more poorly on medium with 6-AU than on medium lacking pyridoxine; excess uracil did not suppress the Pdx- phenotype; snz2/3-sno2/3 mutants were not inhibited by the above conditions. These results lead to the conclusion that 6-AU and Pdx reflect different functions of Snz and Sno proteins.

Interestingly, although 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. Thus, one explanation for this gene distribution is that 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.

Other applications of this invention are to use the sequences as a tool to distinguish fungal or bacterial contamination of tissue culture or as control marker to identify fungal DNA contamination in various purified DNA samples.

Because the 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. Industrial Applicability:

The invention is further illustrated by the following non-limiting examples.

Example 1

Strains and media. The following media were used to cultivate yeast: YPD (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), and nonsupplemented YNB (2% glucose, 0.17% Bacto-yeast nitrogen base without amino acids and ammonium sulfate [Difco], 16.7% succinate buffer). When indicated, supplements were added to the YNB media (supplemented YNB) to the following final concentrations: adenine, 0.06 mg/ml; uracil, tryptophan, and histidine, 0.02 mg/ml; and leucine, 0.03 mg/ml. Solid media contained 2% agar. For each liquid-culture experiment, yeast cells were shaken at 250 rpm in 100 ml of medium at 30°C for the time indicated.

Most of the 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.

To obtain strains with single tryptophan (MW1128), histidine (MW1201), uracil (MW1207), or adenine (MW1203) auxotrophies, 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. To obtain strains that are prototrophic for tryptophan, 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). Example 2

Mutant construction. 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 sno2Δ3snz2Δ3 sno3Δ3snz3Δ3 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 snz1Δ3 sno1Δ3 strain was produced by outward-directed PCR of the snzlΔl 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 sno2Δ3snz2Δ3 sno3Δ3snz3Δ3 strain, and hapioid segregants from tetrads were isolated to obtain the snz, sno sextuple mutant (MW980). The construction of the snzlΔ2 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, Σ1278, 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.

An analysis of the sequences adjacent to the yeast SNZ genes revealed an additional conserved, duplicated gene upstream of each SNZ gene, which we called SNO (for SΛ/Z-proximal ORF). The presence of SN02 and SN03 genes adjacent to SNZ2 and SNZ3 was expected because of the chromosomal duplication that included the entire region. The presence of the SN01 gene adjacent to the more centromeric SNZ1 also included the entire SN01 gene. Like SNZ2 and SNZ3 genes, SN02 and SN03 encode proteins that are almost 100% identical to each other and that are approximately 72% identical to Snol p. The Sno proteins are predicted to have a molecular mass of 21.5 kDa.

On the basis of the microbial genomes that have been entirely sequenced, all species that contain an SNZ gene also contain a SNO gene. In organisms for which the complete genome sequences are available, the proximal location of the SNZ and SNO genes is relatively conserved but the orientations of the two genes differ between eukaryotes and prokaryotes. In yeast, SNZ and SNO genes are adjacent and divergently transcribed, whereas in the bacteria B. subtilis, H. influenzae, and M. leprae the SNZ and SNO homologues are in apparent operons, i.e., they are adjacent and transcribed in the same direction. In the bacterium M. tuberculosis, SNZ and SNO are separated by a single gene, whose product has some homology to thioesterases. In the archaea P. horikoshii and A.fulgidus SNZ and SNO are also in an apparent operon. These observations suggest that there has been strong selective pressure for the retention of the proximal location of SNZ and SNO genes through evolution. M. jannaschii and M. thermoautotrophicum are the two exceptions; for them the chromosomal location is not conserved between SNZ and SNO genes.

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. The SNZ2/3 pattern of expression differs noticeably from that of SNZ1 in cells grown to stationary phase (Fig. 2A). Ethidium bromide-stained rRNAs are presented to show relative loading. Lanes: 1 , early exponential phase (OD₆oo = 0.95); 2, late exponential phase (OD₆oo = 5.0); 3, diauxic shift (OD₆oo = 7.1 ; determined by glucose exhaustion); 4, 24 h after the diauxic shift (OD₆oo = 10.5); 5, stationary phase (5 days after inoculation); 6, stationary phase (8 days after inoculation). SNZ2/3 mRNAs accumulate slightly before the diauxic shift, decrease in abundance at the diauxic shift, and increase for a short period after the diauxic shift. Unlike SNZ1 mRNA, SNZ2/3 mRNAs are not detectable in Northern analysis of total RNA from stationary-phase cells.

