Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-07-07
2003-03-04
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S462000, C435S463000, C435S473000, C435S091210, C435S091200
Reexamination Certificate
active
06528257
ABSTRACT:
FIELD OF INVENTION
The present invention relates to an improved and efficient method for the simultaneous monitoring of individual mutants of a microbe in mixed populations. Such mutants are distinguished from each other by utilizing the features of the mutated genes themselves. More particularly, by the method of this invention even mutants having subtle quantitative phenotypes, or those without plate screens can be readily monitored quantitatively. This would facilitate in understanding the role of a large number of novel genes identified by the systematic sequencing of microbial genomes, many of which may have only subtle quantitative phenotypes. It would also readily allow the parallel screening of large number of mutated genes for their role under conditions which by their very nature can not have a plate screen; examples include virulence genes of pathogenic microbes, and genes conferring stress tolerance to yeast cells during fermentation in liquid broths. This method has pronounced application in discovering the functions of genes of large number of microbes of medical, industrial and agricultural importance.
BACKGROUND OF THE INVENTION
Isolation and study of mutants impaired in normal cellular phenomena is a standard way to dissect them out at the genetic and biochemical levels, and finally to understand them at the molecular level. The conventional approach to screen mutants is on solid media set in petri-plates. If a mutant has increased growth or survival (positive phenotype) compared to the normal wild-type cells, then it can be easily selected on a plate, even from amongst a lawn of wild-type cells. It can also be enriched from a mixed population in a liquid broth by repeated selection. However, if a mutant cell shows reduced growth or survival (negative phenotype) under the selection condition, then it can not be identified from among a mixed population of cells in liquid broths; yet, a large number of such potential mutant cells can be allowed to form isolated colonies on solid media under non-selective conditions, replica-plated on to selective media and then growth assessed. On the other hand, if there is no plate screen for the phenotype, i.e. if the phenotype does not show up on solid media, then screening by replica plating is not possible. One such example is mutants impaired in stress tolerance under fermentation conditions in liquid broths. Another example is from pathogenic microbes, where to isolate mutants impaired in virulence, one has to individually test potential mutants for pathogenicity in the host organism, which is extremely laborious. Thus, conventional mutant screening methods are very inadequate to identify genes whose mutant phenotype does not show up on solid media.
Four methods have been reported which partially redress this problem, though they remain quite laborious (Hensel et al., 1995,
Science
269: 400-403; Shoemaker et al., 1996,
Nature genetics
14: 450-456; Smith et al., 1995,
Proc. Natl. Acad. Sci. USA
92: 6479-6483; Cormack et al.,
Science
285: 578-582, 1999). In the first method, known as ‘signature tagged transposon mutagenesis’ (Hensel et al., 1995,
Science
269: 400-403), random mutants are generated by insertional inactivation of genes by transposons. Prior to this step, the transposons are uniquely marked with random sequence tags. Thus, the mutated genes are tagged with transposons, which in turn are tagged with unique, but random sequence tags; the mutants could be individually monitored in mixed populations by means of the sequence tags. This method was developed and used for identifying bacterial virulence genes. While this is a major advance for screening mutants having negative phenotypes in mixed populations, it suffers from three disadvantages, namely, need for prior introduction of sequence tags, poor sensitivity (only about 100 mutants can be pooled together and screened), and inability to monitor the phenotypes quantitatively.
In the second method known as ‘molecular bar coding’ (Shoemaker et al., 1996,
Nature genetics
14: 450-456), each mutated gene is marked with a unique and known sequence tag. This is carried out by replacing the coding sequence of a gene with a selectable marker and a sequence tag, by transformation with PCR (polymerase chain reaction) products having small regions of homology to genes being deleted. Once a large collection of strains are created with each mutated for a single gene and also carrying a unique sequence tag, all of them can be individually monitored in mixed populations by means of their sequence tags. This method is very powerful and can facilitate the quantitative monitoring of the fate of thousands of mutants simultaneously under any selection condition. However, the initial construction of the set of mutants is extremely laborious and time consuming. It is also very expensive, since for each gene to be deleted, a set of long oligos have to be custom synthesized. A prerequisite of this method is that the nucleotide sequence of the genes being deleted should be known. Thus, if the aim is to delete all the genes of a microbe, then the entire sequence of its genome should be determined in the first place. Another important requirement is that the microbe should have a good homologous recombination system for efficiently replacing the native genes with the deleted versions having minimal length of sequence homology. The last requirement may turn out to be insurmountable for a large number of microbes. At present only yeast
Saccharomyces cerevisiae
has been taken up to be studied by this method; the construction of deletion strains is currently being carried out by a large collaboration involving eight American and European laboratories. However, this method is unlikely to be used for studying many important microbes particularly due to the lack of an efficient homologous recombination system, and also due to the cost, time and labor involved.
In the third method a variation of molecular bar coding is used. Here, to begin with, 96 different isogenic parent strains are constructed by introducing unique sequence tags for each (Cormack et al.,
Science
285: 578-582, 1999). These are then mutated by random insertion of a transforming DNA in the genome. Then pools of 96 mutants each are made, where each mutant in the pool has a unique sequence tag. These are then distinguished from each other in mixed populations by hybridization. The limitation of this method is the need for initial introduction of unique sequence tags, and the need for doing large number of hybridizations. In our method there is no need for introduction of sequence tags, making it more economical, less laborious and faster than existing methods.
In the fourth method known as ‘genetic footprinting’ (Smith et al., 1995,
Proc. Natl. Acad. Sci. USA
92: 6479-6483), random population of mutants are obtained by transposon mutagenesis. However, the mutants are not uniquely marked with any specific sequence tag. Instead, the fate of each mutant during selection is individually analyzed by PCR, with a gene-specific primer and a transposon specific primer. If a particular PCR product corresponding to a mutant gene is present in the starting population of cells, but absent or reduced in the population subjected to selection, then that would indicate that mutants in that gene do not survive the selection. Though with this method one can identify the genes conferring subtle quantitative phenotypes (Smith et al., 1996,
Science
274: 2069-2074), it suffers from the need for gene-specific primers and from the need to do individual PCR reactions for each gene of interest. These two requirements make this method extremely laborious and expensive—to comprehensively identify all the genes providing some benefit to a microbe under a selection condition, it is necessary to do several thousand PCR reactions. Since so many reactions have to be done for each selection condition, this method is extremely labor intensive, time consuming and costly. Besides, prior sequence information is necessary for designing gene-specific
Ganesan Kaliannan
Sharma Vishva Mitra
Council of Scientific & Industrial Research
Katcheves Konstantina
Ketter James
Kilpatrick & Stockton LLP
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