WO1999060111A9 - Estimation de cibles assistee genetiquement et utilisee dans la conception de medicaments antibacteriens - Google Patents
Estimation de cibles assistee genetiquement et utilisee dans la conception de medicaments antibacteriensInfo
- Publication number
- WO1999060111A9 WO1999060111A9 PCT/US1999/010919 US9910919W WO9960111A9 WO 1999060111 A9 WO1999060111 A9 WO 1999060111A9 US 9910919 W US9910919 W US 9910919W WO 9960111 A9 WO9960111 A9 WO 9960111A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- gene
- nonessential
- essential
- gate
- bacteria
- Prior art date
Links
- 238000011156 evaluation Methods 0.000 title abstract description 4
- 229940124350 antibacterial drug Drugs 0.000 title description 3
- 238000007876 drug discovery Methods 0.000 title description 2
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 93
- 238000000034 method Methods 0.000 claims abstract description 17
- 239000013612 plasmid Substances 0.000 claims description 19
- 241000894006 Bacteria Species 0.000 claims description 15
- 230000006798 recombination Effects 0.000 claims description 10
- 238000005215 recombination Methods 0.000 claims description 10
- 239000003550 marker Substances 0.000 claims description 9
- 230000006801 homologous recombination Effects 0.000 claims description 8
- 238000002744 homologous recombination Methods 0.000 claims description 8
- 108010077805 Bacterial Proteins Proteins 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 238000005259 measurement Methods 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 241000193996 Streptococcus pyogenes Species 0.000 abstract description 21
- 230000001580 bacterial effect Effects 0.000 abstract description 13
- 238000004458 analytical method Methods 0.000 abstract description 8
- 244000052616 bacterial pathogen Species 0.000 abstract description 8
- 230000010354 integration Effects 0.000 abstract description 6
- 241000191967 Staphylococcus aureus Species 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000010230 functional analysis Methods 0.000 abstract description 3
- 244000000059 gram-positive pathogen Species 0.000 abstract description 3
- 230000007918 pathogenicity Effects 0.000 abstract description 3
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 10
- 230000003115 biocidal effect Effects 0.000 description 9
- 239000012634 fragment Substances 0.000 description 9
- 238000012360 testing method Methods 0.000 description 8
- 230000009466 transformation Effects 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 101150101115 speB gene Proteins 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 229960003276 erythromycin Drugs 0.000 description 5
- 229930027917 kanamycin Natural products 0.000 description 5
- 229960000318 kanamycin Drugs 0.000 description 5
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 5
- 229930182823 kanamycin A Natural products 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000002759 chromosomal effect Effects 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 238000012252 genetic analysis Methods 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 239000013615 primer Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000005018 casein Substances 0.000 description 2
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 2
- 235000021240 caseins Nutrition 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 125000001475 halogen functional group Chemical group 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000007320 rich medium Substances 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 101710092462 Alpha-hemolysin Proteins 0.000 description 1
- 108700003860 Bacterial Genes Proteins 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 101710097670 Extracellular cysteine protease Proteins 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 101710182223 Toxin B Proteins 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 210000003578 bacterial chromosome Anatomy 0.000 description 1
- 230000008436 biogenesis Effects 0.000 description 1
- 239000006161 blood agar Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000033077 cellular process Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 1
- 229960000268 spectinomycin Drugs 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
Definitions
- the present invention is directed to methods for gene analysis in
- homology should allow assignment of potential function to many gene products.
- GATE Genetics-Assisted Targeted Evaluation
- GATE is a robust procedure for gene knock-out analysis in bacteria that is rapid, reproducible, and scalable to accommodate analysis of large numbers of genes.
- a further attribute of GATE is its potential for functional genetic analysis in a broad range of pathogenic bacteria, including both Gram-positive and Gram-negative organisms.
- GATE holds several advantages over prior art methods for determining whether or not a gene is likely to be essential for bacterial growth. Typically, simple failure to obtain transformants with a plasmid construct designed to produce either gene disruption or gene replacement mutations at a locus of interest is interpreted as evidence that the gene is essential for bacterial growth. However, failure to obtain transformants is not in itself sufficient for assignment of gene function, due to lack of adequate positive controls for successful transformation and plasmid integration.
