Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-09-30
2001-06-26
Arthur, Lisa B. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S252100, C435S320100, C536S023100, C536S024300
Reexamination Certificate
active
06251596
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to N-myristoyl transferase of the fungus
Aspergillus fumigatus
and its use in identifying antifungal agents.
BACKGROUND OF THE INVENTION
The enzyme N-myristoyl transferase (NMT) is responsible for cotranslational modification of a variety of fungal proteins. NMT catalyzes the attachment of a 14-carbon saturated fatty acid to the N-terminal glycine residue of cellular proteins. This modification is thought to be irreversible and essential for the full biological activity of myristoylated proteins.
SUMMARY OF THE INVENTION
The invention is based on the discovery of an NMT gene in the fungus
Aspergillus fumigatus
. The Aspergillus NMT coding sequence is depicted in
FIG. 1
as SEQ ID NO:1, with the amino acid sequence represented by SEQ ID NO:2. The NMT genomic sequence is depicted in
FIG. 2
as SEQ ID NO:3.
The NMT gene of the invention is essential for survival of Aspergillus. Accordingly, the NMT nucleic acid sequence of the invention, and the NMT polypeptide of the invention, are useful targets for identifying compounds that are inhibitors of Aspergillus. Such inhibitors attenuate fungal growth by inhibiting the activity of the essential NMT polypeptide, or by inhibiting transcription or translation. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding Aspergillus NMT polypeptides or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of NMT-encoding nucleic acids (e.g., fragments of at least 15 nucleotides (e.g., at least 18, 20, or 25 nucleotides)).
The invention features a nucleic acid molecule which is at least 65% (or 75%, 85%, 95%, or 98%) identical to the nucleotide sequence shown in SEQ ID NO:1, or SEQ ID NO:3, or the nucleotide sequence of the cDNA insert of the plasmid deposited with ATCC as Accession Number (the “cDNA of ATCC PTA-1663”), or a complement thereof.
The invention features a nucleic acid molecule which includes a fragment of at least 300 (e.g., 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1200, 1400, 1600, or 1770) nucleotides of the nucleotide sequence shown in SEQ ID NO:1, or SEQ ID NO:3, or the nucleotide sequence of the cDNA ATCC PTA-1663, or a complement thereof.
The invention also features a nucleic acid molecule which includes a nucleotide sequence encoding a protein having an amino acid sequence that is at least 65% (or 75%, 85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID NO:2 or the amino acid sequence encoded by the cDNA of ATCC PTA-1663.
Also within the invention is a nucleic acid molecule that encodes a fragment of a polypeptide having the amino acid sequence of SEQ ID NO:2, the fragment including at least 15 (25, 30, 50, 100, 150, 300, 400, or 450) contiguous amino acids of SEQ ID NO:2 or the polypeptide encoded by the cDNA of ATCC Accession Number PTA-1663.
In other embodiments, the invention features an isolated NMT protein having an amino acid sequence that is at least about 65% (e.g., 75%, 85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID NO:2; and an isolated NMT protein which is encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 65% (e.g., 75%, 85%, or 95%) identical to SEQ ID NO:1 or the cDNA of ATCC PTA-1663; and an isolated NMT protein which is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 or the non-coding strand of the cDNA of ATCC PTA-1663.
Another embodiment of the invention features NMT nucleic acid molecules which specifically detect Aspergillus NMT nucleic acid molecules relative to nucleic acid molecules encoding other N-myristoyltransferases. For example, in one embodiment, an Aspergillus NMT nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or the cDNA of ATCC PTA-1663, or a complement thereof. In another embodiment, the Aspergillus NMT nucleic acid molecule is at least 300 (e.g., 400, 500, 700, 900, 1100, or 1300) nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, the cDNA of ATCC PTA-1663, or a complement thereof. In another embodiment, the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of an Aspergillus NMT nucleic acid.
Another aspect of the invention provides a vector, e.g., a recombinant expression vector, comprising an NMT nucleic acid molecule of the invention. In another embodiment the invention provides a host cell containing such a vector. The invention also provides a method for producing NMT protein by culturing, in a suitable medium, a host cell of the invention containing a recombinant expression vector such that a NMT protein is produced.
Another aspect of this invention features isolated or recombinant NMT proteins and polypeptides. Preferred NMT proteins and polypeptides possess at least one biological activity possessed by naturally occurring Aspergillus NMT, e.g., an ability to catalyze transfer of myristate from myristoyl-CoA to the N-terminal glycine residue of a polypeptide. It is not necessary that the NMT polypeptide have an N-myristoyltransferase activity that is equivalent to that of the wild-type Aspergillus NMT. For example, the NMT polypeptide can have 20, 50, 75, 90, 100, or an even higher percent of the wild-type activity.
Now that the Aspergillus NMT gene, which is essential for survival, has been identified, nucleic acids encoding Aspergillus NMT and Aspergillus NMT proteins can be used to identify antifungal agents. Such antifungal agents can readily be identified with high throughput assays to detect inhibition of NMT activity. This inhibition can be caused by small molecules binding directly to the NMT polypeptide or by binding of small molecules to other essential polypeptides in that pathway.
In an exemplary, but not the only assay, a compound is tested for its ability to inhibit Aspergillus NMT in an assay of NMT activity. NMT activity can be assayed by measuring incorporation of labeled myristate (e.g. [
3
H]myristate) in culture. The effect of a test compound can be determined by adding the test compound to the culture containing the labeled myristate, then comparing the level of labeled myristate in the culture with the level obtained in control cultures. Now that the
Aspergillus fumigatus
NMT gene has been identified, it can readily be cloned into various host cells (e.g., fungi,
E. coli
or yeast) for carrying out such assays in whole cells). Similarly, conventional in vitro assays of NMT activity can be used with the NMT of the invention.
A suitable NMT activity assay has been described by Stone et al., Genes and Dev. 5:1969-1981 (1991), which is incorporated herein by reference. Briefly, [
3
H]myristate is added to cell cultures to specifically label myristoylated proteins, which can be separated by SDS-PAGE and visualized by autoradiography. The level of myristoylation can subsequently be quantitated by using conventional methods to measure incorporation of [
3
H]myristate.
In an alternative assay, a promoter that responds to depletion of the NMT polypeptide by upregulation or downregulation is linked to a reporter gene. To identify a promoter that is up- or down-regulated by the depletion of the NMT polypeptide, the gene encoding Aspergillus NMT is deleted from the genome and replaced with a version of the gene in which the sequence encoding the NMT protein is operably linked to a regulatable promoter. The cells containing this regulatable genetic construct are kept alive by the NMT produced from the genetic construct containing the regulatable promoter. However, the regulatable promoter allows the expression of NMT to be reduced to a level that causes growth inhibition. Total RNA p
Bulawa Christine E.
Cook W. James
Arthur Lisa B.
Fish & Richardson PC
Millennium Pharmaceuticals Inc.
Souaya Jehanne
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