Because the positions of the 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). Lanes: 1 , early exponential phase (OD₆₀₀ = 1.4); 2, late exponential phase (OD₆oo = 6.6); 3, diauxic shift (OD₆oo = 7.1); 4, 22 h after the diauxic shift (OD₆₀₀ = 10.5); 5, 48 h after the diauxic shift (OD₆oo = 19.6); 6, stationary phase (5 days after inoculation); 7, stationary phase (8 days after inoculation), BCY1 was used as a control. Autoradiographs were exposed for 3 days (SNZ2 and -3, SN02 and -3, BCY1, and SNZ1) or 5 days (SN01). Likewise, 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. For these analyses, we have used 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. 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.

Example 5

Snz and Sno proteins interact. In order to determine if Snz proteins interact as a higher-order complex, we analyzed extracts of wild-type and various snz mutants via nondenaturing gradient polyacrylamide gel electrophoresis. This method involves running samples on a 4-to-20% acrylamide gel for 20 h through a matrix of decreasing pore size until proteins stop at an impassable acrylamide percentage. Because of the long run, proteins that migrate slowly due to low charge still run to the limiting pore size of the gel. A protein's migration on these gels is based solely on shape and size, allowing specific size comparisons, as opposed to that of proteins analyzed by continuous nondenaturing electrophoresis, where protein migration is based on size, charge, and shape. The analysis revealed that 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). However, the complex is only present when Snzl p is present (Fig. 4). Thus, we conclude that Snzl p is part of a protein complex in stationary-phase cells.

To learn more about the Snzl p complex, we used two-hybrid analysis to identify proteins that might interact with Snzl p. We first examined whether Snzl p could interact with itself by cloning the SNZ1 ORF in frame with the GΛL4-binding domain (GAL4-bd) and GAL4 activation domain (GAL4- ad). By themselves, the Gal4-Snz1 activation or binding domain fusion proteins did not activate GALl-lacZ or allow growth on media lacking histidine or adenine. When cotransformed into yeast cells, the activation and binding domain fusion proteins activated the transcription of a GALl-lacZ reporter gene and allowed strain PJ69-4A, which is conditionally auxotrophic for histidine and adenine, to grow in the absence of these supplements. These results suggest that Snzl p could function in the cell as a dimer or higher-order oligomer.

To identify other proteins that interact with Snzl p, 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. Based on β-galactosidase assays, the interaction between Snzl p and Snz2p is not as strong as the interaction between Snzl p and Snzl p. However, the interactions between Snzl p and Snol p and between Snzlp and Snzlp are equally strong. These results suggest that Snzlp and Snolp may function in the cell as an oligomeric complex. The apparent physical interaction between Snzl p and Snolp, the coregulation of their genes, and their conserved proximity on the chromosome provide strong support for the hypothesis that these two proteins are involved in the same pathway.

Example 6

To determine whether limitation for specific nutrients affects the regulation of SNZ genes, auxotrophic cells (MW644) that have all wild-type SNZ and SNO genes were transferred from rich, glucose-based medium to nonsupplemented, nitrogen-limiting medium (YNB 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). These results indicate that under specific starvation conditions as well as in cells grown to stationary phase, SNZ and SNO genes are coregulated and that SNZ1 and SNZ2 and -3 are controlled by distinct regulatory mechanisms.

To determine whether the accumulation of SNZ1 mRNA in cells incubated in YNB was due to starvation for specific nutrients or a general nitrogen starvation signal, we examined SNZ1 expression in auxotrophic, W303-derived cells (MW644) transferred from rich, glucose-based medium to YNB medium supplemented with auxotrophic requirements (Fig. 6). Under these conditions, SNZ1 mRNA does not accumulate, suggesting that SNZ1 induction is not due to general nitrogen limitation but to the absence of one or more auxotrophic requirements.

To identify the nutrient limitation responsible for the dramatic increase in SNZ1 expression, we evaluated SNZ1 mRNA accumulation in W303-derived strains that are auxotrophic for a single nutrient (Fig. 6). SNZ1 mRNA accumulated in the trρ1-1 (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. Surprisingly, 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 OD₆₀o 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. These results indicate that an imbalance between nucleotide levels could have an effect on SNZ1 mRNA accumulation. The difference in SNZ1 mRNA accumulation in the Ieu2-3, 112 trp1-1 ura3-1 ade2-1 his3-11, 15 can1-100 (MW644) cells versus the ura3-1 (MW1207) or ade2-1 (MW1203) cells incubated in YNB medium with supplements cannot be directly determined because of the additional auxotrophies in the MW644 cells which could affect the SNZ1 mRNA levels.

