Patent Application: US-8740202-A

Abstract:
the present invention provides novel isolated and purified nucleic acid encoding , or complementary to , a canine pept1 . the present invention also provide a method for determining canine pept1 - transportability of a peptide , or method for determining a peptide with beneficial nutritional property in an animal . the present invention further provides a dietary composition for an animal comprising a peptide identified by the method described above .

Description:
this invention relates to peptide amino acid absorption in the dog , and more particularly , to separate , whole or partial - length , complementary dna encoding putative canine low - affinity , high - capacity h + / peptide transport proteins ( cpept1 ), mrna transcripts corresponding to cpept1 , characterization of cpept1 by glycylsarcosine ( glysar ) uptake , identification of dipeptides , tripeptides , and tetrapeptides well recognized by cpept1 , and the effect of supplemental peptide substrate on the transport capacity of cpept1 . the invention also provides a pet food composition comprising at least one dipeptide , tripeptide , or tetrapeptide that provides enhanced uptake of amino acids by pept1 . a typical canine diet for use in the present invention may also , for example , contain about 20 to about 30 % crude protein , about 10 to about 20 % fat , and about 10 % total dietary fiber . however , no specific ratios or percentages of these or other nutrients are required . the inventors have discovered a method for identifying peptides ( e . g . dipeptides , tripeptides , or tetrapeptides ) that increase transport of amino acids by pept1 using mdck cells , particularly when incubated with lactalbumin hydrolysate and assayed at optimum time post - seeding , as indicated in example 2 . in order that the invention may be more readily understood , reference is made to the following examples which are intended to illustrate the invention , but not limit the scope thereof . initial attempts ( over 150 ) to partially clone the putative canine pept1 cdna by reverse transcriptase - polymerase chain reaction ( rt - pcr ) methodology failed . the source of mrna was canine liver tissue that had been frozen for about 6 months ( supplied by dr . randal buddington , mississippi state university ) and oligomer primers were based on the rabbit pept1 sequence . subsequently , frozen canine “ mid ” small intestine ( jejunal ) tissue segments became available ( supplied by dr . buddington ) and a partial length cdna of about 780 base pairs ( bp ) was cloned by rt - pcr . total rna was isolated from jejunal epithelium scraped from intestinal sections using a standard acidic phenol - chloroform protocol . one μg of mrna was isolated from total rna using poly a tract system ® ( promega , madison , wis .) and reversed transcribed using murine leukemia virus reverse transcriptase ( perkin elmer , foster city , calif .) and oligo ( dt ) primers ( gibco brl , grand island , n . y .). successful pcr reactions were 50 μl and contained 1 μm mgcl 2 and taq polymerase ( perkin elmer ). twenty - five thermal cycles of 94 ° c . for 1 min , 40 ° c . for 45 sec , and 72 ° c . for 1 min were used . the cycles were preceded by a 55 sec denaturization of the rt product at 95 ° c ., followed by a 10 min extension of rt - pcr products at 72 ° c . more than 150 rt - pcr reactions testing ten different primer sets were required to achieve this protocol . the resulting cdna using primer set 4 ( fig1 ) was ta - cloned into the pcr ® ii vector ( invitrogen , carlsbad , calif . ), plasmid - containing colonies selected by blue / white screening , and amplified following instructions of the manufacturer . restriction analyses of recovered pcr ® ii / cdna plasmids revealed that four of fifty - six clones contained cdna consistent with rabbit pept1 cdna ( fig2 ). northern blot analysis of cpept1 expression in dog tissue and mdck cells the potential expression of cpept1 mrna by canine kidney , small intestinal epithelium , and immortalized kidney distal tubule epithelial cells ( madin - darby canine kidney , mdck ) was evaluated by northern analyses using cdna derived from canine jejunal epithelium ( fig3 ). rna were subjected to 1 % gel electrophoresis in the presence of 0 . 02 m formaldehyde , transferred by downward capillary action to 0 . 45 - μm nylon membranes ( hybond - n , amersham , arlington heights , ill . ), and covalently cross - linked by ultra - violet light . cdna were randomly labeled with [ 32 p ]- ctp using a kit ( gibco brl ), purified through sephadex - 50 columns ( amersham pharmacia , piscataway , n . j . ), and hybridized with blots at 56 ° c . for 18 h . the blots were then washed 2 times at 56 ° c . for 15 min and once at 57 ° c . for 10 min . autoradiographs were exposed to blots at 80 ° c . for 24 h and the size of the transcript determined by regression of hybridized bands against the migration distance of 18s ( 1 . 9 kb ) and 28s ( 4 . 9 kb ) rna . each canine tissue - derived cdna ( ta - clone 26 , fig3 a ; ta - clone 6 , fig3 b ) hybridized to three mrna species in dog kidney , dog small intestinal epithelium , and mdck cells . to confirm identification of pept1 mrna by these canine cdnas , rna isolated from dog kidney and liver tissues were probed for expression of pept1 mrna using a full - length rabbit pept1 cdna ( fig4 ; rabbit pept1 cdna supplied by drs . f . leibach and v . ganapathy , medical college of georgia ). the results also demonstrated the expression of the same three pept1 mrna species by dog tissues , indicating that the full - length rabbit pept1 cdna and the cdna derived from canine tissue in the present study identified the same transcripts . the mean / sd of transcript sizes calculated from these three blots were 4 . 2 / 0 . 22 , 2 . 75 / 0 . 26 , and 1 . 46 / 0 . 42 kb , respectively . collectively , these data indicate that liver , intestinal epithelial , and mdck cells express the same size and number of pept1 transcripts . in comparison , various tissues of chicken , sheep , cow , pig , rabbit , rat , human , and caco2 cells are reported to express a single transcript , with the principle difference in size being between chicken ( 1 . 9 ) and mammalian species ( 2 . 8 , 2 . 8 , 2 . 9 , 2 . 9 , 3 . 0 , 3 . 1 , 2 . 9 , respectively partial cloning and sequence identification of canine pept1 ( cpept1 ) cdna from mdck cells to confirm the positive northern analysis , identification of pept1 mrna expression using cdna generated from dog small intestinal epithelium , rt - pcr methodologies were used to generate a pept1 cdna from mdck cells . the target cdna region was a subset of the cdna generated by rt - pcr from canine small intestine ( bp 83 to 887 of rabbit pept1 ). accordingly , pcr primers that corresponded to bp 259 to 619 of rabbit pept1 ( genbank acc . no . u06467 ) were used to generate a partial - length “ canine pept1 ” ( cpept1 ) cdna from mrna isolated from mdck cells . rna was collected from cells that were plated at 30 , 000 cm 2 on rat tail collagen - coated dishes and cultured for 3 days in 10 % fetal calf serum / dmem . reverse transcription of 5 μg of total rna by superscript ® ii reverse transcriptase ( gibco - brl ) was performed using random and oligo ( dt ) primers , per instructions of the manufacturer ( gibco - brl ). all pcr reactions contained 2 mm mgcl 2 and thermal cycling using taq polymerase included 30 cycles at 94 ° c . for 2 min , 55 ° c . for 1 min , and 72 ° c . for 2 min . the cycles were preceded by a 10 min denaturization of the rt product at 94 ° c ., followed by a 10 min extension of rt - pcr products at 72 ° c . more than one hundred rt - pcr reactions were required to achieve this protocol . the resulting cdna of about 380 bp was ta - cloned , into the site of pcr ® ii vector ( as described above ), amplified , bacterial colonies evaluated by blue / white screening , and pcr ® ii / cdna plasmids evaluated for cdna by eco ri / pst i restriction analysis ( as described above ). restriction analyses of recovered plasmids revealed that six of thirty - six clones contained cdna consistent with rabbit pept1 cdna . two of the confirmed plasmids were amplified in bacteria , recovered , and sent for sequencing by the university of florida dna sequencing core facility ( gainesville ). sequence comparisons of this 380 bp cdna ( fig5 ) to pept1 sequences of other species using blast 2 . 0 . 14 . software ( blast @ ncbi . nlm . nih . gov ) revealed that the canine sequence shares sequence homology of 79 % to rabbit ( bp 259 to 640 ; genbank acc . no . 473375 ), 83 % to rat ( bp 213 to 593 ; genbank acc . no . d50664 . 1 ), 83 % to mouse ( bp 213 to 589 ; genbank acc . no . af205540 ), and 87 % to human ( bp 285 to 665 ; genbank ace . no . 473375 and u13173 ) pept1 sequences . as seen in fig3 and 5 , mdck cells express a canine homolog of mammalian pept1 mrna . potential expression of pept1 transport activity ( h + - dependent , dipeptide inhibitable , low - affinity dipeptide transport ) by confluent mdck cells was evaluated using whole - cell transport techniques and glycylsarcosine ( glysar ) as a model dipeptide substrate . cells were seeded at 60 , 000 cells / cm 2 into 24 - well trays that had been coated with rat tail collagen or poly - l - lysine and cultured ( 95 % o 2 : 5 % co 2 at 37 ° c .) for 3 d in media consisting of dulbecco &# 39 ; s modified eagle medium / 10 % fetal calf serum / 1 % antimicrobial antibacterial medium . absorption ( pmols / mg protein ) of [ 3 h ]- glycyl - l - sarcosine ( glysar ; 6 mci / ml , moravek biochemicals , brea , calif .) was determined using the 24 - well cluster tray method and representative scintillation counting . before transport , cells were incubated at 37 ° c . for 30 min in 25 mm hepes / tris ( ph 7 . 5 ), 140 mm nacl , 5 . 4 mm kcl , 1 . 8 mm cacl 2 , 0 . 8 mm mgso 4 , and 5 mm glucose ( uptake buffer ) to normalize intracellular amino acid and peptide pools . transport was initiated by the addition of 0 . 25 ml of uptake buffer that contained 2 . 88 μm glysar . after 30 min of uptake at 37 ° c ., transport was terminated by rapid washing of cells with 4 × 2 ml 4 ° c . uptake buffer . cellular protein was precipitated with 10 % trichloroacetic acid and the supernatant recovered and counted to determine radioactivity ( 3 h ) content . cellular protein was then solubilized in 0 . 2 n naoh and 0 . 2 % sds and quantified by the lowry procedure , using bovine serum albumin as a standard . the amount of h + - dependent glysar absorbed was calculated as the difference between uptake in ph 6 . 0 and ph 7 . 5 uptake buffers . the amount of competitor substrate - inhibitable glysar uptake was calculated as the quotient of glysar uptake in the absence and presence of 10 mm competitor substrate ( dipeptide or amino acid ) multiplied by 100 %. glysar uptake in the presence of an intracellularly h + gradient ( extracellular ph of 6 . 0 ) was 2 . 3 - fold higher in cells plated on collagen , and 1 . 7 - fold higher when grown on poly - l - lysine , than uptake in ph 7 . 5 medium ( table 1 ). h + - dependent uptake of glysar by mdck cells was inhibited by 88 or 92 % by the presence of 10 mm leutrp or trpleu when grown on collagen , and 87 or 92 % when grown on poly - l - lysine , respectively ( table 1 ). to preliminarily characterize the kinetic parameters of peptide transport by mdck cells , the uptake of glysar in media that contained ph 6 . 0 and 0 . 00064 , 0 . 0025 , 0 . 010 , 0 . 04 , 0 . 160 , 0 . 640 , 2 . 56 , or 10 . 2 mm of glysar was measured fig6 ). total glysar uptake was by a relatively low - affinity mechanism ( apparent k m of about 4 . 