Patent Application: US-24002403-A

Abstract:
the invention relates to the na + dependent glutamate transporter glast - 1 and a splice variant thereof . a novel splice variant has been bound which lacks exon 3 resulting in a loss of about 46 amino acids . the protein is altered in such a way that indicates altered function of the transporter . indeed , the inventors have surprisingly determined that the splice variant has a reversed transport direction as compared to glast - 1 . thus , the invention provides materials and methods relating to the splice variant glast - 1a including the amino acid and nucleic acid sequence ; materials and methods relating to the detection in vivo or in vitro of the glast - 1a ; and materials and methods relating to the modulation of excitatory amino acids signalling .

Description:
rat tibia were dissected from wistar rats and epiphyses removed . diaphyses were placed into 1 . 5 ml centrifuge tubes and marrow flushed from cavity by centrifugation at 1000 rpm for 30 seconds . bone was then snap frozen in liquid nitrogen and dismembrated ( 2000 rpm for 3 minutes at approximately 120 ° c .) rat tibia using 1 ml trizol ® reagent ( gibco , brl ). total rna extracted from following manufacturers instructions . rna was precipitated with 0 . 5 % v / v isopropanol and 0 . 05 % tack . resin ( biogenesis ). rna was also extracted from 100 mg of whole rat brain as above . contaminating gnomic dna was removed from all rnas using dnase ( promega ) following manufacturers instructions . rna concentration was estimated using a spectrophotometer ( pharmacia , biotech ) measuring wavelength at 260 nm and 280 nm , where 1 unit of absorbance at 260 is equivalent to 40 μg / ml of rna , the 260 / 280 absorbance ratio was used to determine purity of rna and accuracy of reading . 2 . 5 μg of oligo dt ( 15 ) primed rna was reverse transcribed using superscript ™ ii ( gibco , brl ) according to manufacturers instructions . pcr primers designed to sequences in exons 1 ( down stream tccaccagtcacagaatcaga ) and 10 ( upstream gagtcagaagaaagggcaaac ) of the published glast - i sequence ( genbank accession number s59158 ) were used to amplify the glast - 1 cdna . pcr was performed using advantage dna polymerase ( clontech ) for 40 cycles at 95 ° c . for 1 minute , 63 ° c . for 1 minute and 72 ° c . for 2½ minutes . amplicons were incubated at 95 ° c . for 20 minutes to inactivate proof reading enzyme and adenosine overhangs added by adding 5u of taq polymerase ( ags gold : hybaid ) and incubating for 20 minutes at 72 ° c . amplicons were then cloned into pcr ®- xl - topo ( invitrogen ) following manufacturers instructions . transformed plasmids were purified ( wizard ®- sv plus miniprep kit promega ) and inserts sequenced using m13 vector primers and forward and reverse sequencing primers designed to published sequence ( accession no . s59158 ). primers were designed to specifically amplify the glast - 1a splice variant . the forward primer ( cagcgctgtca ttgtgggaatggc ) was designed to prime across the exon 2 - 4 boundary and the reverse primer was designed to the 3 ′ end of exon 4 ( aggaaggcatctgcggcagtcacc ). this reaction was performed using taq polymerase ( ags gold : hybaid ) for 40 cycles at 95 ° c . for 1 minute , 58 ° c . for 1 minute and 72 ° c . for 2 minutes . hydrophobicity plots were performed using tm pred at web address : http :// www . embnet . org / software / tmpred_form . html rt - pcr of bone rna using primers to exons 1 and 10 of the published glast - 1 sequence ( storck , 1992 ) yielded an amplicon of the expected 2201 bp for this molecule ( fig1 ). sequence analysis confirmed that this bone - derived pcr product contained the complete open reading frame of the glast - 1 mrna previously thought to be exclusively expressed in the central nervous system of both