- the present invention provides methods for determining the essentiality
- the recombination cassette is capable of
- cassette comprising a second selectable marker flanked on its 5 ' and 3 ' termini by
- the recombination cassette is capable of being integrated into the genome of
- step (b) individually culturing the transformed culture produced in step (a)
- step (b)
- step (d) comparing the measurements made in step (c), wherein the lack of
- the selectable markers are antibiotic resistance genes
- the selective conditions are individual cultures comprising each antibiotic specified by the cognate resistance gene.
- Figure 1 is a schematic diagram showing the use of the GATE assay for target validation in the discovery of new antibacterial drugs.
- Figure 2 depicts the advantages of using Streptococcus pyogenes group A bacteria in GATE assays of the present invention.
- Figure 3 is a schematic diagram showing the gene disruption strategy using GATE according to the present invention.
- Figure 4 shows the use of the speB locus encoding the Streptococcus erythrogenus toxin B gene used as an internal standard for classifying test genes in S. pyogenes.
- Figure 5 shows the analysis of selected ORFs in S. pyogenes. Colony counts for each cotransformation of the control gene and a test gene are shown.
- Figure 6 shows the use of the GATE technology of the present invention in the Gram-positive pathogen Staphylococcus aureus.
- Successful disruption of the structural gene for ⁇ -hemolysin (hla) gene was evidenced by the loss of hemolysis on blood agar and was confirmed by PCR analysis.
- Gene analysis using GATE is performed by cotransformation of pathogenic bacteria with two independent integrative plasmids which contain a 300- 500 bp internal fragment of either (i) the target gene, whose mutant phenotype is being investigated, or (ii) a nonessential gene for which there is an easily scored mutant phenotype.
- Each integrative plasmid is marked with a different antibiotic resistance determinant so that transformants bearing gene disruptions at either the target locus or at the nonessential control locus can be distinguished by selection on appropriate antibiotic media.
- Gene disruptions are created by a single recombination event between the plasmid-encoded internal gene fragment and the homologous chromosomal sequence, resulting in duplication of truncated copies of the target gene. Determination of gene essentiality/nonessentiality is made by comparing the number of antibiotic-resistant transformants generated by plasmid integration events which inactivate the target with those formed using the control gene.
- a nonessential scorable gene as an internal standard for plasmid transformation and homologous recombination is a key feature of this method. This provides a benchmark against which to assess the dispensability of a given target gene for bacterial growth. If it is possible to obtain plasmid integration with a target gene construct at a frequency comparable to that obtained with the control gene, it is inferred that the target gene is nonessential for growth. If, on the other hand, it is not possible to obtain plasmid integration at the target locus when in the same transformation mixture gene disruption at the control locus is readily obtained, it is inferred that the target is essential for growth.
- Suitable candidates for nonessential scorable genes are easily identifiable in most pathogenic bacteria.
- GATE was performed in Streptococcus pyogenes using as an internal control a nonessential gene, speB, encoding an extracellular cysteine protease whose activity in bacterial colonies is readily assayed on Petri dishes containing casein as a protease substrate.
- speB nonessential gene
- a gene disruption mutation in the structural gene for a secreted lipase (geh) whose expression can be scored conveniently on an indicator medium was used.
- GATE can be applied to any pathogenic bacterium that undergoes homologous recombination and for which gene delivery procedures (e.g., electrotransformation, natural competence, or conjugation) can be established. GATE is well-suited for analysis of large numbers of genes. The approach requires only a modest amount of DNA sequence information in order to create a gene disruption, because homologous recombination can proceed efficiently in many bacteria using cloned internal gene fragments as short as 300-500 bp.
- gene delivery procedures e.g., electrotransformation, natural competence, or conjugation
- a series of integrative vectors (such as, e.g., derivatives of Stratagene's pCR-ScriptTMCam) which (i) permit rapid cloning of blunt- end, PCR-generated gene fragments, and (ii) contain antibiotic-resistance markers selectable in a wide variety of Gram-positive bacteria (such as, e.g., erythromycin, kanamycin, or spectinomycin resistance genes) has been constructed.
- GATE also incorporates high through-put methods for verifying the genotypes of mutant strains. PCR procedures have been developed that utilize crude lysates of individual bacterial colonies and thus permit the determination as to whether or not a gene disruption event occurred at the correct chromosomal location.