0 mm ) and high uptake velocity . collectively , these characteristics of glysar uptake are consistent with functional activity of pept1 expressed by other species , as opposed to high - affinity , h + - dependent uptake by pept2 ( μm k m ). accordingly , it is concluded that mdck cells possess pept1 - like activity , consistent with detection of pept1 mrna by rt - pcr ( fig1 , 5 ) and northern blot analyses ( fig3 ). separate partial - length canine pept1 cdnas ( cpept1 ) were generated by rt - pcr analyses from dog small intestinal epithelium ( n = 2 ; fig1 ) and immortalized canine kidney cells ( mdck cells , n = 1 ). the mdck cdna was sequenced ( fig5 ) and found to share 79 to 87 % sequence identity with pept1 mrna expressed by other mammalian species . northern blot analyses using the intestinal epithelium - derived rt - pcr cdna confirmed expression of canine pept1 ( cpept1 ) by dog tissues ( liver , n = 3 ; kidney , n = 3 ; small intestine n = 1 ) and mdck cells ( n = 2 ). the identification of mrna transcripts corresponding to pept1 using partial - length canine - derived pept1 cdna ( fig3 ) was confirmed by hybridization to full - length rabbit cdna ( fig4 ). characterization of glysar uptake by mdck cells demonstrated that mdck cells express pept1 - like activity ( table 1 , fig5 ), confirming detection of pept1 mrna expression by mdck cells and use of mdck cells as a model to characterize the function of canine pept1 . experimental model of mdck cells for evaluating the effects of various peptide and drug substrates , and hormones and / or growth factors , on the expression of pept1 activity example 1 above showed that ( 1 ) a canine homolog of pept1 ( cpept1 ) mrna cloned from epithelia of the mid small intestine ( jejunum ) shares high sequence identity with pept1 expressed by several other species , ( 2 ) canine liver , kidney , and jejunal epithelium express a similar pattern of cpept1 mrna , and ( 3 ) mdck cells are capable of h + - dependent peptide uptake . accordingly , mdck cells are an appropriate model to evaluate the biochemical characteristics of cpept1 . the specific goals of this research were to ( 1 ) characterize the functional activity of low - affinity h + - dependent glysar uptake ( pept1 activity ) by mdck cells and ( 2 ) identify di - and tripeptides that are well recognized by cpept1 ( cpept1 ), especially those that contain tryptophan and leucine . previous research ( brandsch et al ., 1994 , biochem j . 299 : 253 - 260 ) briefly reported that h + - dependent peptide uptake by mdck cells was greater when cells were grown in a medium that contained lactalbumin hydrolysate ( lhm ) versus one that contained free amino acids ( dmem ). therefore , in an attempt to establish the most sensitive model possible for evaluating peptide transport systems in mdck cells , the potential influences of lhm ( peptide - containing ) versus dmem ( peptide - lacking ) media , and subconfluent versus confluent initial cell plating densities were compared . mdck cells were seeded at either 60 , 000 cells / well ( subconfluent ) or 120 , 000 cells / well ( confluent ) in dmem and , after 1 d , cultured in dmem or lhm media for 1 , 2 , 3 , or 5 d . the amount of protein ( index of cell growth ) and glysar uptake ( index of peptide uptake capacity ) expressed by each well of cells was then determined . as seen in fig7 the amount of cellular protein increased ( p & lt ; 0 . 05 ) for both seeding densities and media with time of culture . a time × media interaction was observed , which reflects the greater protein content of cells grown in dmem at day 6 , as compared to those grown in lhm . at days 2 , 3 , or 4 , however , no difference in protein content was observed . the uptake of [ 3 h ]- glysar ( 2 . 88 μm , 5 μci / ml ) by the mdck cells described in fig7 was measured in the presence ( ph 6 . 0 uptake buffer ) and absence ( ph 7 . 4 uptake buffer ) of an extracellular - to - intercellular h + ( proton ) gradient . a representative graph ( fig8 ) compares the uptake of glysar by cells seeded at 60 , 000 / well and cultured in the lhm or dmem . for both culture media , glysar uptake in the presence of ph 6 . 0 was greater ( p & lt ; 0 . 01 ) than that in ph 7 . 4 buffer and displayed a quadratic ( p & lt ; 0 . 01 ) response to length of culture , reflecting a buffer × day of culture interaction ( p & lt ; 0 . 01 ). dmem - cultured cells seeded at 120 , 000 / well displayed almost identical uptake characteristics as just described for cells seeded at 60 , 000 / well . in contrast , glysar uptake in the presence of ph 6 . 0 buffer at day 3 by lhm - cultured cells was only 28 % larger ( quantitatively ) than that observed by dmem - cultured cells seeded at 60 , 000 / well . to further refine the analysis of media influence on the peptide transport capacity of mdck cells plated at 60 , 000 or 120 , 000 cells per well , the h + - dependent glysar uptake was calculated as the arithmetic difference between uptake in ph 6 . 0 and ph 7 . 4 buffers ( fig9 ). despite the comparable protein contents of cells observed at day 3 ( fig7 ), cells seeded at 60 , 000 and grown in lhm media demonstrated about 60 % greater capacity for glysar uptake as did cells grown in dmem ( fig9 ; day × media interaction , p & lt ; 0 . 01 ). for all cells , the capacity for glysar uptake per mg of cellular protein was decreased at day 6 . this difference was the result of a lesser uptake at ph 6 . 0 by the lhm - cultured cells , and not the result of a larger ph 7 . 4 uptake . the results of this experiment indicate that culturing cells in media that contains peptides does not increase growth rate but does increase the capacity for peptide uptake if cells are seeded at 60 , 000 / well and cultured for 2 days in lhm . as such , these data are consistent with the induction of pept1 expression by culture peptide - containing medium and describe an optimal set of culture conditions for characterizing h + - dependent peptide transport activity of the canine pept1 transporter . these data also confirm , and more thoroughly describe , the stimulating effect of lhm versus dmem media on peptide transport proteins that was initially reported by brandsch et al . ( 1994 ). using the maximal uptake - stimulating culture parameters determined in experiment 3 , the effect of an extracellular - to - intracellular ph gradient on glysar uptake was further evaluated to determine a ph level at which maximal glysar uptake could be achieved , but which would replicate physiologic conditions ( fig1 ). as expected , the presence of a ph gradient stimulated ( p & lt ; 0 . 001 ) h + - dependent glysar uptake , in a quadratic ( p & lt ; 0 . 01 ) fashion . uptake at ph 5 . 5 or 6 . 0 was about 2 . 7 times greater than that achieved at ph 7 . 5 . these results are consistent with the data in fig8 and 9 and known h + - dependence of mammalian peptide transport proteins . accordingly , the use ph 6 . 0 buffers for the characterization of h + - dependent glysar uptake was incorporated into the standard experimental conditions . to determine the appropriate time period to measure initial ( linear ) rates of glysar uptake , a by - minute time - course experiment was performed . as seen in fig1 , h + - dependent glysar ( 100 μm ) uptake increased linearly for 1 h and then slowed ( quadratic response , p & lt ; 0 . 01 ). glysar uptake in ph 6 . 0 buffer at 3 . 75 , 7 . 5 , 15 , 30 , 60 and 120 min was about 2 , 2 . 1 , 2 . 25 , 2 . 65 , 2 . 79 , and 2 . 62 times more ( p & lt ; 0 . 001 ), respectively , than uptake from ph 7 . 4 buffer . because uptake was proportional to time of uptake through 1 h , future experiments were conducted using a 30 - min time period . to confirm that h + - dependent glysar uptake was saturable , and therefore mediated , the uptake of glysar from ph 6 . 0 and 7 . 4 uptake buffers containing 0 . 025 , 0 . 1 , 0 . 4 , 1 . 6 , 6 . 4 , or 25 . 6 mm glysar was evaluated ( fig1 ). uptake of glysar was greatest ( p & lt ; 0 . 001 ) from the ph 6 . 0 buffers , at all concentrations . h + - dependent glysar uptake was saturable , consistent with an apparent k m for glysar of about 1 . 1 mm . these values are consistent with our preliminary trials that estimated a k m of 1 . 1 mm for glysar uptake by mdck cells using only ph 6 . 0 uptake buffer and indicate that h + - dependent glysar uptake is predominately , if not completely , a result of low affinity ( mm ) h + / peptide cotransporter activity ( pept1 ). as a comparative value , the reported k m of for glysar uptake by the pept1 - expressing caco - 2 cells also is 1 . 1 mm . it is of interest also to note that glysar uptake in the absence of a ph gradient ( ph 7 . 4 buffers ) also displayed linear ( p & lt ; 0 . 01 ) and quadratic ( p & lt ; 0 . 001 ) components , ( 1 ) reflects that the ph “ 7 . 4 ” buffer was in fact slightly acidic , ( 2 ) represents the activity of the putative basalateral peptide transporter running in “ reverse ”, or ( 3 ) indicates the presence of a non - characterized peptide transport system . as a result of this experiment , subsequent h + - dependent peptide transport trials were conducted using 100 μm glysar , a value well below the k m but one that will result in increased transport activity , and thus , sensitivity . characteristic hallmarks of low affinity h + / peptide cotransport activity , classically defined using membrane vesicles of several species , and more recently by functional expression studies using human , rat , and rabbit pept1 cdna , is the recognition of some , but not all , β - lactam antibiotics . in addition , pept1 recognition of cefadroxil is low ( the k 1 of cefadroxil inhibition of glysar uptake by pept1 is 3 mm ), whereas recognition of cefadroxil by pept2 is high ( the k i of cefadroxil inhibition of pept2 transport of glysar is 30 μm ). to determine whether mdck cpept1 activity shared these functional features , the uptake of 100 μm glysar in the absence and presence of ph 7 . 5 and ph 6 . 0 buffer , and , in ph 6 . 0 buffers , the presence of 1 mm additional glysar ( self - inhibitor control ), 3 mm penicillin - g , 30 μm cefadroxil , or 3 mm cefadroxil was compared ( fig1 ). h + - dependent glysar uptake was not inhibited by penicillin - g or 30 μm cefadroxil , but was inhibited about 76 % by 3 mm cefadroxil . as expected , the presence of 1 mm glysar self - inhibited 100 μm glysar uptake by 64 %. these results indicate that h +- dependent uptake of glysar by mdck cells is by pept1 activity . other hallmarks of pept1 function are the decreased ability of gly - containing peptides to inhibit glysar , in proportion to their length , and sensitivity to inhibition by carnosine ( β - ala - his ). to determine if cpept1 activity behaves as reported for other pept1 activities , the relative abilities of 1 mm gly ([ 3 h ]- gly free amino acid control ), glygly , [ gly ] 4 , or [ gly ] 5 to inhibit h + - dependent 100 μm glysar was determined ( fig1 ). gly ( 5 . 0 %) and [ gly ] 5 ( 7 . 3 %) did not influence uptake , whereas glygly inhibited and [ gly ] 4 tended to inhibit uptake by 63 and 23 %, respectively . this pattern of gly - containing peptides to inhibit glysar uptake in an inverse proportion to the number of glycyl residues in the canine mdck cell model is consistent with pept1 activities reported for other species . similarly , glysar uptake was inhibited 50 % by 1 mm carnosine ( data not shown but listed in table 2 below ). together with the molecular identification of pept1 mrna expression in mdck cells using full - length rabbit cdna and our canine rt - pcr product ( see example 1 data ), the above biochemical characterization data indicate that h + - dependent glysar uptake activity in mdck cells is consistent with the low - affinity , high - capacity of the pept1 transport protein . collectively , the above experiments resulted in the generation of an experimental regimen for the culture and determination of h + - dependent peptide transport activity in mdck cells , with which to evaluate the relative substrate preferences of canine pept1 ( cpept1 ). accordingly , the following general regimen was used to perform a series of experiments that evaluated the relative abilities of candidate di - ( primarily ) and tri - peptides to inhibit glysar uptake by endogenously expressed cpept1 in mdck cells : 1 . sixty thousand cells / well were plated into collagen - coated 24 - well trays and cultured at 37 ° c . in an atmosphere of 95 % air / 5 % co 2 in dmem / 10 % fcs that contained antibiotics for 1 day . 