Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2001-11-20
2004-06-08
Smith, Lynette (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S006120, C435S007100, C435S007200, C435S007310, C435S193000, C435S254100, C435S254210, C435S325000, C435S362000, C435S069100, C435S320100, C536S023100, C536S023740, C514S019300, C424S078310, C424S078310, C424S189100
Reexamination Certificate
active
06746837
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates to recombinant DNA technology. In particular, the invention concerns the cloning of nucleic acid encoding a multiple drug resistance protein of
Aspergillus nidulans.
BACKGROUND OF THE INVENTION
Multiple drug resistance (MDR) mediated by the human mdr-1 gene product was initially recognized during the course of developing regimens for cancer chemotherapy (Fojo et al., 1987,
Journal of Clinical Oncology
5:1922-1927). A multiple drug resistant cancer cell line exhibits resistance to high levels of a large variety of cytotoxic compounds. Frequently these cytotoxic compounds will have no common structural features nor will they interact with a common target within the cell. Resistance to these cytotoxic agents is mediated by an outward directed, ATP-dependent pump encoded by the mdr-1 gene. By this mechanism, toxic levels of a particular cytotoxic compound are not allowed to accumulate within the cell.
MDR-like genes have been identified in a number of divergent organisms including numerous bacterial species, the fruit fly
Drosophila melanogaster, Plasmodium falciparum
, the yeast
Saccharomyces cerevisiae, Caenorhabditis elegans, Leishmania donovanii
, marine sponges, the plant
Arabidopsis thaliana
, as well as
Homo sapiens
. Extensive searches have revealed several classes of compounds that are able to reverse the MDR phenotype of multiple drug resistant human cancer cell lines rendering them susceptible to the effects of cytotoxic compounds. These compounds, referred to herein as “MDR inhibitors”, include for example, calcium channel blockers, antiarrhythmics, antihypertensives, antibiotics, antihistamines, immuno-suppressants, steroid hormones, modified steroids, lipophilic cations, diterpenes, detergents, antidepressants, and antipsychotics (Gottesman and Pastan, 1993,
Annual Review of Biochemistry
62:385-427). Clinical application of human MDR inhibitors to cancer chemotherapy has become an area of intensive focus for research.
On another front, the discovery and development of antifungal compounds for specific fungal species has also met with some degree of success. Candida species represent the majority of fungal infections, and screens for new antifungal compounds have been designed to discover anti-Candida compounds. During development of antifungal agents, activity has generally been optimized based on activity against
Candida albicans
. As a consequence, these anti-Candida compounds frequently do not possess clinically significant activity against other fungal species such as
Aspergillus nidulans
. However, it is interesting to note that at higher concentrations some anti-Candida compounds are able to kill other fungal species such as
A. nidulans
and
A. fumigatus
. This type of observation suggests that the antifungal target(s) of these anti-Candida compounds is present in
A. nidulans
as well. Such results indicate that
A. nidulans
may possess a natural mechanism of resistance that permits them to survive in clinically relevant concentrations of antifungal compounds. Until the present invention, such a general mechanism of resistance to antifungal compounds in
A. nidulans
has remained undescribed.
SUMMARY OF THE INVENTION
The invention provides, inter alia, isolated nucleic acid molecules that comprise nucleic acid encoding a multiple drug resistance protein from Aspergillus nidulans, herein referred to as atrD, vectors encoding atrD, and host cells transformed with these vectors.
In another embodiment, the invention provides a method for determining the fungal MDR inhibition activity of a compound which comprises:
a) placing a culture of fungal cells, transformed with a vector capable of expressing atrD, in the presence of:
(i) an antifungal agent to which said fungal cell is resistant, but to which said fungal cell is sensitive in its untransformed state;
(ii) a compound suspected of possessing fungal MDR inhibition activity; and
b) determining the fungal MDR inhibition activity of said compound by measuring the ability of the antifungal agent to inhibit the growth of said fungal cell.
In still another embodiment the present invention relates to strains of
A. nidulans
in which the atrD gene is disrupted or otherwise mutated such that the atrD protein is not produced in said strains.
In yet another embodiment, the present invention relates to a method for identifiying new antifungal compounds comprising the use of atrD gene disruption or gene replacement strains of
A. nidulans.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides isolated nucleic acid molecules that comprise a nucleic acid sequence encoding atrD. The cDNA (complementary deoxyribonucleic acid) sequence encoding atrD is provided in the Sequence Listing as SEQ ID NO: 1. The amino acid sequence of the protein encoded by atrD is provided in the Sequence Listing as SEQ ID NO: 2.
Those skilled in the art will recognize that the degenerate nature of the genetic code enables one to construct many different nucleic acid sequences that encode the amino acid sequence of SEQ ID NO: 2. The cDNA sequence depicted by SEQ ID NO: 1 is only one of many possible atrD-encoding sequences. Consequently, the constructions described below and in the accompanying examples for the preferred nucleic acid molecules, vectors, and transformants of the invention are illustrative and are not intended to limit the scope of the invention.
All nucleotide and amino acid abbreviations used in this disclosure are those accepted by the United States Patent and Trademark Office as set forth in 37 C.F.R. §1.822(b)(1994).
The term “vector” refers to any autonomously replicating or integrating agent, including but not limited to plasmids, cosmids, and viruses (including phage), comprising a nucleic acid molecule to which one or more additional nucleic acid molecules can be added. Included in the definition of “vector” is the term “expression vector”. Vectors are used either to amplify and/or to express deoxyribonucleic acid (DNA), either genomic or cDNA, or RNA (ribonucleic acid) which encodes atrD, or to amplify DNA or RNA that hybridizes with DNA or RNA encoding atrD.
The term “expression vector” refers to vectors which comprise a transcriptional promoter (hereinafter “promoter”) and other regulatory sequences positioned to drive expression of a DNA segment that encodes atrD. Expression vectors of the present invention are replicable DNA constructs in which a DNA sequence encoding atrD is operably linked to suitable control sequences capable of effecting the expression of atrD in a suitable host. Such control sequences include a promoter, an optional operator sequence to control transcription, a sequence encoding suitable MRNA ribosomal binding sites, and sequences which control termination of transcription and translation. DNA regions are operably linked when they are functionally related to each other. For example, a promoter is operably linked to a DNA coding sequence if it controls the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.
The term “MDR inhibition activity” refers to the ability of a compound to inhibit the MDR activity of a host cell, thereby increasing the antifungal activity of an antifungal compound against said host cell.
In the present invention, atrD may be synthesized by host cells transformed with vectors that provide for the expression of DNA encoding atrD. The DNA encoding atrD may be the natural sequence or a synthetic sequence or a combination of both (“semi-synthetic sequence”). The in vitro or in vivo transcription and translation of these sequences results in the production of atrD. Synthetic and semi-synthetic sequences encoding atrD may be constructed by techniques well known in the art. See Brown et al. (1979)
Methods in Enzymology
, Academic Press, N.Y., 68:109-151. atrD-encoding DNA, or portions thereof, may be generated using a conventional DNA synthesizing apparatus such as the Applied Biosystems Model 380A,380B,
Baskar Padma
Slusher Stephen A.
Smith Lynette
Stichting voor de Technische Wetenschappen
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