Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving fixed or stabilized – nonliving microorganism,...
Reexamination Certificate
2000-12-01
2003-02-25
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving fixed or stabilized, nonliving microorganism,...
C435S008000, C435S252300, C435S252330
Reexamination Certificate
active
06524810
ABSTRACT:
BACKGROUND OF THE INVENTION
The use of bacteria with a signal-generating metabolic activity as indicators of toxicity is well established. UK patent number GB 2005018 describes a method of assaying a liquid sample for toxic substances which involves contacting a suspension of bioluminescent microorganisms with a sample suspected of containing a toxic substance and observing the change in the light output of the bioluminescent organisms as a result of contact with the suspected toxic substance. Furthermore, a toxicity monitoring system embodying the same assay principle, which is manufactured and sold under the Trade Mark Microtox®, is in routine use in both environmental laboratories and for a variety of industrial applications. An improved toxicity assay method using bioluminescent bacteria, which can be used in a wider range of test conditions than the method of GB 2005018, is described in International patent application number WO 95/10767.
The assay methods known in the prior art may utilize naturally occurring bioluminescent organisms, including
Photobacterium phosphoreum
and
Vibrio fischeri.
However, recent interest has focused on the use of genetically modified microorganisms which have been engineered to express bioluminescence. These genetically modified bioluminescent microorganisms usually express lux genes, encoding the enzyme luciferase, which have been cloned from a naturally occurring bioluminescent microorganism (E. A. Meighen (1994) Genetics of Bacterial Bioluminescence.
Ann. Rev. Genet.
28: 117-139; Stewart, G. S. A. B. Jassin, S. A. A. and Denyer, S. P. (1993), Engineering Microbial bioluminescence and biosensor applications. In Molecular Diagnosis. Eds R. Rapley and M. R. Walker Blackwell Scientific Pubs/Oxford). A process for producing genetically modified bioluminescent microorganisms expressing lux genes cloned from
Vibrio harveyi
is described in U.S. Pat. No. 4,581,335.
The use of genetically modified bioluminescent microorganisms in toxicity testing applications has several advantages over the use of naturally occurring microorganisms. For example, it is possible to engineer microorganisms with different sensitivities to a range of different toxic substances or to a single toxic substance. However, genetically modified microorganisms are subject to marketing restrictions as a result of government legislation and there is major concern relating to the deliberate release of genetically modified microorganisms into the environment as components of commercial products. This is particularly relevant with regard to toxicity testing which is often performed in the field rather than within the laboratory. The potential risk from release of potentially pathogenic genetically modified microorganisms into the environment where they may continue to grow in an uncontrollable manner has led to the introduction of legal restrictions on the use of genetically modified organisms in the field in many countries.
It has been suggested, to avoid the problems discussed above, to use genetically modified bioluminescent microorganisms which have been treated so that they retain the metabolic function of bioluminescence but can no longer reproduce. The use of radiation (gamma-radiation), X-rays or an electron beam to kill bioluminescent cells whilst retaining the metabolic function of bioluminescence is demonstrated in International Patent Application Number WO 95/07346.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide an alternative method of killing bioluminescent cells whilst retaining the metabolic function of bioluminescence which does not require the use of radiation and, as such, can be easily carried out without the need for specialized radiation equipment and containment facilities and without the risk to laboratory personnel associated with the use of radiation.
Accordingly, in a first aspect the invention provides a method of making a non-viable preparation of prokaryotic or eukaryotic cells, which preparation has a signal-generating metabolic activity, which method comprises contacting a viable culture of cells with signal-generating metabolic activity with a member of the bleomycin/phleomycin family of antibiotics.
Bleomycin and phleomycin are closely related glycopeptide antibiotics that are isolated in the form of copper chelates from cultures of
Streptomyces verticillus.
They represent a group of proteins with molecular weights ranging from 1000 to 10000 kda that are potent antibiotics and anti-tumour agents. So far more than 200 members of the bleomycin/phleomycin family have been isolated and characterised as complex basic glycopeptides. Family members resemble each other with respect to their physicochemical properties and their structure, indicating that functionally they all behave in the same manner. Furthermore, the chemical structure of the active moiety is conserved between family members and consists of 5 amino acids, L-glucose, 3-O-carbamoyl-D-mannose and a terminal cation. The various different bleomycin/phleomycin family members differ from each other in the nature of the terminal cation moiety, which is usually an amine. A preferred bleomycin/phleomycin antibiotic for use in the method of the invention is phleomycin D1, sold under the trade name Zeocin™.
Bleomycin and phleomycin are strong, selective inhibitors of DNA synthesis in intact bacteria and in mammalian cells. Bleomycin can be observed to attack purified DNA in vitro when incubated under appropriate conditions and analysis of the bleomycin damaged DNA shows that both single-stranded and double-stranded cleavages occur, the latter being the result of staggered single strand breaks formed approximately two base pairs apart in the complementary strands.
In in vivo systems, after being taken up by the cell, bleomycin enters the cell nucleus, binds to DNA (by virtue of the interaction between its positively charged terminal amine moiety and a negatively charged phosphate group of the DNA backbone) and causes strand scission. Bleomycin causes strand scission of DNA in viruses, bacteria and eukaryotic cell systems.
The present inventors have surprisingly found that treatment of a culture of cells with signal-generating metabolic activity with a bleomycin/phleomycin antibiotic renders the culture non-viable whilst retaining a level of signal-generating metabolic activity suitable for use in toxicity testing applications. In the context of this application the term non-viable is taken to mean that the cells are unable to reproduce. The process of rendering cells non-viable whilst retaining signal-generating metabolic activity may hereinafter be referred to as ‘inactivation’ and cells which have been rendered non-viable according to the method of the invention may be referred to as ‘inactivated’.
Because of the broad spectrum of action of the bleomycin/phleomycin family of antibiotics the method of the invention is equally applicable to bacterial cells and to eukaryotic cells with signal generating metabolic activity. Preferably the signal-generating metabolic activity is bioluminescence but other signal-generating metabolic activities which are reporters of toxic damage could be used with equivalent effect.
The method of the invention is preferred for use with bacteria or eukaryotic cells that have been genetically modified to express a signal-generating metabolic activity. The examples given below relate to
E. coil
which have been engineered to express bioluminescence by transformation with a plasmid carrying lux genes. The eukaryotic equivalent would be cells transfected with a vector containing nucleic acid encoding a eukaryotic luciferase enzyme (abbreviated luc) such as, for example, luciferase from the firefly
Photinus pyralis.
A suitable plasmid vector containing cDNA encoding firefly luciferase under the control of an SV40 viral promoter is available from Promega Corporation, Madison Wis., USA. However, in connection with the present invention it is advantageous to use recombinant cells containing the entire eukaryotic luc operon so as to avoid t
Isles Katherine
Lewington Jay
Primrose Sandy
Azur Environmental
Kilpatrick & Stockton LLP
Slobodyansky Elizabeth
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