2 . the media was removed and cells were cultured in lhm / 10 % fcs / antibiotics for 1 day . 3 . the media was removed and cells cultured in lhm / 10 % fcs ( no antibiotics ) for 20 h . 4 . the media was removed and cells cultured for 30 min in air at 37 ° c . in depletion medium ( 25 mm hepes / tris ( ph 7 . 5 ), 140 mm nacl , 5 . 4 mm kcl , 1 . 8 mm cacl2 , 0 . 8 mm mgso 4 , and 5 mm glucose , to normalize intracellular nutrient pools . 5 . transport was initiated by replacing depletion medium with uptake medium ( depletion medium adjusted to ph 6 . 0 or kept at ph 7 . 4 ) that contained 100 μm glysar ( at a specific activity of 5 μci / ml , with [ 3 h ]- glysar supplying 2 . 88 % of total glysar substrate ) and ( or ) 1 mm of inhibiting peptide . an inhibitory substrate concentration of 1 mm was selected because the literature indicates that typical k m values for pept1 ranges from 0 . 5 to 5 mm . therefore , by selecting an inhibitor concentration of 1 mm ( not expected to completely inhibit uptake ), our goal was to more finely delineate the relative abilities of candidate inhibitors than if the typical 5 mm inhibitor concentration ( expected to achieve close to 100 % inhibition of glysar uptake ) was used . candidate peptides were selected based on their containing trp , leu , met , and ( or ) arg , substrates . in total , 23 inhibitory peptides and 2 drug compounds were screened using this protocol . to determine the potential of trp and leu absorption as dipeptides by cpept1 , the ability of trpleu versus leutrp dipeptides to inhibit 100 μm glysar uptake was evaluated ( fig1 ). the presence of either trpleu or leutrp in the ph 6 . 0 uptake buffer abolished h + - dependent glysar uptake by 117 % or 114 %, respectively . in contrast , neither leu nor trp significantly influenced h + - dependent glysar uptake . these results indicate that a lesser concentration of inhibitor would be required to delineate the relative recognition of trpleu and leutrp by cpept1 . with regard to the mechanism of h + - independent glysar uptake observed throughout these experiments , it is of interest to note that trpleu and leutrp inhibited h + - independent glysar uptake by 36 % and 46 %, respectively . to further evaluate the potential of trp to be absorbed in the form of peptides by cpept1 , the ability of trptrp , trpgly , and trpglygly to inhibit glysar uptake was compared ( fig1 ). as observed for trpleu ( fig1 ), trptrp abolished h + - dependent glysar uptake and inhibited h + - independent uptake by about 22 %. trpgly abolished h + - dependent glysar uptake but did not influence h + - independent uptake . the tripeptide trpglygly also significantly inhibited glysar uptake , but to a lesser extent ( 73 %) than did trptrp or trpgly . to determine the relative potential of other amino acids ( met , arg , lys , phe , for example ) to be absorbed in the peptide - bound form , additional glysar competitive inhibition experiments were conducted using the above - described regimen and a variety candidate peptides at 1 mm . the results of these experiments are summarized in table 2 , which also includes those experiments described in fig1 , 14 , 15 , and 16 for comparative purposes . the inhibitors are listed within groupings in order of their relative ability to inhibit 100 μm of glysar uptake . in addition to the listed peptides , the constituent free amino acids were tested within the appropriate experiment to evaluate whether the peptide - bound or free amino acid was responsible for any affect on glysar uptake . as expected , the presence of 1 mm constituent free amino acid did not influence glysar uptake . inhibition percentages of 50 % indicate that the inhibitor substrate was recognized at least as well as was glysar , given that the k m of glysar was determined to be about 1 mm ( fig1 ) and that the substrate was present at 1 mm . of the 19 treatment peptides evaluated , eleven abolished h + - dependent glysar uptake , with seven of these also displaying the ability to inhibit h + - independent glysar uptake . of the remaining eight peptides tested , four displayed greater than 80 % inhibition while four inhibited glysar uptake by 50 % or less . these results indicate that a wide variety of peptides of nutritionally important constituent amino acids are recognized by cpept1 . overall , the observation that cpept1 activity was sensitive to a number of substrates is typical of pept1 function . however , what was surprising was the large number of peptides that completely inhibited glysar uptake . to establish a more sensitive relative inhibitory order among peptides that inhibited glysar uptake by more than 80 %, and , therefore , a more accurate potential for recognition , fourteen peptides were re - screened for their ability to inhibit 100 μm glysar uptake using the same cell culture and transport regimen but using only 10 % of the previous inhibitor concentration ( 100 μm ). the data from an experiment to directly compare the ability of 100 μm trp - containing peptides are shown in fig1 . all trp - containing peptides inhibited h + - dependent glysar uptake . however , trpleu inhibited more ( 92 %) than did leutrp ( 58 %), trptrp ( 62 %), or trpgly ( 45 %). these values and the results of other experiments comparing the relative ability of leu -, met -, and arg - containing peptides are listed in table 3 . overall , four of the peptides inhibited glysar uptake by at least 80 %, six by more than 40 %, and four less than 40 %, thus establishing a relative ranking for recognition by cpept1 . among the five trp - containing peptides ( fig1 , table 3 ), trpleu demonstrated the greatest ability to inhibit glysar uptake . trpleu also demonstrated the greatest ability to inhibit glysar uptake ( 94 %) among the leu - containing peptides . among the met - containing substrates that were directly compared within the same experiment , the neutral peptides , metmet and metphe , inhibited more glysar uptake than did the anionic ( metglu ) or cationic ( metlys ) carboxyl residues . interestingly , as a group the arg peptides demonstrated the least inhibitory ability , seemingly in keeping with the apparent lesser recognition by pept1 of substrates with charged residues . however , it is of interest to note that 100 μm argleu demonstrated a much greater ability to inhibit glysar uptake than did leuarg ( 49 versus 8 . 9 %). to confirm the relative ranking of trpleu & gt ; trptrp inhibition of glysar ( tables 2 and 3 ), michaelis - menton constants for substrate inhibition ( k i ) of glysar uptake by trpleu and trptrp were generated by graphical analyses of ic 50 experiments ( fig1 ). in keeping with the results achieved in the 100 μm - inhibition studies , trpleu inhibited glysar uptake at lower concentrations than did trptrp ( k 1 = 0 . 2 versus 0 . 75 μm , respectively ). collectively , the results of cpept1 competitive inhibition trials using mdck cells indicate that trpleu is better recognized by cpept1 than any other tested peptide . the results also indicate that a number of trp -, leu , and met - containing peptides also are well recognized by cpept1 . ultimately , in the intestinal environment , it is the combination of recognition by the transporter and relative resistance of the peptide to luminal and membrane - bound peptidases that will determine how much of a given peptide will be absorbed . in this regard , there is some evidence to suggest that gly - x peptides are more resistant than other peptides , especially by blood and renal peptidases . if so , then glyleu may be a better candidate substrate than trpleu to supply leu . similarly , tripeptides , as a group , are thought to be relatively resistant to hydrolysis . thus , more trpglygly may prove to be absorbed in larger amounts by the intestine than trpleu . an important result of this set of experiments was the establishment of a sensitive experimental regimen / model to evaluate potential affecters of peptide transport capacity . accordingly , this experimental model of mdck cells grown in lhm affords an opportunity to evaluate the effects of various peptide and drug substrates , and hormones and ( or ) growth factors , on the expression of pept1 . thus , the culture of mdck cells in lhm versus dmem results in an increase of h + - dependent glysar uptake ( k m = 1 . 1 mm ) that is consistent with mammalian pept1 - like activity . using this stimulated model , the ability of twenty - three di - and tripeptides at 1 mm , and fourteen at 100 μm , extracellular concentrations were screened for their ability to inhibit 100 μm glysar uptake , as an indicator of recognition by pept1 . of the trp - and ( or ) leu - containing peptides evaluated , trpleu ( k i = 0 . 2 μm ) and leutrp ( k 1 = 0 . 75 μm ) demonstrated the greatest ability to inhibit glysar uptake , with trpleu demonstrating a relatively higher affinity ( lower k 1 ) for pept1 . of the met - containing peptides evaluated , four ( metmet , metphe , leumet , metleu ) appear particularly well recognized by pept1 . in contrast , as a group , arg - containing peptides displayed the least inhibition of pept1 activity . overall , these results indicate that cpept1 is capable of recognizing a variety of di - and tripeptides , including , for example , those that contain leucine and tryptophan . experimental model to determine whether the h + / peptide transport capacity expressed by mdck cells is sensitive to substrate regulation examples 1 and 2 above demonstrated that madin - darby canine kidney ( mdck ) cells express pept1 mrna and characterized h + - dependent biochemical properties . therefore , mdck cells were chosen as the experimental model to determine whether the h + / peptide transport capacity expressed by mdck cells is sensitive to substrate regulation . research from example 2 demonstrated that mdck cells grown in lactalbumin hydrolysate medium ( lhm ) had elevated levels of peptide uptake capacity . accordingly , to avoid potential confounding effects of the peptide - containing lhm and individual treatment peptides , dmem ( contains no peptides ) and not lhm was selected as the appropriate medium to test the influence of extracellular peptides on canine pept1 functional capacity of mdck cells . glyphe was selected as a substrate because it has been reported to increase brush border membrane content of pept1 , ( shiraga t , miyamoto k , tanaka h , yamamoto h , taketani y , morita k , tamai i , tsuji a , takada e . cellular and molecular mechanisms of dietary regulation on rat intestinal h + / peptide transporter pept1 . gastroenterology 1999 ; 116 : 354 - 362 ), whereas phe and gly were tested as constituent free amino acid treatment controls . carnosine was selected because of its high content in meat - based diets . cell culture . all cells were plated ( 60 , 000 / 2 cm 2 well ) and cultured ( 95 % air / 5 % co 2 , 37 ° c .) for 24 h in dulbecco &# 39 ; s modified eagle media / 10 % fetal calf serum ( fcs )/ 1 % antibiotic / antimicrobial solution ( abam ) ( dmem media ). following these initial common culture conditions , cells then were cultured in dmem , or dmem that contained 10 mm of carnosine , glyphe , phe , or gly . media were changed every 24 h . media treatments ( n = 8 ) were as follows : uptake measurements . the measurement of [ 3 h ] glysarcosine uptake was performed by using a 24 - well cluster tray method ( kilberg m s . measurement of amino acid transport by hepatocytes in suspension and monolayer culture . methods enzym 1989 ; 173 : 564 - 575 . matthews j c , aslanian a , mcdonald k k , yang w , malandro m s , novak d a , kilberg m s . an expression system for mammalian amino acid transport using a stably maintained episomal vector . anal biochem 1997 ; 254 : 208 - 214 ), and used in examples 1 and 2 . cells were cultured for 30 min in air at 37 ° c . in depletion medium ( 25 mm hepes / tris ( ph 7 . 5 ), 140 mm nacl , 5 . 