Patent Application: US-36284299-A

Abstract:
tandem pore domain weak inward rectifying k + channel nucleic acids and proteins that have been isolated from drosophila melanogaster and leptinotarsa are described . the twik channel nucleic acids and proteins can be used to genetically modify metazoan invertebrate organisms , such as insects , coelomates , and pseudocoelomates , or cultured cells , resulting in twik channel expression or mis - expression . the genetically modified organisms or cells can be used in screening assays to identify candidate compounds which are potential pesticidal agents or therapeutics that interact with twik channel proteins . they can also be used in methods for studying twik channel activity and identifying other genes that modulate the function of , or interact with , the twik channel gene .

Description:
table iv further summarizes the blast results for each twik channel amino acid sequence . the “% identity ” column indicates , for each twik channel amino acid sequence , the longest stretch of contiguous amino acids that is novel with respect to prior art sequences as determined by blast . thus , for example , any contiguous stretch of 8 amino acids of the twik2 amino acid sequence ( seq id no : 2 ) is novel over sequences in public databases . the “% similarity ” column shows the longest stretch of contiguous amino acids for which there are no sequences contained within public database sharing 100 % sequence similarity . for example , for any contiguous stretch of 15 amino acids of the twik2 channel protein , there are no sequences in the public databases that share 100 % sequence identity . twik expression analysis was carried out by in situ hybridization to drosophila embryonic and larval tissues for the drosophila twiks , using the cdnas to generate antisense probes on drosophila embryos and larval tissue . antisense dig - labeled rna probes were synthesized using the roche ( nj ) dig rna labeling kit and t7 rna polymerase according to the in situ hybridization protocol of bdgp methods page ( http :// www . fruitfly . org / methods /). the results showed that twik2 is expressed in the progenitor and developing muscle cells of the embryo and larval musculature . twik3 is expressed in the developing esophagus and hindgut of the embryo . twik6 is expressed in a region of the gut , in band of cells around the larval proventriculus . this most likely corresponding to the described imaginal ring that will give rise to adult tissues . twik7 is highly expressed in neuronal tissues : during late differentiation of the embryonic central nervous system , throughout the larval cns during 1 st instar and in the brains of 3 rd instar larvae , particularly the optic lobes . cdnas encoding twik channels of interest are cloned into baculovirus - compatible vectors ( e . g . pie - 1 series , novagen , madison , wis .) or mammalian cell culture - compatible vectors ( e . g . pcdna , invitrogen , carlsbad , calif .) using standard methods . the resultant constructs are transiently transfected into insect or mammalian cell cultures , respectively . the transiently transfected cell lines are typically used 24 - 48 hours following transfection for electrophysiology studies . whole cell recordings , using the voltage clamp technique , are taken on the transfected cells and compared with recordings taken from cells transfected with vector only . the cells are injected with a current of designated intensity for brief periods ( up to 200 milliseconds ). a resultant current , generated by the cell in returning to the resting potential , is recorded . the amplitude and duration of this current indicates the presence of a functional channel in the transfected cells . current injections are repeated with increasing amperage — the resultant channel - generated current should increase to reflect the increased injected current . a channel - blocking compound may then be applied to the cell culture in the concentration range of about 1 × 10 − 9 to 1 × 10 − 6 m to determine the effect on the channel activity . if the compound inhibits channel activity , a resultant current is not generated in response to the injected current . the compound may be washed out of the cell culture and another compound tested in series . in a second type of experiment , the resting potential of transfected cells is compared to that of control cells . for potassium leak channels e . g . the twiks , a depolarized resting potential is observed in the transfected cells . a series of compounds can then be tested for interactions with the expressed channel by applying each compound and measuring any resultant changes in cellular resting potential . the present invention is not to be limited in scope by the specific embodiments described herein . indeed , various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings . such modifications are intended to fall within the scope of the appended claims . the disclosure of each reference cited herein , including patents , patent applications , and other references , is hereby incorporated herein by reference in its entirety for all purposes . his glu ala pro lys thr arg gly arg asn arg ser ser gly tyr asp thr his pro arg pro arg ala pro lys ile pro gln gly phe val ala ile leu arg lys his glu tyr leu glu val gly glu ser arg asp glu val ile lys pro arg ile ser glu ala glu gln gln ser glu leu met leu ala glu gln ile leu thr ala glu gly val arg arg ser his ser ser ile ser glu ala lys pro lys val thr leu trp gln arg leu lys asn leu phe arg arg lys lys lys ile gln ala lys gly glu asp val gly leu leu phe arg tyr thr glu gly ala ala glu asn ile tyr lys val ser his asn met arg glu glu asp trp lys ser leu ala 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