Strains derived from another common laboratory strain, S288C, were examined to determine whether SNZ1 induction in YNB medium was a strain-specific phenomenon. As in W303-derived strains, SNZ1 is induced when S288C strains with multiple auxotrophies, e.g., MW751 (his3Δ200 leu2Δl lys2Δ202 trp1Δ63 ura3-52), are incubated in YNB medium but is not induced in a prototrophic strain (MW481) (data not shown). We conclude from these results that 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.

To further investigate Snz and Sno protein function, we evaluated the phenotypes of snz and sno mutants. Yeast strains carrying snzl, snol, snz2,3 sno2,3, and snz1,2,3 sno1,2,3 mutations are viable and grow at rates indistinguishable from those of wild-type cells on rich, glucose-based medium (data not shown). To determine if Snz and Sno proteins were essential under other conditions, we tested these mutants for their ability to survive and grow under a variety of different medium and temperature regimes. None of the snz sno mutants exhibited changes in growth rates or viability during growth to stationary phase on glucose medium, acetate-based medium, or nitrogen-limiting medium (YNB medium) at 30 and 37°C, compared with control cells.

Since SNZ1 mRNA accumulates in ura3 and ade2 mutants starved for uracil and adenine, we tested snzl, snol, snz2,3 sno2,3, and snz1,2,3 sno1,2,3 mutants for sensitivity to 6-AU, an inhibitor of both pyrimidine and purine biosynthesis. 6-AU sensitivity was evaluated on SC medium lacking uracil and supplemented with 6-AU to a final concentration of 30.0 μg/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 μg/ml. When indicated uracil was added to the SC medium to a final concentration of 30.0 μg/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 snz1Δ2 and snzlΔ2 snolΔ3, 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; snz1Δ2 strain, MW926; snz1Δ3sno1Δ3 strain, MW908; snz1Δ2 YC- plac19 strain, MW1435; control B MW1071 : sextuple snz1,2,3Δ3 sno1,2,3Δ3 mutant strain, MW980; control C, MW1283; snz2,3Δ3 sno2,3Δ3 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.

The addition of uracil to the medium or in the introduction of URA3 on a 2μm plasmid suppresses the 6-AU growth inhibition of snzl strains (data not shown). However, the introduction of the centromeric plasmid YCplad 9 (URA3 TRP1) to the snz1Δ2 mutant does not suppress the 6-AU sensitivity (Fig. 7A). Thus, one copy or a few copies of plasmid-borne URA3 are not sufficient to suppress the 6-AU sensitivity of snzl mutant strains. However, snzlΔ2 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 snzlΔ2 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 snz1Δ2 mutant allele results in an alteration of SN01 expression.

We examined complementation by comparison of sno1-1/SN01 heterozygous diploid and sno1-1 hapioid strains which both contain a single gene disrupted by transposon mutagenesis (Fig. 1). To ensure that the 6-AU sensitivity was not due to the URA3 gene used to disrupt SN01, and ssa4::URA3 mutant was used as a control. 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. To determine whether the 6-AU sensitivity of snzl mutants was due to inhibition of IMP dehydrogenase or OMP decarboxylase or both, we examined the growth of snzl mutants in SC medium containing MPA, a specific inhibitor of IMP dehydrogenase. The growth of snzl mutants was not affected by MPA (data not shown), suggesting that the sensitivity occurs through an effect of Snzl p on pyrimidine biosynthesis.