4 mm kcl , 1 . 8 mm cacl2 , 0 . 8 mm mgso4 , and 5 mm glucose ), to normalize intracellular nutrient pools before transport . the transport assays are initiated by replacing depletion medium with uptake medium ( depletion medium adjusted to ph 6 . 0 ) that contained 100 μm glysar ( 5 μci / ml , with [ 3 h ]- glysar supplying 2 . 88 % of total glysar ). after a 30 minute incubation period , transport was terminated with four rinses of 4 ° c . depletion medium ( ph 7 . 5 ). two hundred and twenty μl of 10 % trichloroacetic acid was added to each well , and the radioactivity of the supernatant quantified by liquid scintillation counting . the cells of each well are solubilized in 0 . 2 n naoh / 0 . 2 % sds and the protein quantified by using the modified lowry assay , using bovine serum as a standard . id . peptide uptake will be reported as pmol * mg − 1 protein * 30 min − 1 . uptake measurements were taken after 24 , 48 , and 72 hours of culture in treatment media . results . the previous research characterizing h + - dependent peptide transport by mdck cells ( example 2 above ) clearly showed that transport velocity is dependent on protein content . therefore , to make a valid comparison of various treatment parameters on glysar uptake , the protein content of compared treatment groups must not differ . accordingly , the influence of culture media on mdck cellular protein was evaluated ( fig1 ). all media treatments supported cellular growth from 1 to 3 d and no difference in protein content among treatments was observed . similarly , no difference in uptake velocity ( capacity ) was observed among treatment groups , for any culture period ( fig2 ). the results from trial 1 suggest that either canine pept1 is not sensitive to substrate regulation or that the substrates and ( or ) stimulation time were inadequate to influence h + - dependent peptide uptake in mdck cells . again , dmem was selected as the basal medium to allow the effect of individual peptides on peptide transport activity to be evaluated . to evaluate the latter two possibilities , a second trial was conducted that included a culture period of 9 d . glysar was added as another potential affecter of h + - dependent peptide transport capacity because 10 mm glysar it is reported capable of stimulating increased pept1 activity ( adibi s . the oligopeptide transporter pept1 in human intestine : biology and function . gastroenterology 1997 ; 113 : 332 - 340 ) in caco - 2 cells . glypro was added as a treatment because of its high content in muscle tissue , thus is likely to be abundant in meat - based diets . cell culture . the mdck cell line was maintained as described previously in the methods section of trial 1 . following initial and common culture conditions , cells were cultured in dmem , or dmem that contained 10 mm glysar , glypro , glyphe , or carnosine . media were changed every 24 h . media treatments ( n = 8 ) were as follows : uptake measurements . the measurement of [ 3 h ] glysarcosine uptake was performed by using the 24 - well cluster tray method as previously described in the methods section of trial 1 . peptide uptake will be reported as pmol * mg − 1 protein * 30 min − 1 . uptake measurements were taken after 4 , 12 , 24 , 36 , 72 , 120 , 168 , and 216 hours of culture in treatment media . results . protein content in all treatment groups increased linearly from 4 to 216 h ( 9 d ) of culture , for all treatment groups ( fig2 ). however , within a culture period , protein contents of treatment groups did not differ . over the 216 - h culture period , protein increased about 4 . 5 times , from about 40 to 220 μg / well . in contrast to trial 1 results , media treatment did influence glysar uptake capacity ( fig2 ). in addition , a treatment × time effect was observed that represents differences in the time of culture required for glysar and carnosine treatment stimulation of glysar uptake capacity . specifically , glysar containing dmem culture treatment resulted in an increase in glysar uptake capacity of about 30 % over dmem control media by 24 h of culture time . this level of increase was maintained through 216 h . in contrast , culture in carnosine - containing media did not result in a significant ( 23 %) increase of glysar uptake capacity over that by dmem - cultured cells until 72 h of culture . this stimulation then steadily increased to 291 % over 216 h of culture . the nature of stimulated uptake between the two peptide substrates also differed . that is , the magnitude of carnosine - stimulated glysar uptake was essentially constant from 72 to 216 h , whereas that for glysar culture decreased during this period . collectively , these data indicate that h + - dependent peptide transport in cultured mdck cells can be stimulated by at least two of pept1 substrates , glysar and carnosine . the data from trial 2 indicate that h + - dependent glysar uptake capacity by fed mdck cells can be upregulated by the inclusion of 10 mm glysar for at least 24 h and 10 mm carnosine for at least 72 h . it is of equal interest to understand if h + - dependent glysar uptake capacity is sensitive to nutrient deprivation and ( or ) stimulation by glucocorticoids . a preliminary study indicates that fasting increases the expression of pept1 in rat small intestine epithelia . thamotharan m , bawani s , zhou x , adibi s . functional and molecular expression of intestinal oligopeptide transporter ( pept1 ) after a brief fast . metabolism 1999 ; 48 : 681 - 684 . to initiate investigation of potential influence of fasting and glucocorticoids on mdck cells expression of glysar uptake capacity , the h + - dependent uptake of glysar was evaluated over a 72 period of nutrient deprived or fed and cultured with dexamethasone ( dex ) and compared to that by cells cultured in dmem or dmem that contained insulin ( negative control ) ( trial 3a ). the “ nutrient deprived ” treatment actually contained 5 mm glucose and appropriate salts to ensure adequate basal metabolic conditions . although recruitment of pept1 protein and activity appears sensitive to insulin - stimulated recruitment from cytosolic vesicles in caco - 2 cells ( thamotharan m , bawani s , zhou x , adibi s . hormonal regulation of oligopeptide transporter pept1 after a brief fast . am j physiol 1999 ; 276 : c821 - 826 , mdck cells are reported to be insensitive to insulin , likely as an inability to express the insulin receptor . hofmann c , crettaz m , bruns p , hessel p , hadawi g . cellular responses elicited by insulin mimickers in cells lacking detectable plasma membrane insulin receptors . j cell biol 1985 ; 27 : 401 - 414 . in contrast to the lack of insulin sensitivity , igf - i is known to stimulate dna synthesis and cell proliferation in mdck cells . sukegawa i , hizuka n , takano k , asakawa k , shizume k . characterization of igf - 1 receptors on mdck cell line . endocrinol japan 1987 ; 34 ( 3 ): 339 - 346 . mouzon s h , kahn r . insulin - like growth factor - mediated phosphorylation and proto - ontogeny induction in mdck cells . mol endocrinol 1991 ; 5 : 51 - 60 . the understanding that mdck cells are apparently insensitive to insulin stimulation yet are sensitive to igf - i stimulation appears to be a paradox given that the supraphysiologic levels of both substrates employed in the perspective studies and the known ability of insulin to cross react with the igf - i receptor . accordingly , another trial ( trial 3b ) was conducted to evaluate the influence of increasing igf - i concentrations on h + - dependent glysar uptake by mdck of the same plating stock . cell culture . mdck cells were maintained as described in trial 1 , except that cells were cultured for only 1 d before transport trials were performed . following initial and common culture conditions , cells were cultured in a “ nutrient depleted ” buffer ( hepes / tris ( ph 7 . 5 ), 140 mm nacl , 5 . 4 mm kcl , 1 . 8 mm cacl 2 , 0 . 8 mm mgso 4 ) that contained 5 mm glucose as an energy source , but that lacked amino acid or vitamin sources . in contrast , cells cultured in dmem , or dmem that contained 5 nm dex , 500 nm dex , 5 nm insulin , or 500 nm insulin , were adequately nourished . media treatments ( n = 4 ) were as follows : uptake measurements . the measurement of [ 3 h ] glysarcosine uptake was performed by using the 24 - well cluster tray method as previously described in the methods section of trial 1 . peptide uptake is reported as pmol * mg − 1 protein * 30 min − 1 . uptake measurements were taken after 30 min and 4 h of culture in treatment media . trial 3b was conducted in the same manner as described for trail 3a , except that cells were cultured in dmem or dmem that contained 1 nm igf - 1 , 5 nm igf - 1 , 25 nm igf - 1 , or 100 nm igf - 1 . uptake measurements were taken after 30 min and 4 h of culture time . media treatments ( n = 4 ) were as follows : results . protein content of the treatments within trails 3a or 3b did not differ . after 4 h of culture , however , the capacity for h + - dependent peptide uptake was reduced 35 % in cells deprived of nutrients but adequate in energy ( fig2 ). in contrast , dexamethasone had no effect on glysar uptake . as expected , and consistent with the concept that mdck cells are insulin - insensitive , the presence of insulin for 4 h had no effect on glysar uptake capacity . similarly , culture of cells with increasing amounts of igf - i elicited no significant stimulation of h + - dependent glysar uptake ( fig2 ). quantitatively , however , 1 to 25 nm of igf - i tended to increase glysar uptake capacity by 10 to 15 %. given the noted restrictions of trail 3 , and the low number of observations ( n = 4 ) results from trial 3a and 3b suggest that h + - dependent uptake of glysar by mdck is sensitive to nutrient deprivation and , perhaps , igf - i . [ 0214 ] pept1 sequence clone12 ( 5 th round ; seq id no : 11 ) primer pair is gsp3 - 4 ; gsp3 - 1r using regular rt - pcr catcttcttcatcgtggt caatgagttctgtgaaagattttcctactatg gaatgagagcactcctgattctgtacttcagacggttcatcgggtgggac gataatctgtccacggccatctaccacacgtttgtggctctgtgctacct gacgccgatcctcggcgcactgatcgcagactcctggctgggaaagttca agacaatcgtgtcactctccattgctacacaattggacaggcggtcactg cagtaagctcaattaatgacctcacagactataacaaagatggaactcct gacaatctgtccgtgcatgtggcactgtccatgattggcctggccctgat agctctgggaactggaggaataaagccctgtgtgtctgcatttggtggag accagtttgaagagggccaggaaaaacaaagaaacagattcttttccatc ttttatttggccattaatgctggaagcttgatttccactattgtcactcc catgctcagagttcacgaatgtggaatttacagtcagaaagcttgttacc cactggcatttggggttcctgctgctctcatggccgtatctctgattgta tttgtcattggcagtggaatgtacaagaagtttcagccccagggtaatgt catgggtaaagttgtcaagtgcattggttttgccctcaaaaataggttta ggcaccggagtaagcagtttcccaagagggagcactggctggactgggct aaagagaaatacgatgagcggctcatctctcaaattaagatggtcacaaa agtgatgttcttgtacatcccactcccaatgttctgggccctgtttgacc agcagggctccaggtggacactgcaagcaacagctatgagtgggaaaatt ggacttcttgaagttcagccagatcagatgcagactgtgaatgccatctt gattgtcgtcatggtccccatcatggatgccgtggtgtaccctctgattg caaaatgtggcttcaatttcacctccttgaagaggatgacagttggaatg ttcctggcttccatggccttcgtgatggcggcgattgttcagctggaaat tgataaaactcttccagtcttccccaaacaaaatgaagtccaaatcaaag tactgaatataggaaatggtgccatgaatgtatcttttcctggagcggtg gtgacagttagccaaatgagtcaatcagatggatttatgacttttgatgt agacaaactgacaagtataaacatttcttccactggatcaccagtcattc cagtgacttataactttgagcagggccatcgccatacccttctagtatgg gcccccaataattaccgagtggtaaaggatggcctt aaccagaagcc agaaaaagggag initiale denatur - an - amplifi - denaturat ation nealing cation extension cooling temp 94 ° c . 94 ° c . 55 ° c . 72 ° c . 72 ° c . 4 ° c . min . 10 min 2 min 1 . 5 min 2 min 10 min inf . cycle 1 35 1 [ 0216 ] clone37 beginning ( 6th round ; seq id no : 12 ) primer pair is gsp3 - 9 ; auap using 3 ′ race protocol gccatcgccatacccttcta gtatgggcccccaataattaccgagtggta aaggatggccttaaccagaagccagaaaaaggagaaaatggaatcagatt tataaatagtcttaatgagagcctcaacatcaccatgggcgacaaagttt atgtgaatgtcaccagtcacaatgccagcgagtatcagttctttttcttt tctttgggcacaaaaaacattacaataagttcaacacaacgatctcacaa aattgtacaaaagttctccaatcatccaaccttgaatttggtagtgcata tacctatgtaatcggaacgcagagcactggctgccctgaattgcatatgt ttgaagatatttcacccaacacagttaacatggctctgcagatcccgcag tacttcctcatcacctgcggcgaggtggttttctctgtcacaggactgga gttctcatattctcaggccccctccaacatgaagtcggtgcttcaggcgg gatggctgctgacagtggcttgttggcaacatcattgtgctcattgtggc aggagcaggccagttcagtgaacagtgggctgaatacatcctatttgcgg cattgcttctggttgtctgtgtaatatttgccatcatggcccggttttac acttacgtcaatccagcagagattg initiale denatur - an - amplifi - denaturat ation nealing cation extension cooling temp 94 ° c . 