rats and humans ( tanaka , 1993 ). eco ri restriction digest of cloned exon 1 - 10 pcr products yielded two different sized inserts ( fig2 a ). comparison of dna sequence data revealed a novel variant of glast - 1 mrna that does not possess exon 3 ( fig2 b ). this variant has been called glast - 1a . rt - pcr , using an upstream primer to the exon 2 - 4 boundary and a downstream primer to exon 4 to specifically amplify glast - 1a , demonstrated that it is expressed in brain as well as bone ( fig2 c ). transmembrane ( tm ) prediction of the first four exons of glast - 1 reveals that it has three hydrophobic regions that may correspond to tm domains ( fig3 a ). interestingly tm prediction of the hypothetical protein without exon three reveals that there are only two hydrophobic regions which would correspond to just two transmembrane domains ( fig3 b ). loss of exon three alters the n - terminal region from three potential tm domains ( glast - 1 ) to two ( glast - 1a ) which may result in reorientating the c - terminal ( fig4 ). immunoblot analysis was used to confirm the presence of glast - 1 protein expression in long bones and to identify glast isoforms present in rat cerebellum . lyophilized fractions were dissolved in sample buffer ( 8m urea , 2m thiourea , 5 % ( w / v ) sds , 25mm tris - hci ( ph 7 . 5 ), 1 % ( w / v ) bromophenol blue and 5 % ( v / v ) β - mercaptoethanol ) to a final concentration of 10 mg / ml and incubated at 60 ° c . for 15 minutes . 50 μg of - each extract were resolved on 7 . 5 % or 10 % sds - polyacrylamide gels and subsequently transferred to polyvinyldifluoride membrane ( immobilon - pvdf , millipore ). 5 μl of prestained sds - page protein standards ( bio - rad laboratories ) were also resolved on each gel and the mobilities of these standards ( molecular weights 28 . 5 kda to 113 kda ) were used to determine molecular weight of glast isoforms . non - specific binding sites on the membrane were blocked by incubating in 1 % ( w / v ) skimmed milk powder in tbs ( 0 . 05m tris - hs1 , ph 8 . 0 , 0 . 15m nacl ) for 30 minutes . membranes were incubated sequentially with an antibody preparation that recognises amino acids 24 - 40 of the rat glast - 1 protein ( kindly provided by wilhelm stoffel , university of cologne [ 5 ]), diluted 1 : 1000 in tbs containing 0 . 2 % ( v / v ) tween 20 ( tbs - tween ) and horse - radish peroxidase conjugated anti rabbit igg diluted 1 : 1000 with tbs - tween . an additional blot was incubated without primary antibody to control for non - specific binding of secondary antibody . membranes were washed extensively in between incubations with tbs - tween . specific binding of the anti glast - 1 antibody was detected by enhanced chemiluminescence on hyperfilm - ecl ( amersham , uk ). birch , m . a ., a . patton j . et al ( 1997 ) j . bone miner . res . 12 : s411 gafvelin , g . m . sakaguch et al ( 1997 ). j . biol . chem ., 272 ( 10 ): 6119 - 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[ 26 ] said , s . i . et al ( 1996 ) proc natl acad sci usa 93 , 4688 - 92 . val gln asn ile thr lys glu asp val lys ser tyr leu phe arg asn ile ile ile his pro gly lys gly thr lys glu asn met tyr arg glu arg asn met phe pro pro asn leu val glu ala cys phe lys gln phe lys thr ser tyr glu lys arg ser phe lys val pro ile gln ala asn phe val ile gly asn met lys glu gln gly gln ala leu arg glu phe phe asp ser leu asn glu ala ile met arg leu val ala val ile met glu met glu asp met gly val ile gly gly gln leu ala met tyr thr leu leu tyr phe leu val thr arg lys asn pro trp val phe ile gly ala thr leu pro ile thr phe lys cys leu glu glu asn asn gly val asp lys arg ile thr arg phe val leu pro val gly ala thr ile asn glu his leu ser arg his glu leu lys asn arg asp val glu met gly asn ser val ile glu glu asn glu met lys lys pro tyr gln leu ile