Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-11-14
2001-05-08
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S091100, C435S091200, C536S023100, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06228579
ABSTRACT:
BACKGROUND OF THE INVENTION
Genes that encode proteins essential for microbial growth or viability are useful targets for antibiotics because inhibition of such proteins by an antibiotic can reduce or eliminate the spread of infectious disease in a patient. The emergence of bacteria resistant to multiple antibiotics has led to renewed interest in isolating variants of known antibiotics and also in identifying new required genes and the corresponding gene products that could serve as new targets for novel antibiotics.
Approximately fifteen different bacterial proteins encoded by essential genes have been used as targets for antibiotics. Such target proteins include ribosomal proteins, gyrase, RNA polymerase and proteins involved in the synthesis of the peptidoglycan layer and its precursors. However, there are estimated to be more than 4000 putative genes in the genome of the bacterium
Escherichia coli
and it is not known how many of these genes are required for growth and/or viability. The discovery of additional required genes could facilitate the search for new antibiotics.
Microbial genes required or essential for growth and/or viability have been identified by two major techniques. One approach is to determine the nucleotide sequence of the genome of a microbial species of interest. Sequence information is then compared to sequences in computer databases to identify possible functions for the putative gene sequences. To prove that a putative gene identified only by sequence comparisons is in fact an essential gene, however, a null or “knockout” mutation must be made in the gene. It must be shown that a microorganism containing the null mutation cannot survive unless it is complemented by a wild type allele of the gene. Such an approach is time consuming and, in some species, can be difficult.
A second approach is to isolate conditionally lethal mutants by means such as chemical mutagenesis. A common type of conditional lethal mutation is a temperature-sensitive mutation, in which the mutant is non-viable at higher temperature such as 42° C. but is viable at a lower temperature such as 30° C. The mutation is then mapped and identified by cloning and genetic complementation. The mutagenesis techniques employed are sometimes not completely random, thus reducing the likelihood of identifying a required gene that does not have a “hot spot” for the mutagen used. Moreover, products of some required genes may not be amenable to the formation of conditional lethal mutants. Such essential genes would not be identified by this approach.
SUMMARY OF THE INVENTION
The present invention is based on the discovery that microbial genes and their encoded proteins essential for growth and/or viability can be identified by introduction into a microorganism of an exogenous nucleic acid having sequence similarity to the microbial gene. The exogenous nucleic acid can be, for example, an antisense RNA that is derived from a chromosomal fragment cloned downstream of an inducible promoter. The endogenous gene can be identified as required by the inhibition of proliferation of microorganisms containing or expressing the exogenous nucleic acid, compared to the proliferation of microorganisms that do not contain or are not expressing the nucleic acid. The invention facilitates the rapid, efficient identification of microbial genes not previously known to be required for growth or viability.
It will be evident to those skilled in the art that the invention can be applied to any organism for which it is feasible to 1) isolate and fragment the chromosome of the organism, 2) re-introduce exogenous nucleic acid fragments of the organism or fragments substantially similar to those of the organism back into the organism and 3) culture the organism without the exogenously added nucleic acids.
The present invention advantageously permits the rapid, inexpensive identification of microbial genes that heretofore have not been identified as required for survival. The method comprises the steps of introducing an exogenous nucleic acid into a microorganism and determining the proliferation of the microorganism relative to the proliferation of the microorganism when the exogenous nucleic acid is not present or not expressed. The exogenous nucleic acid has substantial sequence identity to an endogenous microbial gene. The exogenous nucleic acid can be operably linked to a regulatory element effective for controlling expression of the nucleic acid, either in sense orientation or in antisense orientation to the regulatory element. The regulatory element can control expression via chemical induction of the exogenous nucleic acid. The microorganism can be, for example, a bacterium, either gram-negative or gram-positive, a fungus, or an Archaebacteria.
The novel genes and the proteins encoded by such genes are promising targets for new antimicrobial agents. A method for identifying such agents comprises the steps of contacting an agent with a microorganism having a proliferation gene and determining the effect of the agent on the proliferation gene or a product of the proliferation gene. The proliferation gene can be endogenous to the microorganism, or can be from another source microorganism. The effect of the agent on the proliferation gene can be, for example, on transcription or on translation of the proliferation gene.
Specific genes and their mRNAs identified by the novel method can be targets for antisense polynucleotide inhibitors, and these antisense polynucleotides can have therapeutic value. A method for inhibiting expression of a microbial proliferation gene comprises contacting a microorganism having a proliferation gene with an antisense polynucleotide having substantial sequence identity to the proliferation gene.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
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Xie, Y., et al., “
Forsyth R. Allyn
Zyskind Judith W.
Fish & Richardson PC
Fredman Jeffrey
San Diego State University Foundation
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