94 ° c . 52 ° c . 72 ° c . 72 ° c . 4 ° c . min . 10 2 min 1 . 5 min 2 10 inf . cycle 1 30 1 [ 0218 ] merge sequence ( seq id no : 8 ) is : catcttcttcatcgtggtcaatgagttctgtgaaagattttcctatggaa tgagagcactcctgattctgtacttcagacggttcatcgggtgggacgat aatctgtccacggccatctaccacacgtttgtggctctgtgctacctgac gccgatcctcggcgcactgatcgcagactcctggctgggaaagttcaaga caatcgtgtcactctccattgtctacacaattggacaggcggtcactgca gtaagctcaattaatgacctcacagactataacaaagatggaactcctga caatctgtccgtgcatgtggcactgtccatgattggcctggccctgatag ctctgggaactggaggaataaagccctgtgtgtctgcatttggtggagac cagtttgaagagggccaggaaaaacaaagaaacagattcttttccatctt ttatttggccattaatgctggaagcttgatttccactattgtcactccca tgctcagagttcacgaatgtggaatttacagtcagaaagcttgttaccca ctggcatttggggttcctgctgctctcatggccgtatctctgattgtatt tgtcattggcagtggaatgtacaagaagtttcagccccagggtaagtcat gggtaaagttgtcaagtgcattggttttgccctcaaaaataggtttaggc accggagtaagcagtttcccaagagggagcactggctggactgggctaaa gagaaatacgatgagcggctcatctctcaaattaagatggtcacaaaagt gatgtcttgtacatcccactcccaatgttctgggccctgtttgaccagca gggctccaggtggacactgcaagcaagcaacagctatgagtgggaaaatt ggacttcttgaagttcagccagatcagatgcagactgtgaatgccatctt gattgtcgtcatggtccccatcatggatgccgtggtgtaccctctgattg caaaatgtggcttcaatttcacctccttgaagaggatgacagttggaatg ttcctggcttccatggccttcgtgatggcggcgattgttcagctggaaat tgataaaactcttccagtcttccccaaacaaaatgaagtccaaatcaaag tactgaatataggaaatggtgccatgaatgtatcttttcctggagcggtg gtgacagttagccaaatgagtcaatcagatggatttatgacttttgatgt agacaaactgacaagtataaacatttcttccactggatcaccagcattcc agtgacttataactttgagcagg gccatcgccatacccttcta gtatggg cccccaataattaccgagtggtaaaggatggccttaaccagaagccagaa aaaggagaaaatggaatcagatttataaatagtcttaatgagagcctcaa catcaccatgggcgacaaagtttatgtgaatgtcaccagtcacaatgcca gcgagtatcagttcttttctttgggcacaaaaaacattacaataagttca acacaacagatctcacaaaaffgtacaaaagttctccaatcatccaaccf fgaatttggtagtgcatatacctatgtaatcggaacgcagagcactggct gccctgaattgcatatgtttgaagatafftcacccaacacagttaacatg gctctgcagatcccgcagtacttcctcatcacctgcggcgaggtggtttt ctctgtcacaggactggagttctcatattctcaggccccctccaacatga agtcggtgcttcaggcgggatggctgctgacagtggct tgttggcaacat cattgtgctcattgtggcaggagcaggccagttcagtgaacagtgggctgc aatacatcctatttgcggcattgcttctggttgtctgtgtaatatttgcc atcatggccczgttttacacttacgtcaatccagcagagattg [ 0219 ] multiple alignment of nucleotide full length sequences sequence 1 : xm_007063 homosapiens 3045 bp sequence 2 : ay027496ovis 2829 bp sequence 3 : d50306rat 2900 bp sequence 4 : nm_053079 musmusculus 3128 bp sequence 5 : u13808 oryctolaguscunic 2709 bp sequence 6 : ay029615 gallusgallus 2914 bp sequence 7 : sequencetosubmitgenbak 1840 bp start of pairwise alignments aligining . . . sequences ( 4 : 5 ) aligned . score : 65 sequences ( 1 : 2 ) aligned . score : 65 sequences ( 2 : 3 ) aligned . score : 66 sequences ( 3 : 4 ) aligned . score : 88 sequences ( 4 : 6 ) aligned . score : 48 sequences ( 2 : 4 ) aligned . score : 64 sequences ( 1 : 3 ) aligned . score : 67 sequences ( 3 : 5 ) aligned . score : 66 sequences ( 4 : 7 ) aligned . score : 80 sequences ( 2 : 5 ) aligned . score : 77 sequences ( 3 : 6 ) aligned . score : 48 sequences ( 5 : 6 ) aligned . score : 51 sequences ( 1 : 4 ) aligned . score : 76 sequences ( 3 : 7 ) aligned . score : 81 sequences ( 5 : 7 ) aligned . score : 79 sequences ( 2 : 6 ) aligned . score : 50 sequences ( 6 : 7 ) aligned . score : 70 sequences ( 1 : 5 ) aligned . score : 67 sequences ( 2 : 7 ) aligned . score : 83 sequences ( 1 : 6 ) aligned . score : 49 sequences ( 1 : 7 ) aligned . score : 85 guide tree file created : [/ net / nfs0 / vol1 / production / w3nobody / tmp / 999267 . 834538 - 239427 . aln ] start of multiple alignment there are 6 groups aligning . . . group 1 : sequences : 2 score : 48218 group 2 : sequences : 3 score : 43200 group 3 : sequences : 2 score : 42027 group 4 : sequences : 5 score : 39817 group 5 : sequences : 6 score : 30418 group 6 : sequences : 7 score : 33857 alignment score 249395 clustal - alignment file created [/ net / nfs0 / voll / production / w3nobody / tmp / 999267 . 834538 - 239427 . aln ] your multiple sequence alignment : 999267 . 834538 - 239427 . aln clustal w ( 1 . 81 ) multiple sequence alignment ( seq id no : 3 ) d50306rat ----------------------------------- ctgaactcctgcttg 15 ( seq id no : 4 ) nm_053079 musmusculus -------------------------------------------------- ( seq id no : 1 ) xm_007063 homosapiens -------------------------------------------------- ( seq id no : 2 ) ay027496ovis ----- gaaacaacatctttagcacggattcctcccacctggactcctcgc 45 ( seq id no : 5 ) u13707 oryctolaguscunic ------------------------------------------------ ( seq id no : 6 ) sequencetosubmitgenbak ------------------------------------------------ ( seq id no : 6 ) ay029615 gallusgallus gctctctgtccgtccctcggtccctccgtccctccgtccccgcgcggccg 50 d50306rat ccagtcgccggtcaggagcctcggagccacaatggggatgtccaagt 65 nm_053079 musmusculus --- gtcgcccgtccggagccttggagccaccacaatggggatgtccaagt 47 xm_007063 homosapiens -------------------------------------- gaatgtccaaat 12 ay027496ovis tcgccagtcgcagggagccctcggagccgccagcatgggaatgtccgtgc 95 u13707 oryctolaguscunic ------------------------------ caccatgggaatgtctaagt 95 sequencetosubmitgenbak -------------------------------------------------- ay029615 gallusgallus ccagcagcgtgccggccccatggctgcaaaaagtaagagtaagggccgat 100 d50306rat ct --- cggggttgctttggctacccattgagcatcttcttcatcgtggtc 112 nm_053079 musmusculus ct --- cggggttgcttcggttacccgttgcatcttcttcatcgtggtc 94 xm_007063 homosapiens ca --- cacagtttctttggttatcccctgagcatcttcttcatcgtggtc 59 ay027496ovis cg --- aagagctgcttcggttaccccttagcatcttcttcatcgtggtc 142 u13707 oryctolaguscunic ca --- ctgagctgcttcggctatcccctgagcatcttcttcatcgtggtc 67 sequencetosubmitgenbak ------------------------------ catcttcttcatcgtggct 19 ay029615 gallusgallus cagtgccgaactgctttggctaccccttgagcatcttcttcatcgtcatc 150 *************** ** d50306rat aatgaattctgtgaaagattctcctactatgggatgcgagctctcctggt 162 nm_053079 musmusculus aatgaattctgtgaaagattctcctactatggcatgcgagcactcctggt 144 xm_007063 homosapiens aatgagttttgcgaaagattttcctactatggcatgcgagcacttctggt 109 ay027496ovis aatgagttctgcgaaaggttctcttactatggaatgcgagcaatcctgat 192 u13707 oryctolaguscunic aatgagttctgcgaaaggttctcctactatggaatgcgagcaatcctgat 117 sequencetosubmitgenbak aatgagttctgtgaaagattttcctactatggaatgcgagcaatcctgat 69 ay029615 gallusgallus aatgagttctgcgagaggttctcctactatggcatgcgagcaatgctcgt 200 ***** ** ** ** ** ** ** ******** *** **** * ** * d5030grat tctgtacttcaggaacttccttggctgggatgatgacctctccacggcca 212 nm_053079 musmusculus tctgtacttcaggaacttcctcggctgggacgacaatctctccacggcca 194 xm_007063 homosapiens tctgtacttcacaaatttcatcagctgggatgataacctgtccaccgcca 159 ay027496ovis cctgtacttccaacgtttcctgggctggaacgacaacctgggcaccgcca 242 u13707 oryctolaguscunic tctgtacttcagaaacttcatcggctgggacgacaacctgtccacggtca 167 sequencetosubmitgenbak tctgtacttcagacggttcatcgggtgggacgataatctgtccacggcca 119 ay029615 gallusgallus attgtatttcaagtacttcctgcggtgggatgacaacttttctacagcca 250 **** *** *** * * *** * ** * * ** * ** d50306rat tctaccatacgtttgttgccctctgctacctgactccaattcttggagct 262 nm_053079 musmusculus tttaccatacgttcgttgccctctgctacctgactccaattcttggagct 244 xm_007063 homosapiens tctaccatacgtttgtggctctgtgctacctgacgccaattctcggagct 209 ay027496ovis tctatcacacgttcgtcgccctgtgctacctgacgcccatcctcggagct 292 u13707 oryctolaguscunic tctaccacacgttcgtcgcgctgtgctacctcacgcccattctcggagct 217 sequencetosubmitgenbak tctaccacacgtttgtggctctgtgctacctcacgcccattctcggagct 169 ay029615 gallusgallus tctaccacacgtttgttgctctgtgctacttgacgcccatcctgggagcg 300 * ** ** ***** ** ** ** ****** * ** ** ** ** ** ** d50306rat ctgatcgcagactcgtggctggggaagttcaagacaattgtctgactatc 312 nm_053079 musmusculus ctgatcgcagactcgtggctggggaagttcaagacaattgtttcactatc 294 xm_007063 homosapiens cttatcgccgactcgtggctgggaaagttcaagacaattgtttcactctc 259 ay027496ovis ctcatcgccgactcctggctggggaagttcaagaccattgtgtcgctgtc 342 u13707 oryctolaguscunic ctcatcgccgacgcgtggctggggaagttcaagaccatcgtgtcgctgtc 267 sequencetosubmitgenbak ctgatcgcagactcctggctgggaaagttcaagacaatcgtgtcactctc 269 ayo29615 gallusgallus ctcattgcagactcatggctgggaaagtttaagaccattgtctccctgtc 350 ** ** ** *** * ******** ***** ***** ** ** * ** ** d50306rat catcgtctacacgatcggacaggccgtcatctcagtgagctcaattaatg 362 nm_053079 musmusculus catcgtctacacgattggacaagcagtcatctcggtgagctcaattaatg 344 xm_007063 homosapiens cattgtctacacaattggacaagcagtcacctcagtaagctccattaatg 309 ay027496ovis catcgtctacaccattgggcaggtagtcatcgctgtgagctcaattaatg 392 u13707 oryctolaguscunic catcgtctacaccatcggacaagcagtcacctccctcagctccgtcaatg 317 sequencetosubmitgenbak cattgtctacacaattggacaggcggtcactgcagtaagctcaattaatg 269 ay029615 gallusgallus cattgtctatacaattgggcaggcagtcatggctgtaagctccataaacg 400 *** ***** ** ** ** ** * **** * * ***** * ** * d50306rat accttacagaccatgaccacgacggcagtcctaacaaccttcctttgcac 412 nm_053079 musmusculus acctcacagaccacgaccacaatggcagtcctgacagccttcccgtgcac 394 xm_007063 homosapiens acctcacagaccacaaccatgatggcacccccgacagccttcctgtgcac 359 ay027496ovis acctcactgacttcaaccatgatggaaccccaaacaatatttctgtgcac 442 u13707 oryctolaguscunic agctcacagacaacaaccatgacgggacccccgacagcctccctgtgcac 367 sequencetosubmitgenbak acctcacagactataacaaagatggaactcctgacaatctgtccgtgcat 319 ay029615 gallusgallus acatgacagatcaaaacagagatggcaatcctgataatattgcggtgcac 450 * * ** ** ** * ** * ** * * * * **** d50306rat gtagcactgtccatgatcggcctggccctgatagcccttggtacaggagg 462 nm_053079musmusculus gtagcactgtccatggttggcctggccctgatagcccttggtacaggagg 444 xm_007063 homosapiens gtggtgctgtccttgatcggcctggccctgatagctctcgggactggagg 409 ay0274696ovis gtggcactctccatgattggcctggtcctgatagctctgggtaccggagg 492 u13707 oryctolaguscunic gtggcggtgtgcatgatcggcctgctcctgatagccctcgggacaggagg 417 sequencetosubmitgenbak gtggcactgtccatgattggcctggccctgatagctctgggaactggagg 369 ay029615 gallusgallus attgccctgtctatgactggcttgattctcatcgcgcttggaactggtgg 500 * * * * ** *** ** ** ** ** ** ** ** ** ** d50306rat aatcaagccctgtgtgtctgcatttggtggcgatcagtttgaagagggtc 512 nm_053079 musmusculus aatcaagccctgtgtgtctgcgtttggtggcgatcagtttgaagagggtc 494 xm_007063 homosapiens aatcaaaccctgtgtgtctgcgtttggtggagatcagtttgaagagggcc 459 ay027496ovis gataaagccttgcgtgtctgcatttggcggagatcagtttgaagagggcc 542 u13707oryctolaquscunic aatcaagccctgtgtgtctgcctttggcggcgatcagtttgaggagggcc 467 sequencetosubmitgenbak aataaagccctgtgtgtctgcatttggtggagaccagtttgaagagggcc 419 ay029615 gallusgallus gatcaaaccttgtgtctcagcatttggtggggatcagtttgaagaacatc 550 ** ** ** ** ** ** ** ***** ** ** ******** ** * d50306rat aggaaaaacagcgaaaccggttcttttccatcttttatttggctatcaac 562 nm_053079 musmusculus aggaaaaacagcgaaaccggttcttttccatcttttatttggctatcaac 544 xm_007063 homosapiens aggagaaacaaagaaacagatttttttccatcttttacttggctattaat 509 ay027496ovis aggaaaagcaaaggaacagatttttttccatcttttatttggccattaat 592 u13707 oryctolaguscunic aggaaaagcaaagaaaccggtttttttccatcttttacttggccattaac 517 sequencetosubmitgenbak aggaaaaacaaagaaacagattcttttccatcttttatttggccattaat 469 ay029615 gallusgallus aggaaaaacaaagaagtagattcttctctatcttttatttgtccattaat 600 **** ** ** * * * ** ** ** ******** *** * ** ** d50306rat gcaggaagcctgctctccacgatcatcactcccatactcagagttcagca 612 nm_053079 musmusculus gggggaagcctgctctccacgatcatcactcccatactcagagttcaaca 594 xm_007063 homosapiens gctggaagtttgctttccacaatcatcacacccatgctcagagttcaaca 559 ay027496ovis gctggaagtttgctttctactatcatcacccccatgctcagagttcaggt 642 u13707 oryctolaguscunic gctgggagtctgctgtccacaatcatcacccccatggtcagagttcaaca 567 sequencetosubmitgenbak gctggaagcttgatttccactattgtcactcccatgctcagagttcacga 519 ay029615 gallusgallus gctggaagtctcatatccactataatcaccccaattctcagagctcaaga 650 * ** ** * * ** ** ** **** ** ** ****** *** d50306rat gtgcggaatccacagccaacaagcttgttacccactggcctttggggttc 662 nm_053079 musmusculus gtgcggaatccacagtcaacaagcttgttacccactggccttcggggttc 644 xm_007063 homosapiens atgtggaattcacagtaaacaagcttgttacccactggcctttggggttc 609 ay027496ovis atgcggaattcacagtaagcaagcttgttaccccctggcctttggggttc 