Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-08-06
2002-03-19
Kemmerer, Elizabeth (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C435S252300, C435S173300, C536S023100, C536S024310
Reexamination Certificate
active
06358701
ABSTRACT:
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not applicable.
REFERENCE TO MICROFICHE APPENDIX
Not applicable.
FIELD OF THE INVENTION
The present invention relates in part to isolated nucleic acid molecules (polynucleotides) which encode
Ctenocephalides felis
(flea) glutamate gated chloride channels. The present invention also relates to recombinant vectors and recombinant hosts which contain a DNA fragment encoding
C. felis
glutamate gated chloride channels, substantially purified forms of associated
C. felis
glutamate gated chloride channels, associated mutant proteins, and methods associated with identifying compounds which modulate associated
Ctenocephalides felis
glutamate gated chloride channels, which will be useful as insecticides.
BACKGROUND OF THE INVENTION
Glutamate-gated chloride channels, or H-receptors, have been identified in arthropod nerve and muscle (Lingle et al, 1981,
Brain Res.
212: 481-488; Horseman et al., 1988,
Neurosci. Lett.
85: 65-70; Wafford and Sattelle, 1989,
J. Exp. Bio.
144:449-462; Lea and Usherwood, 1973,
Comp. Gen. Parmacol.
4: 333-350; and Cull-Candy, 1976,
J. Physiol.
255:449-464).
Additionally, glutamate-gated chloride channels have been cloned from the soil nematode
Caenorhabditis elegans
(Cully et al., 1994,
Nature
371: 707-711; see also U.S. Pat. No. 5,527,703) and
Drosophila melanogaster
(Cully et al., 1996,
J. Biol. Chem.
271: 20187-20191).
Invertebrate glutamate-gated chloride channels are important targets for the widely used avermectin class of anthelmintic and insecticidal compounds. The avermectins are a family of macrocyclic lactones originally isolated from the actinomycete
Streptomyces avermitilis.
The semisynthetic avermectin derivative, ivermectin (22,23-dihydro-avermectin B
1a
), is used throughout the world to treat parasitic helminths and insect pests of man and animals. The avermectins remain the most potent broad spectrum endectocides exhibiting low toxicity to the host. After many years of use in the field, there remains little resistance to avermectin in the insect population. The combination of good therapeutic index and low resistance strongly suggests that the glutamate-gated chloride (GluCl) channels remain good targets for insecticide development.
It would be advantageous to identify additional invertebrate genes encoding encoding GluCl channels in order to allow screening to identify novel GluCl channel modulators that may have insecticidal, mitacidal and/or nematocidal activity for animal health or crop protection. The present invention addresses and meets these needs by disclosing isolated nucleic acid molecules which express a
Ctenocephalides felis
GluGl channel wherein expression of flea GluCl cRNA in
Xenopus oocytes
results in an active GluCl channel.
SUMMARY OF THE INVENTION
The present invention relates to isolated nucleic acid molecules (polynucleotides) which encode novel invertebrate GluCl channel proteins, especially nucleic acid molecules which encode a functional
C. felis
GluCl (herein, “CfGluCl”) channel.
The present invention also relates to isolated nucleic acid fragments of CfGluCl which encode mRNA expressing a biologically active CfGluCl channel. Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode cRNA which express a functional
C. felis
GluCl channel in a eukaryotic cell, such as Xenopus oocytes, so as to be useful for screening for agonists and/or antagonists of
C. felis
GluCl activity.
The isolated nucleic acid molecule of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA), including but not limited to messenger RNA (mRNA) encoding a biologically active
C. felis
GluCl channel and complementary RNA (cRNA) transcribed from a recombinant expression vector comprising a DNA molecule which encodes a full-length or biologically active portion of the full-length
C. felis
GluCl channel.
A preferred aspect of the present invention is disclosed in
FIGS. 1A-B
and SEQ ID NO:1, an isolated cDNA molecule encoding a
C. felis
GluCl channel, CfGluCl-1.
The present invention relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification, especially a nucleic acid molecule encoding a
C. felis
GluCl channel, CfGluCl, such as the cDNA molecule disclosed in
FIGS. 1A-B
and set forth in SEQ ID NO:1.
The present invention also relates to a substantially purified form of a
C. felis
GluCl channel protein and especially the
C. felis
GluCl channel disclosed in FIG.
2
and set forth in SEQ ID NO:2.
The present invention relates to a substantially purified membrane preparation which comprises a
C. felis
GluCl channel and is essentially free from contaminating proteins, including but not limited to other
C. felis
source proteins or host proteins from a recombinant cell which expresses CfGluCl. Especially preferred is a membrane preparation which comprises
C. felis
GluCl channel disclosed in FIG.
2
and set forth in SEQ ID NO:2. To this end, the present invention also relates to a substantially purified membrane preparation which is purified from a recombinant host, whether a recombinant eukaryotic or recombinant prokaryotic host, wherein a recombinant vector expresses a
C. felis
GluCl channel. Especially preferred is a membrane preparation which comprises a recombinant form of the
C. felis
GluCl channel, CfGluCl, disclosed in FIG.
2
and set forth in SEQ ID NO:2, referred to as CfGluCl-1.
The present invention also relates to biologically active fragments and/or mutants of a
C. felis
GluCl channel protein, including but not limited to the CfGluCl protein disclosed in FIG.
2
and set forth in SEQ ID NO:2, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for a biologically active channel which is useful in screening for agonists and/or antagonists of
C. felis
GluCl channel activity.
The present invention also relates to an isolated nucleic acid molecule (polynucleotide) which encodes a truncated form of the flea GluCl channel protein (herein, “tr-CfGluCl”), as exemplified in FIG.
3
and set forth in SEQ ID NO:3. Co-expression of tr-CfGluCl in
Xenopus oocytes
with CfGluCl is shown to inhibit glutamate-gated channel activity.
The present invention also relates to isolated nucleic acid fragments of tr-CfGluCl-1 (SEQ ID NO:3) which encodes cRNA expressing a biologically active form of tr-CfGluCl, including but not limited to inhibition or promotion of CfGluCl channel activity in the target cell type. Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations from the truncated form.
Again, any such truncated nucleic acid molecule (as compared to CfGluCl) may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA), including but not limited to messenger RNA (mRNA) or complementary RNA (cRNA) transcribed from a recombinant expression vector comprising a DNA molecule which encodes a truncated version of the full-length
C. felis
GluCl channel.
A preferred aspect of this portion of the invention is disclosed in
Brochu Richard
Cohen Charles J.
Cully Doris F.
Etter Adrian
Paress Philip S.
Basi Nirmal S.
Heber Sheldon O.
Kemmerer Elizabeth
Merck & Co. , Inc.
Tribble Jack L.
LandOfFree
DNA molecules encoding Ctenocephalides felis glutamate gated... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with DNA molecules encoding Ctenocephalides felis glutamate gated..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and DNA molecules encoding Ctenocephalides felis glutamate gated... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2835213