- GATE has been successfully implemented in a functional genetic analysis performed on 26 predicted genes in the Gram-positive bacterial pathogen S. pyrogenes. These targets predominantly included S. pyrogenes homologs of genes of known function in other bacteria, e.g., genes involved in essential cellular processes such as transcription, translation, DNA replication, and cell wall biogenesis. Representative genes which are suspected to be nonessential for growth in rich medium, including S. pyrogenes homologs of virulence factors known in other bacteria, have also been examined. In a few cases, functional analysis was performed on genes which were essential in certain bacteria and nonessential in others, such that the outcome of the experiment in S. pyogenes could not be predicted in advance.
- integrative vectors bearing an internal fragment of the target gene and the speB nonessential scorable control gene were introduced into S. pyogenes by electroporation and gene disruption events at target or control loci were selected for on rich antibiotic media. Inferences as to gene function in vitro were made based on colony counts as described above. It was possible to readily distinguish essential and nonessential loci: 16 of the 26 predicted genes that were evaluated clearly encoded functions essential for bacterial growth,
- GATE as an approach to functional analysis is the fact that, in addition to classifying genes according to their essential or nonessential character for growth, mutant strains can be assayed later for alternative phenotypes in vitro such as, e.g., growth under conditions of oxidative stress, altered osmolarity or pH, or iron deprivation. This procedure can be used to identify functions involved in pathogenicity.
- the present invention is described below in working examples which are intended to further describe the invention without limiting its scope.
- integrative plasmid vectors were used that contain different antibiotic resistance markers (Tao, et al., 1992). These plasmids cannot replicate in S. pyogenes because they do not contain a streptococcal origin of replication (Tao, et al. , 1992). Integrative plasmids used in this study consisted of the PCR Script vector (Stratagene) which had been modified by the addition of antibiotic resistance markers.
- kanamycin and erythromycin resistance genes were generated by PCR amplification of plasmids pSF151 and pVA891.1, respectively (Tao, et al., 1992) using Pfu polymerase (Stratagene).
- Pfu polymerase (Stratagene).
- PCRScript-kan conferring kanamycin-resistance, the following oligonucleotide primers were used: 5 ' -GATCCCATGGGCGAACCATT-3 ' and 5 ' -GATCCC ATGGAATTCCTCGT-3 ' .
- PCRScript-erm conferring erythromycin-resistance
- the oligonucleotide pr imers w ere 5 ' -GATC C C A TGGC GA A A T GAT A - 3 ' and 5 ' -GATCCC ATGGGGCGCTAGGG-3 ' .
- PCR-generated antibiotic resistance genes were ligated into the Nco I site of the PCRScript vector.
- a 300-500 bp internal fragment corresponding to each gene of interest was generated by PCR amplification with Pfu polymerase (Stratagene) using S. pyogenes genomic DNA template (S.
- pyogenes strain 2F-3MB-1512 and gene-specific primers were designed using DNA sequence data in the public domain (Streptococcus pyogenes genome sequence database, University of Oklahoma).
- the PCR reaction mixtures contained 300 ng of genomic DNA template, 100 ng of each primer, 0.2mM dNTPs, and 2.5 units of Pfu polymerase. The reactions were amplified for 30 cycles (94°C, 1 " ; 50°C, 1 ' ; 72°C, 3'). Each gene fragment was ligated into the Sma I site of PCRScript-erm in the presence of Srfl to prevent vector recircularization.
- S. pyogenes An internal gene fragment of the nonessential control gene (speB) was ligated into the Sma I site of PCRScript-kan in the same manner. Cotransformation of S. pyogenes was performed to determine whether a particular gene is essential or nonessential. Ten micrograms of each plasmid were combined, ethanol precipitated, and dried under vacuum. Transformation of S. pyogenes was by electroporation (McLaughlin and Ferretti, 1992). When preparing electrocompetent cells, 100 ml of early log phase cultures were harvested by centrifugation at 3000 g for 10 minutes at 4°C. The supernatant was saved and cell pellets were resuspended in 5 ml of the spent media.
- the cell suspension was subjected to heat shock at 42° C for 9 minutes, after which cells were collected by centrifugation and washed 2X in 10 ml of 15% sterile glycerol. Final pellets were resuspended in 0.6 ml of 15% glycerol.
- 200 ml of electrocompetent cells were added directly to the tube containing the dried plasmid DNA pellet.