692 u13707 oryctolaguscunic atgtggaattcacgttaaacaagcttgctacccactggcctttgggattc 617 sequencetosubmitgenbak atgtggaatttacagtcagataagcttgttaccactggcatttggggttc 569 ay029615 gallusgallus atgtggcattcacagcagacagcagtgctacccgctggcatttggagttc 700 ** ** ** ** * ** ***** ***** ** ** *** d5030grat cggcagctctcatggctgttgccctaattgtgtttgtcctcggcagtgga 712 nm_053079 musmusculus cagcggctctcatggctgttgccctaattcgtgtttgtccttggcagtgga 694 xm_007063 homosapiens ctgctgctctcatggctgtagccctgattcgtgtttgtccttggcagtggg 659 ay027496ovis ctgctgcactcatggctgtatctctgatccgtgtttgtcattggcagtgga 742 u13707 oryctolaguscunic ctgctatcctcatggctgtatccctgatccgtgttcatcatcggcagtggg 667 sequencetosubmitgenbak ctgctgctctcatggccgtatctctgattcgtatttgtcattggcagtgga 619 ay029615 gallusgallus ccgctgccctcatggctgtttcattagttgtgttcatagctggaagtgga 750 * ** ******** ** * * * ** ** * ** ***** d50306rat atgtacaagaagtttcagccccagggcaacatcatgggcaaagtggccaa 762 nm_053079 musmusculus atgtacaagaagttccagccccagggcaacatcatgggcaaagtggccaa 744 xm_007063 homosapiens atgtacaagaagttcaagccacagggcaacatcatgggtaaagtggccaa 709 ay027496ovis atgtacaagaaggtccagccccagggtaacatcatgtctaaagttgccag 792 u13707 oryctolaguscunic atgtacaagaagttcaagccgcaggggaacatcctgagcaaagtggtgaa 717 sequencetosubmitgenbak atgtacaagaagtttcagccccagggtaatgtcatgggtaaagttgtcaa 669 ay029615 gallusgallus atgtacaaaaaagttcaaccgcaaggcaatataatggttcgagtttgtaa 800 ******** ** * * ** ** ** ** * ** *** * d50306rat gtgcattggctttgccatcaaaaacaggtttcggcaccgaagtaaggcat 812 nm_053079 musmusculus gtgcattggttttgccatcaaaaacaggtttcggcaccgaagtaaggcat 794 xm_007063 homosapiens gtgcatcggttttgccatcaaaaatagatttaggcatcggagtaaggcat 759 ay027496ovis gtgcattgggtttgccatcaaaaataggattagccatcggagtaagaaat 842 u13707 oryctolaguscunic gtgcatctgctttgccatcaaaaataggtttaggcgccgcagtaagcagt 767 sequencetosubmitgenbak gtgcattggttttgccctcaaaaataggtttaggcaccggagtaagcagt 719 ay029615 gallusgallus atgcattggatttgccattaaaaacaggtttcggcatcgcagcaaagagt 850 ***** * ****** * ***** ** ** * ** ** ** ** * d50306rat ttcccaagagggaacactggctggactgggctaaagagaaatacgatgag 862 nm_053079 musmusculus atcccaagagggagcactggctggactgggctaaagagaaatacgacgag 844 xm_007063 homosapiens ttcccaagagggagcactggctggactgggctaaagagaaatacgatgag 809 ay027496ovis ttcctaagagggagcactggctggactgggctagcgagaaatatgatgag 892 u13707 oryctolaguscunic ttcccaagagggcgcactggctggactgggctaaggagaaatacgacgag 817 sequencetosubmitgenbak ttcccaagagggagcactggctggactgggctaaagagaaatacgatgag 769 ay029615 gallusgallus atcccaaaagagagcactggctagactgggcaagcgagaagtatgataaa 900 *** ** ** * ******** ******** * ***** ** ** * d50306rat aggctcatctcgcagattaagatggtgacgaaggtgatgttcctgtacat 912 nm_053079musmusculs cggctcatctcacagattaagatggtcacgaaggtgatgttcctgttcat 894 xm_007063 homosapiens cggctcatctcccaaattaagatggttacgagggtgatgttcctgtatat 859 ay027496ovis cggctcatctctcaaattaagatggttacaagggtgatgttcctgtacat 942 u13707 oryctolaguscunic cggcttatcgcgcagatcaagatggttacgagggtgctgttcctgtacat 867 sequencetosubmitgenbak cggctcatctctcaaattaagatggtcacaaaagtgatgttcttgtacat 819 ay029615 gallusgallus cgactgattgctcagaccaagatggtgttgaaggtgcttttcctttacat 950 * ** ** * ** * ******** * *** * *** * * ** d50306rat tcccctccccatgttttgggccttgtttgaccagcagggttccaggtgga 962 nm_053079 musmusculus cccactccccatgttctggggcctgtttgaccaacaagggtccagatgga 944 xm_007063 homosapiens tccactcccaatgttctgggccttgtttgaccagcagggctccaggtgga 909 ay027496ovis tcctctccccatgttctgggccttgtttgatcagcagggctccaggtgga 992 u13707 oryctolaguscunic ccccactccccatgttctgggccttgtttgatcagcagggttccagatgga 917 sequencetosubmitgenbak cccactcccaatgttctgggccctgtttgaccagcagggctccaggtgga 869 ay029615 gallusgallus ccctctcccgatgttctgggcactttttgaccagcagggatcgagatgga 1000 ** ***** ***** **** * ***** ** ** ** ** ** **** d50306rat cactgcaagcaacgaccatcactgggaaaattggaacaattgagattcag 1012 nm_053079 musmusculus cactgcaagcaacgaccatgaatgggaaaattggagcaaatgaaattcag 994 xm_007063 homosapiens cactgcaggcaacaactatgtccgggaaaatcggagctcttgaaattcag 959 ay027496ovis cactgcaagcaacgaccatgagtgggaagattggaatcattgaaatccag 1042 u13707 oryctolaguscunic cgctgcaagcgacgaccatgtccgggagaattggaatccttgaaattcag 967 sequencetosubmitgenbak cactgcaagcaacagctatgagtgggaaaattggacttcttgaagttcag 919 ay029615 gallusgallus cactgcaagccacaactatggatggggactttggagctatgcagattcag 1050 * ***** ** ** * *** *** * *** * * *** d50306rat ccggaccagatgcagacggtgaacgccatcttgattgtcatcatggtccc 1062 nm_053079 musmusculus ccggaccagatgcagacggtgaatgccatcctgaatgtcaacaatggccc 1044 xm_007063 homosapiens cccgatcagatgcagaccgtgaacgccatcctgatcgtgatcatggtccc 1009 ay027496ovis ccggatcagatgcagacggtgaacgccatcctgatcgtcgtcatggtccc 1092 u13707 oryctolaguscunic ccggatcagatgcagactgtgaacaccatcttgattattatcctggtccc 1017 sequencetosubmitgenbak ccagatcagatgcagactgtgaatgccatcttgattgtcgtcatggtccc 969 ay029615 gallusgallus ccagaccaatgcagactgtcaatccaatcctgattataataatggtccc 1100 ** ** ** ******** ** ** * *** *** * * *** d50306rat cattgtggacgccgtggtgtatccgctcattgtggtttcaact 1112 nm_053079 musmusculus caatgtggacgccgttgtgtaccgctcaattgcaaaatgtggtttcaact 1094 xm_007063 homosapiens gatcttcgatgctgtgctgtaccctctcattgcaaaatgtggcttcaatt 1059 ay027496ovis catcgtggatgccgtggtatatcctctgatcgcaaagtgtggtttaaatt 1142 u13707 oryctolaguscunic catcatggacgccgtggtgtatcctctgattgcaaagtgtggcctcaact 1067 sequencetosubmitgenbak catcatggatgccgtggtgtaccctctgattgcaaaatgtggcttcaatt 1019 ay029615 gallusgallus agttgtagatgctgtgatttatcctttaatccagaaatgcaagatcaatt 1150 * ** ** ** * ** * ** ** ** * ** * d50306rat tcacctccctgaagaagatgaccgttgggatgttcctggcatccatggcc 1162 nm_053079 musmusculus tcacatccctgaagaagatgactgttgggatgttcctggcgtccatggcc 1144 xm_007063 homosapiens tcacctccttgaagaagatggcagttggcatggtcctggcctccatggcc 1109 ay027496ovis tcacctccctgaagaagatgaccgtcggcatgtttctggcctccatggct 1192 u13707 oryctolaguscunic tcacctctctgaagaagatgacgattgggatgttcctggcttccatggcc 1117 sequencetosubmitgenbak tcacctccttgaagaggatgacagttggaatgttcctggcttccatggcc 1069 ay029615 gallusgallus ttacgcccctgaggaggatcactgttggcatgttccttgctggtctggct 1200 * ** * *** ** *** * * ** *** * ** ** **** d50306rat tttgtggtggctgcaattgtgcaggtggaaatcgataaactcttccagt 1212 nm_053079 musmusculus tttgtggtggctgcaattgtgcaggtggaaatcgataaaactcttccagt 1194 xm_007063 homosapiens tttgtggtggctgccatcgtgcaggtggaaatcgataaaactcttccagt 1159 ay027496ovis ttcgtggcagctgccatcgtgcaggtggacattgacaaaactctgcccgt 1242 u13707 oryctolaguscunic ttcgtggcagctgcaatcctgcaggtggaaatcgataaaactcttcctgt 1167 sequencetosubmitgenbak ttcgtgatggcggcgattgttcagctggaaattgataaaactcttccagt 1119 ay029615 gallusgallus ttcgttgctgctgctcttttgcaagtgcaaatagataaaactcttccagt 1250 ** ** ** ** * * ** ** * ** ** ******** ** ** d50306rat cttccccagcggaaatcaagttcaaattaaggtcttgaacattggaaaca 1262 nm_053079 musmusculus cttccctggtggaaatcaagtccaaattaaggtcttgaacatcggaaaca 1244 xm_007063 homosapiens cttccccaaaggaaacgaagtccaaattaaagttttgaatataggaaaca 1209 ay027496ovis cttccccaaaggaaatgaagtccaaatcaaagtcctgaatataggaaata 1292 u13707 oryctolaguscunic cttccccaaagccaatgaagtccaaattaagttctgaatgtaggaagtg 1217 sequencetosubmitgenbak cttccccaaacaaaatgaagtccaaatcaaagtactgaatataggaaatg 1169 ay029615 gallusgallus tttccctgcagctggacaggcccaaatcaaaataataaatctaggtgata 1300 **** * * ***** ** * * ** * ** d50306rat atgacatggccgtgtattttcctggaaagaatgtgacagttgcccaaatg 1312 nm_053079 musmusculus ataacatgaccgtgcattttcctggaaaatagtgtgacgcttgcccaaatg 1294 xm_007063 homosapiens ataccatgaatatatctcttcctggagagatggtgacacttggcccaatg 1259 ay027496ovis atagcatgaccgtgtcttttcccggaacgacagcagtgacatgtgaccagatg 1342 u13707 oryctolaguscunic cagaacatgatcatctctcttcctgggcagacggtgacgctcaaccagatg 1267 sequencetosubmitgenbak gtgccatgaatgtatcttttcctggagcggtggtgacagttagccaaatg 1219 ay029615 gallusgallus gcaatgcgaatgt - tacatttctgcctaatcttcagaacgtgactgtcct 1349 * * ** * * * d50306rat tctca --- gacagacacatt - catgactttcgatgtagaccagctgacaa 1358 nm_053079 musmusculus tctca --- gacagacacgtt - catgactttcgatatagacaagctgacaa 1340 xm_007063 homosapiens tctca --- aacaaatgcatt - tatgacttttgatgtaaacaaactgacaa 1305 ay027496ovis tctca --- aacaaacggatt - tctgactttcaacgtagacaacct --- aa 1385 u13797 oryctolaguscunic tctca --- aacgaatgaatt - catgactttcaatgaagacacactgacaa 1313 sequencetosubmitgenbak agtca --- atcagatggatt - tatgacttttgatgtagacaaactgacaa 1265 ay029615 gallusgallus tcccatggagtcaacaggctacaggatgtgtttgagtcttcccagctaaaat 1399 ** * * ** ** * * ** * d50306rat gcataaacgtgtcttctcccgg - atctccaggcgtcaccacggtagctca 1407 nm_053079 musmusculus gcataaacatatcttcctctgg - atccccaggagtcaccacagtagctca 1389 xm_007063 homosapiens ggataaacatttcttctcctgg - atcaccag --- tcactgctgtaactga 1351 ay027496ovis gtataaacatttcttctactgg - aacaccag --- tcactccagtaactca 1431 u13707 oryctolaguscunic gcataacatcacttcc - gg - atcacaag --- tcaccatgatcacacc 1356 sequencetosubmitgenbak gtataaacatttcttccactgg - atcaccag --- tcaccatgatcacacc 1311 ay029615 gallusgallus ctgtaatggtaaattttgggagtgagagtagaagtgaaaatatcgactca 1449 *** * ** * ** * * * d50306rat - tgagtttgagccgggtcaccggcacacccttctagtgtggggccccaat 1456 nm_053079 musmusculus - tgattttgagcagggtcaccggcacaaccttctagtgtgggaacccagt 1438 xm_007063 homosapiens - cgacttcaagcagggccaacgccacacgcttctagtgtgggcccccaat 1400 ay027496ovis - taactttgagtccggccatcgccatacccttctcgtctgggccccaagt 1480 u13707 oryctolaguscunic - cagccttgaggcaggccagcgccacaccctgctggtgtgggcccccaat 1405 sequencetosubmitgenbak - taactttgagcagggccatcgccatacccttctagtatgggcccccaat 1360 ay029615 gallusgallus ataagcagcaatacgcatactgtcaccatcaagaatgcagcagccggcat 1499 * * * * ** * * * * d50306rat ctataccgtgtggtaaa - agacggtcttaaccaaaagccagagaaagggg 1505 nm_053079 musmusculus caataccgtgtggtaaa - agatggtcctaaccaaaagccagagaaagggg 1487 xm_007063 homosapiens cactaccaggtggtaaa - ggatggtcttaaccagaagccagaaaaagggg 1449 ay027496ovis aactaccaagtggtaaa - agatggccttaaccagaagccagaaaaaggga 1529 u13707 oryctolaguscunic aactaccgagtggtcaa - tgacggcctgacccagaagtcagacaaaggag 1454 sequencetosubmitgenbak aattaccgagtggtaaa - ggatggccttaaccagaagccagaaaaaggag 1409 ay029615 gallusgallus tgtttctagcttgcggtctgataatttcacatcaaaaccagaagaaggaa 1549 * * * * ** * ** **** **** d50306rat agaacggaatcagattcgtcagcacccttaacgagatgatcaccatcaaa 1555 nm_053079 musmusculus agaacggaatcaggtttgtcaacacccttaacgagatggtcaccaacaaa 1537 xm_007063 homosapiens aaaatggaatcagatttgtaaatacttttaacgagctcatcaccatcaca 1499 ay027496ovis gaaatggaatcagattcgttaatgcttttggcgagagcttcggcgtcaca 1579 u13707 oryctolaguscunic aaaatggaatcaggtttgtgaacacttacagccagcccatcaacgtcacg 1504 sequencetosubmitgenbak aaaatggaatcagatttataaatagtcttaatgagagcctcaacatcacc 1459 ay029615 gallusgallus agaatctagtcaggtttgtaaataatttgcctcagacagtcaacatcact 1599 ** * **** ** * * ** ** * ** d50306rat atgagtggaaaagtgtacgaaaatgtcaccagtcacag - cgccagcaact 1604 nm_053079 musmusculus atgagtgggaaagtatatgaaaaattcacaagtcacaa - cgccagcggct 1586 xm_007063 homosapiens atgagtgggaaagtttatgcaaacatcagcagctacaa - tgccagcacat 1548 ay027496ovis atggatggggaagtttacaacaatgtctccggtcacaa - tgccagtgaat 1628 u13707 oryctolaguscunic atgagcgggaaagtttacgaacacatcgccagctacaa - tgccagcgagt 1553 sequencetosubmitgenbak atgggcgacaaagtttatgtgaatgtcaccagtcacaa - tgccagcgagt 1508 ay029g15 gallusgallus atgggtgacacgacttttg - gaatactggaagagacaagtatcagtaatt 1648 *** * * * * *** *** * d50306rat atcagtttttcccttctggccaaaaagactacacaataaacaccacaga - 1653 nm_053079 musmusculus acaagttcctcccttctggcgaaaagcagtacacaataaacaccacggc - 1635 xm_007063 homosapiens accagttttttccttctggcataaaaggcttcacaataagctcaacaga - 1597 ay027496ovis atctttttttctcttctggcgtaaagagcttcacaataaactcaccaga - 1677 u13707 oryctolaguscunic atcagtttttcacttctggagtaaagggcttcaccgtcagctcggcagg - 1602 sequencetosubmitgenbak atcagttcttttctttgggcacaaaaaacattacaataagttcaacacaa 1558 ay029615 gallusgallus acagtccgttctcaggaggaagaacatatgatatagtgataactgcagg - 1697 * * * ** ** * * * * * d50306rat -- gattgcaccaaactgttcatctgattttaaatcttccaaccttgactt 1701 nm_053079 musmusculus -- ggtggcaccaacctgtctaactgattttaaatcttccaaccttgactt 1683 xm_007063 homosapiens -- gattccgccacaatgtcaacctaatttcaatactttctaccttgaatt 1645 ay027496ovis -- gatttcacaacagtgtgaaaaacagttcaaaacatcctaccttgaatt 1725 u13707 oryctolaguscunic -- catctcggagcagtgcaggcgggactttgagtctccgtacctggagtt 1650 sequencetosubmitgenbak cagatctcacaaaattgtacaaaagttctccaatcatccaaccttgaatt 1608 ay029615 gallusgallus ----- ttcaactaattgcaaacc - aacttcagag ----- aaattaggata 1736 * ** * * * * * d50306rat