Example 7

Sensitivity of snz and sno mutants to methylene blue. A recent report indicated that a mutant of the SNZ1 orthologue in the ascomycete C. nicotianae was sensitive to singlet oxygen generators, including methylene. To evaluate methylene blue sensitivity, yeast strains were grown overnight in YPD and the growth medium was diluted to a final OD₆oo of 0.25/ml and then diluted by 1/5 for a series of five dilutions. Then 6 μl from each of these dilutions was plated onto YPD medium, supplemented with 37.0 μg of methylene blue per ml and exposed to a light source, when indicated, for 3 days at 25°C. Our results indicated that methylene blue sensitivity is specific to cells carrying snzl or snol mutations (Fig. 7B). Growth of snz and sno mutants and control strains on YPD medium supplemented with methylene blue, in the presence or absence of light. Strains are as follows: control strain, MW740; snzlΔ2 strain, MW925; sno1-1 strain, MW1427; snz1Δ3 sno1Δ3 strain, MW908; heterozygous diploid sno1-1/SN01 strain, MW1359. Surprisingly, the snz1,2,3Δ3 sno1,2,3Δ3, the snz2,3Δ3 sno2,3Δ3 (data not shown), and the snz1Δ3sno1Δ3 alleles are not sensitive to growth on methylene blue (Fig. 7B). Thus, sensitivity to methylene blue is only exhibited when Snzlp or Snol p is absent. These results indicate that 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).

Example 8

To examine the relationship between 6-AU and pyridoxine, exponentially growing cultures (OD₆oo 1-2) were diluted to the same (OD₆oo -25) and then serial dilutions (5μl) were spotted onto medium containing 6-AU or pyridoxine. Plates were incubated for 3 days at room temperature prior to scanning. SnzlΔ2 lacks a functional ORF. Δsnz1-sno1 lacks functional ORFs and ail of the intergenic region. "Sextuple" lacks all three SNZ1 and SN01 ORFs, isogenic wild-type controls. The results are discussed above in the Description of Preferred Embodiments section.

Example 9

Figure 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. The three deletion constructs, MW1254, MW1457, and MW1460 exhibit altered SNZ1 expression both in terms of timing of transcription and in the quantity of message.

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

Although the invention has been described in detail with particular reference to these preferred embodiments, other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover in the appended claims all such modifications and equivalents. The entire disclosures of ail references, applications, patents, and publications cited above are hereby incorporated by reference.

Claims

CLAIMSWhat is claimed is:

1. A probe comprising at least one sequence selected from the group consisting of

SNZ1, SNZ 2, SNZ3, SN01, SN02, SN03, Snzlp, Snz2p, Snz3p, Snolp Sno2p, Sno3p, and fragments thereof, for detecting and determining origin of a foreign organism in a sample.

2. A kit for detecting and determining origin of a foreign organism in a sample, said kit comprising: a Snz/Sno probe; a marker; and a detection method for detecting said probe.

3. The kit of claim 2 wherein said probe comprises at least one sequence selected from the group consisting of SNZ1, SNZ 2, SNZ3, SN01, SN02, SN03, Snzl p, Snz2p, Snz3p, Snol p Sno2p, Sno3p, and fragments thereof.

4. The kit of claim 2 wherein the probe 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.

5. The kit of claim 2 wherein said marker comprises at least one marker selected from the group consisting of fluorescent, radioactive, magnetic, and other markers; and optionally, wherein said 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.

6. An assay for detecting and determining origin of a foreign organism in a sample, said assay comprising the steps of: a) probing the sample with a Snz/Sno probe having a marker; and b) detecting the marker.

7. The assay of claim 6 wherein the step of probing the sample with a Snz/Sno probe having a marker comprises probing the sample with at least one sequence selected from the group consisting of SNZ1, SNZ 2, SNZ3, SN01, SN02, SN03, Snzl p, Snz2p, Snz3p, Snol p Sno2p, Sno3p2p, Sno3p, and fragments thereof; and optionally wherein the step of probing the sample with a Snz/Sno probe having a marker comprises probing with at least one marker selected from the group consisting of fluorescent, radioactive, and other markers; and optionally wherein the step of detecting the marker comprises 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 optionally wherein the step of probing the sample comprises probing at least one source selected from the group consisting of cell culture, DNA sample, and patient specimen.

8. A method for treating a disease with antibiotics, the method comprising the steps of: a) targeting a Snz/Sno target for receiving the antibiotic; and b) administering the antibiotic.

9. The method of claim 8 wherein the step of targeting the Snz/Sno target comprises targeting at least one sequence selected from the group consisting of SNZ1, SNZ 2, SNZ3, SN01, SN02, SN03, Snzlp, Snz2p, Snz3p, Snolp Sno2p, Sno3p2p, Sno3p, and fragments thereof; and optionally wherein the step of targeting the Snz/Sno target comprises targeting the metabolic pathway of the Snz/Sno target.

10. The method of claim 8 wherein the step of administering the antibiotic comprises administering an antibiotic which impedes the metabolic pathway of the target.