- the transformation mixture was transferred to a chilled sterile electroporation cuvet and a single pulse was applied (1.75kV, 25 ⁇ F capacitance, 400W resistance). Cell suspensions were placed on ice for 30 minutes, then transferred to 10 ml THY broth and incubated at 37 ° C/5 % CO 2 for 2-3 hours .
- test gene Colonies were counted to determine whether the test gene is essential or nonessential. If the test gene is nonessential, the number of colonies on the test plates (erythromycin) should be comparable to the control plates (kanamycin). If the test gene is essential, the erythromycin plates should have no colonies, while the control kanamycin plates should contain significant numbers of colonies (typically 200-300) indicating integration into the nonessential control gene.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
L'invention concerne un procédé baptisé GATE (Genetics-Assisted Target Evaluation, ou 'estimation de cibles assistée génétiquement'), destiné à l'analyse des gènes dans des bactéries pathogènes. Le procédé GATE permet de classer les gènes de manière fonctionnelle, selon leur caractère essentiel ou non essentiel en rapport avec la croissance bactérienne ou la pathogénie. La technique de l'invention s'appuie sur un gène non essentiel pouvant être soumis à la notation, utilisé en tant que standard interne par rapport auquel on évalue l'aptitude à la distribution d'un gène cible donné par disruption génique au moyen de l'intégration de type Campbell. Une analyse fonctionnelle a été effectuée sur 26 gènes du pathogène gram positif Streptococcus pyogenes. L'application de ce procédé a permis d'identifier clairement les loci essentiels et non essentiels: seize sur les vingt-six gènes évalués chez S. pyogenes codaient clairement les fonctions essentielles pour la croissance bactérienne, les dix autres fonctions codées n'étant pas essentielles. Le succès du procédé GATE a aussi été démontré vis-à-vis de Staphylococcus aureus.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU46720/99A AU4672099A (en) | 1998-05-15 | 1999-05-13 | Genetics-assisted target evaluation in antibacterial drug discovery |
| CA002328497A CA2328497A1 (fr) | 1998-05-15 | 1999-05-13 | Estimation de cibles assistee genetiquement et utilisee dans la conception de medicaments antibacteriens |
| JP2000549719A JP2002515242A (ja) | 1998-05-15 | 1999-05-13 | 抗生物質薬剤の発見における遺伝学的補助の標的評価 |
| EP99930113A EP1078049A1 (fr) | 1998-05-15 | 1999-05-13 | Estimation de cibles assistee genetiquement et utilisee dans la conception de medicaments antibacteriens |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US8559398P | 1998-05-15 | 1998-05-15 | |
| US60/085,593 | 1998-05-15 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO1999060111A1 WO1999060111A1 (fr) | 1999-11-25 |
| WO1999060111A9 true WO1999060111A9 (fr) | 2000-02-24 |
Family
ID=22192673
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1999/010919 WO1999060111A1 (fr) | 1998-05-15 | 1999-05-13 | Estimation de cibles assistee genetiquement et utilisee dans la conception de medicaments antibacteriens |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP1078049A1 (fr) |
| JP (1) | JP2002515242A (fr) |
| AU (1) | AU4672099A (fr) |
| CA (1) | CA2328497A1 (fr) |
| WO (1) | WO1999060111A1 (fr) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0948646B1 (fr) * | 1996-11-06 | 2007-02-07 | SmithKline Beecham Corporation | Procedes d'identification de genes indispensables a la croissance d'un organisme |
-
1999
- 1999-05-13 AU AU46720/99A patent/AU4672099A/en not_active Abandoned
- 1999-05-13 EP EP99930113A patent/EP1078049A1/fr not_active Withdrawn
- 1999-05-13 JP JP2000549719A patent/JP2002515242A/ja not_active Withdrawn
- 1999-05-13 WO PCT/US1999/010919 patent/WO1999060111A1/fr not_active Application Discontinuation
- 1999-05-13 CA CA002328497A patent/CA2328497A1/fr not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| AU4672099A (en) | 1999-12-06 |
| EP1078049A1 (fr) | 2001-02-28 |
| JP2002515242A (ja) | 2002-05-28 |
| WO1999060111A1 (fr) | 1999-11-25 |
| CA2328497A1 (fr) | 1999-11-25 |
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