cggcagcgcgtacacctacgtgatcagaagtagggcgagtgatggctgcc 1751 nm_053079 musmusculus tggcagcgcgtatacctacgtgatccga --- agggcgagtgatggctgcc 1730 xm_007063 homosapiens tggtagtgcttatacctatatagtccaa --- aggaagaatgacagctgcc 1692 ay027496ovis tggtagtgcgtttacctatgtaatcagc --- agaaagagtgacggttgcc 1772 u13707 oryctolaguscunic tggcagcgcgtacacgtacctgatcacg --- agccaggctactggctgcc 1697 sequencetosubmitgenbak tggtagtgcatatacctatgtaatcgga --- acgcagagcactggctgcc 1655 ay029615 gallusgallus tggtggtgcttatacgatcgtaattaat --- gagtgttctggagatgtga 1783 ** * ** * ** * * d50306rat tggaagtgaaggaattcgaagacatcccacccaacacggtgaacatggcc 1801 nm_053079 musmusculus tggaagtgaaggaatttgaagacatcccacccaacactgtgaacatggct 1780 xm_007063 homosapiens ctgaagtgaaggtgtttgaagatatttcagccaacacagttaacatggct 1742 ay027496ovis ccgaaccaaagattttcgaagacatctcccccaacacagtcagcatggct 1822 u13707 oryctolaguscunic cccaagtgacggagtttgaagatattccgcccaacacaayhaacatggct 1747 sequencetosubmitgenbak ctgaattgcatatgtttgaagatatttcacccaacacagttaacatggct 1705 ay029615 gallusgallus ctcaattaagatacattgaagatatccaacccaatacagtccatatggct 1833 ** * ***** ** **** ** * **** d50306rat ctgcagatcccacagtacttcctcctcacctgcggcgaggtggtcttctc 1851 nm_053079 musmusculus ctgcagatcccacagtacttccttctcacctgcggcgaggtggtcttctc 1830 xm_007063 homosapiens ctgcaaatcccgcagtattttcttctcacctgtggcgaagtggtcttctc 1792 ay027496ovis ctgcagatcccccagtacttcctcctcacctgtggcgaggtggtcttctc 1872 u13707 oryctolaguscunic tggcaaatcccacagtacttcctcatcacctctggcgaggtggtcttctc 1797 sequencetosubmitgenbak ctgcagatcccgcagtacttcctcatcacctgcggcgaggtggttttctc 1755 ay029615 gallusgallus tggcagatccctcagtatttcatacttacatgtggagaagtagtcttctc 1883 *** ***** ***** ** * * ** * ** ** ** ** ***** d50306rat tgtcacaggactggagttctcctattcccaggccccgtctaacatgaagt 1901 nm_053079 musmusculus tgtcacaggactggagttctcttattcccaggctccgtctaacatgaagt 1880 xm_007063 homosapiens tgtcacgggattggaattctcatattctcaggctccttccaacatgaagt 1842 ay027496ovis catcaccggcctggagttctcctattctcaggctccttccaacatgaagt 1922 u13707 oryctolaguscunic catcacgggcctggagttctcctattctcaggctccttccaacatgaagt 1847 sequencetosubmitgenbak tgtcacaggactggagttctcatattctcaggccccctccaacatgaagt 1805 ay029615 gallusgallus tgtcactgggctggagttttcatactcacaggcaccatctaatatgaagt 1933 **** ** **** ** ** ** ** ***** ** ** ** ******* d50306rat ccgtgcttcaggcagoatggcttctaaccgtggccatcggtaatatcatt 1951 nm_053079 musmusculus ccgtgcttcaggcaggctggcttctaactgtggcggtcggcaatatcatt 1930 xm_007063 homosapiens cggtgcttcaggcaggatggctgctgaccgtggctgttggcaacatcatt 1892 ay027496ovis cggtacttcaggcaggatggctgttgaccgtggccgttggcaacatcatc 1972 u13707 oryctolaguscunic cggtgctgcaggaccggtggctgctgacggtggctgtgggcaacatcatt 1897 sequencetosubmitgenbak cggtgcttcaggcgggatggctgctgacagtggct --------------- 1840 ay029615 gallusgallus cagtgctgcaagcaggatggctgctaacagtggctgtcgcataacataatt 1983 * ** ** ** * * ***** * ** ***** d50306rat gtcctcattgtggctgaggcaggccacttcgacaaacagtgggctgagta 2001 nm_053079 musmusculus gtgctcatcgtggcaggggcggggcacttccccaaacagtgggctgagta 1980 xm_007063 homosapiens gtgctcatcgtggcaggggcaggccagttcagcaaacagtgggccgagta 1942 ay027496ovis gtgcttattgtggcaggagcaggccagttcagtgaacagtgggcccagta 2022 u13707 oryctolaguscunic gtgctcatcgtggccggcgcgggccagatcaacaagcagtgggccgagta 1947 sequencetosubmitgenbak -------------------------------------------------- ay029615 gallusgallus gtccttatcgtggctggagcatccaaactcagtgagcagtgggcagaata 2033 d50306rat tgttctgttcgcctccttgctcctggtcgtctgcatcatatttgccatta 2051 nm_053079 musmusculus cattctgtttgcctcattgcttctggttgtctgcgtgatattcgccatca 2030 xm_007063 homosapiens cattctatttgccgcgttgcttctggtcgtctctgtgtaatttttgccatca 1992 ay027496ovis cgttctgtttgcggcattgcttctggtcgtctgtgtaatatttgccatca 2072 u13707 oryctolaguscunic catcctctttgccgccctgctcctggtcgtcgtcatatttgccatca 1997 sequencetosubmitgenbak ----------------------------------------------- ay029615 gallusgallus tgttctctttgctgccttgctttttgcagtttgcattatttttgctgtca 2083 d50306rat tggcccgattctacacctacatcaacccagcagagatcgaggcacagttc 2101 nm_053079 musmusculus tggctcgattctacacctacatcaacccagcagagattgaagcacagttt 2080 xm_007063 homosapiens tggctcggttctatacttacatcaacccagcggagatcgaagctcaattt 2042 ay027496ovis tggctcgattctatacgtatgtcaaccccgcagagattgaagctcagttt 2122 u13707 oryctolaguscunic tggctcgattctatacgtatgtcaacccggccgagatcgaggctcagttt 2047 sequencetosubmitgenbak -------------------------------------------------- ay029615 gallusgallus tggcatatttttatacatatactgatccaaatgaggttgaagcccggctt 2133 d50306rat gatgaggatgagaagaaaaagggcgtagggaaggaa --- aacccgtattc 2148 nm_053079 musmusculus gatgaggatgagaagaaaaagggcataggaaaggaa --- aacccgtattc 2127 xm_007063 homosapiens gatgaggatgaaaagaaaaacagactggaaaagagt --- aacccatattt 2089 ay027496ovis gatgaggatgacaaggaggatgacctggaaaagagt --- aacccatacgc 2169 u13707 oryctolaguscunic gaagaagatgagaagaaaaagaacccagaaaagaac - gacctctaccc 2094 sequencetosubmitgenbak ------------------------------------------------ ay029615 gallusgallus gatgaagaagaaaagaaagaaacaaataaaacaggatccagacttgcacgg 2183 d50306rat ctcg --- ttggaacctgtctcacagacaaacatgtgaagatcagaaagca 2195 nm_053079 musmusculus ttca --- ttggaaccagtctcacagacaaatatgtgaagggcagaaggca 2174 xm_007063 homosapiens catg --- tcaggggccaattcacagpaacagatgtgaaggtcaggaggca 2136 ay027496ovis caag --- ctggacttcgtctcacagacacaaatgtgaatgtcaggaagca 2216 u13707 oryctolaguscunic ctcc --- gtggcgcccgtctcacagacacacagatgtga -- gtctggaggcg 2139 sequencetosubmitgenbak ---------------------------------------------------- ay029615 gallusgallus aaaagaatctgaagctgtctctcagatgtagaag - gtgtattcaagagca 2232 d50306rat agtggagaacataccaagtc -- cagcattcaccatgacctctgccc -- aa 2241 nm_053079 musmusculus aattggagaaagatcaagtt -- caacatgagccctgacctctgtcc -- aa 2220 xm_007063 homosapiens agtggaggatggactgggcc -- c - gcagatgccctgacctctgcccccag 2183 ay027496ovis agcggacgc - ggggctgggc -- cagggtgtgcccaggggtctgtcccatg 2263 u13707 oryctolaguscunic - gtgtagga - ggcccacgcc -- tggcgtgcactgtgacctctgtccga - g 2184 sequencetosubmitgenbak -------------------------------------------------- ay029615 gallusgallus tttgtaaatcatggtagcctgttaactgtccctgcaataacaggaatcag 2282 d50306rat gggacaggaccctccaccacagagtccttgctggagaaagacttcagaca 2291 nm_053079 musmusculus gggacaggacactccaccacagagtccctgatggagaaagacctcagaag 2270 xm_007063 homosapiens gtagcaggacactccattggatggcccctgatg - aggaagacttcagaat 2232 ay027496ovis ggggcaggacactctgttgggtggcctctgatg - gggaagacttcagaac 2312 u13707 oryctolaguscunic ggcgcaggacgtacccctgggcagccccggaag - gggaggacttgagaac 2233 sequencetosubmitgenbak -------------------------------------------------- ay029615 gallusgallus ggtattgctgacatcactgggtaatataccttgtgggagagactaagaaa 2332 d50306rat tgtgagccaaaataataacaaagcaggttttcaggctgacggctgtgaat 2341 nm_053079 musmusculus tgtgagccagaataataacaaagcaggttttctaaccaacagctgtgaac 2320 xm_007063 homosapiens tgggaactaaaccatgaatgc -- tattttcttttttctttttcttttctt 2280 ay027496ovis tgtggaccaaaccaagacagc -- tgctttctc - agcagccggcaatgaac 2359 u13707 oryctolaguscunic tgtgaaccagaccacgaaagc -- tatgttctg - agcagccagtgatgagt 2280 sequencetosubmitgenbak -------------------------------------------------- ay029g15 gallusgallus cactgttctgacttaacatac -- agcctcttgggaagcaagacgaaatg 2379 d50306rat ctgaaactctaggggagccttttt ------------------------------------ 2365 nm_053079 musmusculus ctgaaactctaggggagccttttttatttaaaaaaattttttttttaatt 2370 xm_007063 homosapiens tttttttttt ------- tttttttttttgagacagagttttgctcttgtt 2323 ay027496ovis ctgaaactccaaaagacgtcctttt -------------------------- 2384 u13707 oryctolaguscunic ccaaaactctgaaagaaatcttgtt ------------------------- 2305 sequencetosubmitgenbak -------------------------------------------------- ay029615 gallusgallus attaatctcttgtacagaagctggc ------------------------- 2404 d50306rat --------------------------------------------------- nm_053079 musmusculus ttttaaattttttttatttttatttttttttcgttgtttgtttgtttcga 2420 xm_007063 homosapiens gtccaggctggagtgcaatggcacgatctcagctcactgc --------- a 2364 ay027496ovis -------------------------------------------------- u13707 oryctolaguscunic -------------------------------------------------- sequencetosubmitgenbak -------------------------------------------------- ay029615 gallusgallus -------------------------------------------------- d50306rat -------------------------------------------------- nm_053079 musmusculus gacagggtttctcgtgtgtagcccttggttgtcctggaactcactctgta 2470 xm_007063 homosapiens acctccgcctcccaggttcaagtaattctcctgcctcagcctcccgagtg 2414 ay027496ovis -------------------------------------------------- u13707 oryctolaguscunic -------------------------------------------------- sequencetosubmitgenbak -------------------------------------------------- ay029615 gallusgallus -------------------------------------------------- d50306rat -------------------------------------------------- nm_053079 musmusculus gaccagactggcctcaaactcagaaatccacctgcccctgcccctgcccc 2520 xm_007063 homosapiens gctgggattagcggca ---------------------------------- 2430 ay027496ovis -------------------------------------------------- u13707 oryctolaguscunic -------------------------------------------------- sequencetosubmitgenbak -------------------------------------------------- ay029615 gallusgallus -------------------------------------------------- d50306rat -------------------------------------------------- nm_053079 musmusculus tgcccctgcccctgcccctgcctctgcctctgcctcccaagtgctggatt 2570 xm_007063 homosapiens ------ tgcaccaccacgcccagctatttttgtatttttagtagagat -- 2472 ay027496ovis -------------------------------------------------- u13707 oryctolaguscunic --------------------------------------------------- sequencetosubmitgenbak --------------------------------------------------- ay029615 gallusgallus -------------------------------------------------- d50306rat ----------------------------- aatttgtttttcttgagacaa 2386 nm_053079 musmusculus tggaggcatgcaccaccatgcccagctataattttttttttttaagacag 2620 xm_007063 homosapiens --- ggggtttcaccatgttggccagg - atggtctcgatctcttgacctgg 2518 ay027496ovis -------------------------------------------------- u13707 oryctolaguscunic --------------------------------------------------- sequencetosubmitgenbak --------------------------------------------------- ay029615 gallusgallus --------------------------------------------------- d50306rat ggtatctctgtgtaaccctggctatcctggaactcactctatagaccagg 2436 nm_053079 musmusculus ggattctctgtataagcctgactgccctgcaacttgctctatagaccagg 2670 xm_007063 homosapiens tga --- tctgcccacctcggcctgccaaagtgctgggattacaggcttga 2565 ay027496ovis --------------------------------------------------- u13707 oryctolaguscunic --------------------------------------------------- sequencetosubmitgenbak --------------------------------------------------- ay029615 gallusgallus -------------------------- atcctgaggaaactcctgcagaatttg 2431 d50306rat ctggcctcgaactcacagatatctgtctgtctgcctctgcctcctaagtactgg 2486 nm_053079 musmusculus ctggccttgaactcacagagatctgcctgcctgcctcttcctcctaagtactgg 2720 xm_007063 homosapiens gctaccgcgcccggccgtgaacgctattttctaagcagcc -- agcagtga 2613 ay027496ovis ------------------------ gtttgtttgtttttag -- agaagtct 2408 u13707 oryctolaguscunic ------------------------ g ------------------ aaagtct 2313 sequencetosubmitgenbak -------------------------------------------------- ay029615 gallusgallus cactcttaaaatgtacctcaagctcaataccatagcataa - aaatattga 2480 d50306rat gattcaaggcatgtacggcaactgcccagctaaaatattatttataacat 2536 nm_053079 musmusculus gatttcaggcatgcaccacaactgcccagctaaaatattatttataatat 2770 xm_007063 homosapiens atctaaaactctggaagaagtcttctgtttgaaaggcttatttaagccac 2663 ay027496ovis tatttaaagcgcacac - acacgcacacgcacaca ------------- cat 2444 u13707 oryctolaguscunic tatttaaaacacacac - acacacacacacacaca ------------- cac 2349 sequencetosubmitgenbak -------------------------------------------------- ay029615 gallusgallus aattgcacttggcactattagacactctaaaaagatgtatttt ---- tat 2526 d50306rat gcactttctgggttttttgtttttaaaacatacttttttttttaacactg 2586 nm_053079 musmusculus gcactttctgg ---- tttgtttttg -------- tttttcttttaa - actg 2807 xm_007063 homosapiens acgtacacaca ----- ctgtcttaga ------- gtactgtgagcccaccc 2701 ay027496ovis gcacacacaca ------ cacttttat ---------- aagagtccatactc 2478 u13707 oryctolaguscunic acacacttttc ------ caacactg ------------ acagcctac --- c 2378 sequencetosubmitgenbak -------------------------------------------------- ay029615 gallusgallus actgtatttcaattttataatgtggaggggtggggaaaaaggtgttgcca 2576 d50306rat ggccatttctaacatttctgccacagaagtggatttagctcagattaa -- 2634 nm_053079 musmusculus ggctgtatcttacatttctgccacagaaatgaacttagctcagattaact 2857 xm_007063 homosapiens cacattggtcatcttccctatcacacaaatgatgttattttggactagct 2751 ay027496ovis tgcctgaactccttttcctaacacacaaataaagttattttggactaact 2528 u13707 oryctolaguscunic catgttaactccttctctaccaatgcaaatgctgttattttggactaact 2428 sequencetosubmitgenbak -------------------------------------------------- ay029615 gallusgallus agaaatagtaattgaagccaaactgtctgcgtgacccttctagcctcact 2626 d50306rat ----- ttttgaaaaggtaacagtactgttttttt ----------- tcctt 2668 nm_053079 musmusculus t -- aattttgaaaaggcaatagtattgtttttt --------------- ct 2890 xm_007063 homosapiens t -- aattttgaaatggtaacaaagtttcctactgttcatttct 2799 ay027496ovis tgaatttttgaaatggtggccaagctccatacgt ----------- gcatt 2567 u13707 oryctolaguscunic t - aattttgaacactgtt - ctatgttgcttgtat ----------- tc -- t 2463 sequencetosubmitgenbak -------------------------------------------------- ay029615 gallusgallus gttacttgaaagcaggtcac - atgtgccttaaatt --------- cttttc 2666 d50306rat aatgctctta - tgaaaacaatgttgaa ----------------- tttaca 2700 nm_053079 musmusculus aacagtttta - tgaaaacaatattgaa ----------------- tttaca 2922 xm_007063 homosapiens aatactctta - cgaaaactattctaaaggaggcaggagccaaggccaaaa 2848 ay027496ovis cgcacactctgtgcaaacaatgttaaaggaggcaaaaagtga ---- atgg 2613 u13707 oryctolaguscunic aacatccttaggaaaggcaatgttaagagaggcaggaggcaatgccaaag 2513 sequencetosubmitgenbak -------------------------------------------------- ay029615 gallusgallus tatgtcctta --- agaataataggagaaag ---------------- gttc 2697 d50306rat gagggctt ------- ttttagcagtgtgtagtgagtgtcagctgattcga 2743 nm_053079 musmusculus gagggctt ------- ttttaatagtgtgtaatgagtatcaactgattcaa 2965 xm_007063 homosapiens gtgaacgtacagg -- tttgaaatggctgtgataaggaccagctggtatta 2896 ay027496ovis ttggggcttttga - atagtacgtgttcataataaggaccggctggtatta 2662 u13707 oryctolaguscunic ttgaatatgtaggtgtcagaatggtatataccacatattacttagtatta 2563 sequencetosubmitgenbak -------------------------------------------------- ay029615 gallusgallus ttagatttc ------ tcagattaaaatgt - ctctgctccacatagcagga 2740 d50306rat gctaataaccttacctcggggttttt ---------------------- gt 2771 nm_053079 musmusculus gctaattgctttaccttggggtttttttgtttgtttgtttgtttgtttgt 3015 xm_007063 homosapiens actgataactttacctttgggttttt ---------------------- gt 2924 ay027496ovis actgataactctaccttctgttttta ----------------------- 2688 u13707 oryctolaguscunic actgaaaacctcaactttgaggtttt ---------------------- 2589 sequencetosubmitgenbak ------------------------------------------------ ay029615 gallusgallus acttggacatgcactgtgatgtgctt ---------------------- t 2767 d50306rat ttctttgttttcctggtctcctttgcctgacctctttttaaattatgtgt 2821 nm_053079 musmusculus ttgtttgtttttctagtctcctttgccttacctctttttaaattatgtgt 3065 xm_007063 homosapiens tattttgtttttctagtccct -------- acctgtgtttaaattatggat 2966 ay027496ovis - gttctgttttt - ccattccct ------- acctctttgtaaattatggat 2729 u13707 oryctolaguscunic - gttctattttttccactcctt ------- acctctttttaacctgtggac 2631 sequencetosubmitgenbak -------------------------------------------------- ay029615 gallusgallus atgtgcctattattaactgcccattggtatgttcttaattaattgtgt - t 2816 d50306rat aa --- ttcaaaagactattcaagtgat - ggttagtcatgagtcgt -- gac 2865 nm_053079 musmusculus aa --- ttcaaaagacta ---------------- gtcatgagttgt -- gaa 3094 xm_007063 homosapiens aa --- ctcgaaagacagctcaggtgaa - ggccagtaatgatttttttgaa 3012 ay027496ovis taacctttgaaaaaccactcaggtaaa - ggcaagtcatgattttt - gga 2776 u13707 oryctolaguscunic aa -- ctcaaaaggaccactcagataaa - ggccagtaaagattttt -- ttt 2676 sequencetosubmitgenbak -------------------------------------------------- ay029615 gallusgallus aa ---- tgggatgtccactgaggtgaacagacaatggcaaattatatttt 2862 d50306rat gtttgactggtgtgaagtaaattcttgttcttaag --------------- 2900 nm_053079 musmusculus gtttcactggtctgaaataaattctagttcttaa ---------------- 3128 xm_007063 homosapiens gtttcaatggtgtgaaataaatttctgttctta ----------------- 3045 ay027496ovis gtctcaacggtatgaaataaactctcattctcaagaaaaaaaaaaaaaaa 2826 u13707 oryctolaguscunic gccgttttg -- atgaaataaaataatgttcctaag --------------- 2709 sequencetosubmitgenbak -------------------------------------------------- ay029615 gallusgallus gaataaccaccaagaataaaacttgtgttgtaacaaaaaaaaaaaaaaaa 2912 d50306rat --- nm_053079 musmusculus --- xm_007063 homosapiens --- ay027496ovis aaa 2829 u13707 oryctolaguscunic --- sequencetosubmitgenbak --- ay029615 gallusgallus aa - 2914 alignment of nucleotides full length sequence of canine and human sequence 1 : sequencetosubmitgenbank 1840 bp sequence 2 : xm_0070g3 homosapiens 3045 bp start of pairwise alignments aligning . . . sequences ( 1 : 2 ) aligned . score : 85 guide tree file created : [/ net / nfs0 / vol1 / production / w3nobody / tmp / 305133 . 38341 - 239044 . dnd ] start of multiple alignment there are 1 groups aligning . . . group 1 : sequences : 2 score : 31290 alignment score 10725 clustal - alignment file created [/ net / nfs0 / vol1 / production / w3nobody / tmp / 305133 . 88341 - 239044 . aln ] your multiple sequence alignment : 305133 . 88341 - 239044 . aln clustal w ( 1 . 81 ) multiple sequence alignment ( seq id no : 7 ) sequencetosubmitgenbank ---------------------------------------- catcttcttc 10 ( seq id no : 1 ) xm_007063 homosapiens gaatgtccaaatcacacagtttctttggttatcccctgagcatcttcttc 50 ********** sequencetosubmitgenbank atcgtggtcaatgagttctgtgaaagattttcctactatggaatgagagc 60 xm_007063 homosapiens atcgtggtcaatgagttttgcgaaagattttcctactatggaatgcgagc 100 ***************** ** ************************ **** sequencetosubmitgenbank actcctgattctgtacttcagacggttcatcgggtgggacgataatctgt 110 xm_007063 homosapiens aatcctgattctgtacttcacaaatttcatcagctgggatgataacctgt 150 * ****************** * ****** * ***** ***** **** sequencetosubmitgenbank ccacggccatctaccacacgtttgtggctctgtgctacctgacgccgatc 160 xm_007063 homosapiens ccaccgccatctaccatacgtttgtggctctgtgctacctgacgccaatt 200 **** *********** ***************************** ** sequencetosubmitgenbank ctcggcgcactgatcgcagactcctggctgggaaagttcaagacaatcgt 210 xm_007063 homosapiens ctcggagctcttatcgccgactcgtggctgggaaagttcaagaccattgt 250 ***** ** ** ***** ***** ******************** ** ** sequencetosubmitgenbank gtcactctccattgtctacacaattggacaggtcactgcagtaagct 260 xm_007063 homosapiens gtcgctctccattgtctacacaattggacaagcagtcacctcagtaagct 300 *** ************************** ** ***** ********* sequencetosubmitgenbank caattaatgacctcacagactataacaaagatggaactcctgacaatctg 310 xm_007063 homosapiens ccattaatgacctcacagaccacaaccatgatggcacccccgacagcctt 350 * ****************** * *** * ***** ** ** **** ** sequencetosubmitgenbank tccgtgcatgtggcactgtccatgattggcctggccctgatagctctggg 360 xm_007063 homosapiens cctgtgcacgtggtgctgtccttgatcggcctggccctgatagctctcgg 400 * ***** **** ****** **** *********************** ** sequencetosubmitgenbank aactggaggaataaagccctgtgtgtctgcatttggtggagaccagtttg 410 xm_007063 homosapiens gactggaggaatcaaaccctgtgtgtctgcgtttggtggagatcagtttg 450 *********** ** ************** *********** ******* sequencetosubmitgenbank aagagggccaggaaaaacaaagaaacagattcttttccatcttttatttg 450 xm_007063 homosapiens aagagggccaggagaaacaaagaaacagatttttttccatcttttacttg 500 ************* ***************** ************** *** sequencetosubmitgenbank gccattaatgctggaagcttgatttccactattgtcactcccatgctcag 510 xm_007063 homosapiens gctattaatgctggaagtttgctttccacaatcatcacacccatgctcag 550 ** ************** *** ******* ** **** *********** sequencetosubmitgenbank agttcacgaatgtggaatttacagtcagaaagcttgttacccactggcat 560 xm_007063 homosapiens agttcaacaatgtggaattcacagtaaacaagcttgttacccactggcct 600 ****** *********** ***** * ******************* * sequencetosubmitgenbank ttggggttcctgctgctctcatggccgtatctctgattgtatttgtcatt 610 xm_007063 homosapiens ttggggttcctgctgctctcatggctgtagccctgattgtgtttgtcctt 650 ************************* *** * ******** ****** ** sequencetosubmitgenbank ggcagtggaatgtacaagaagtttcagcccagggtaatgtcatgggtaa 660 xm_007063 homosapiens ggcagtgggatgtacaagaagttcaagccacagggcaacatcatgggtaa 700 ******** ************** **** ***** ** ********** sequencetosubmitgenbank agttgtcaagtgcattggttttgccctcaaaaataggtttaggcaccgga 710 xm_007063 homosapiens agtggccaagtgcatcggttttgccatcaaaaatagatttaggcatcgga 750 *** * ********* ********* ********** ******** **** sequencetosubmitgenbank gtaagcagtttcccaagagggagcactggctggactgggctaaagagaaa 760 xm_007063 homosapiens gtaaggcatttcccaagagggagcactggctggactgggctaaagagaaa 800 ***** ****************************************** sequencetosubmitgenbank tacgatgagcggctcatctctcaaattaagatggtcacaaaagtgatgtt 810 xm_007063 homosapiens tacgatgagcggctcatctcccaaattaagatggttacgagggtgatgtt 850 ******************** ***************** ** * ******** sequencetosubmitgenbank cttgtacatcccactcccaatgttctgggccctgtttgaccagcagggct 860 xm_007063 homosapiens cctgtatattccactcccaatgttctgggccttgtttgaccagcagggct 900 * **** ** ********************* ****************** sequencetosubmitgenbank ccaggtggacactgcaagcaacagctatgagtgggaaaattggacttctt 910 xm_007063 homosapiens ccaggtggacactgcaggcaacaactatgtccgggaaaatcggagctctt 950 **************** ****** ***** ******** *** **** sequencetosubmitgenbank gaagttcagccagatcagatgcagactgtgaatgccatcttgattgtcgt 960 xm_007063 homosapiens gaaattcagcccgatcagatgcagaccgtgaacgccatcctgatcgtgat 1000 *** ******* ************** ***** ****** **** ** * sequencetosubmitgenbank catggtccccatcatggatgccgtggtgtaccctctgattgcaaaatgtg 1010 xm_007063 homosapiens catggtcccgatcttcgatgctgtgctgtaccctctcattgcaaaatgtg 1050 ********* *** * ***** *** ********** ************* sequencetosubmitgenbank gcttcaatttcacctccttgaagaggatgacagttggaatgttcctggct 1060 xm_007063 homosapiens gcttcaatttcacctccttgaagaagatggcagttggcatggtcctggcc 1100 ************************ **** ******* *** ******* sequencetosubmitgenbank tccatggccttcgtgatggcggcgattgttcagctggaaattgataaaac 1110 xm_007063 homosapiens tccatggcctttgtggtggctgccatcgtgcaggtggaaatcgataaaac 1150 *************** *** **** ** ** ** *** ******* ******** sequencetosubmitgenbank tcttccagtcttccccaaacaaaatgaagtccaaatcaaagtactgaata 1160 xm_007063 homosapiens tcttccagtcttccccaaaggaaacgaagtccaaattaaagttttgaata 1200 ******************* *** *********** ***** ****** sequencetosubmitgenbank taggaaatggtgccatgaatgtatcttttcctggagcggtggtgacagtt 1210 xm_007063 homosapiens taggaaacaataccatgaatatatctcttcctggagagatggtgacactt 1250 ******* * ******** ***** ********* * ******** ** sequencetosubmitgenbank agccaaatgagtcaatcagatggatttatgacttttgatgtagacaaact 1260 xm_007063 homosapiens ggcccaatgtctcaaacaaatgcatttatgacttttgatgtaaacaaact 1300 *** **** **** ** *** ******************* ******* sequencetosubmitgenbank gacaagtataaacatttcttccactggatcaccagtcattccagtgactt 1310 xm_007063 homosapiens gacaaggataaacatttcttctcctggatcaccagtcactgctgtaactg 1350 ****** ************** *************** * * ** *** sequencetosubmitgenbank ataactttgagcagggccatcgccatacccttctagtatgggcccccaat 1360 xm_007063 homosapiens acgacttcaagcagggccaacgccacacgcttctagtgtgggcccccaat 1400 * **** ********** ***** ** ******** ************ sequencetosubmitgenbank aattaccgagtggtaaaggatggccttaaccagaagccagaaaaaggaga 1410 xm_007063 homosapiens cactaccaggtggtaaaggatggtcttaaccagaagccagaaaaagggga 1450 * **** ************** *********************** ** sequencetosubmitgenbank aaatggaatcagatttataaatagtcttaatcttaatgagagcctcaacatcacca 1460 xm_007063 homosapiens aaatggaatcagatttgtaaatacttttaacgacgctcatcaccatcacaa 1500 **************** ****** * **** *** * *** ****** * sequencetosubmitgenbank tgggcgacaaagtttatgtgaatgtcaccagtcacaatgccagcgagtat 1510 xm_007063 homosapiens tgagtgggaaagtttatgcaaacatcagcagctacaatgccagcacatac 1550 ** * * ********** ** *** *** *********** ** sequencetosubmitgenbank cagttcttttctttgggcacaaaaaacattacaataagttcaacacaaca 1560 xm_007063 homosapiens cagttttttccttctggcataaaaggcttcacaataagctcaacat --- a 1597 ***** *** *** **** **** * * ******** ****** * sequencetosubmitgenbank gatctcacaaaattgtacaaaagttctccaatcatccaaccttgaatttg 1610 xm_007063 homosapiens gattccgccacaatgtcaacctaatttcaatactttctaccttgaatttg 1647 *** * * * * *** * * ** * * * * ************ sequencetosubmitgenbank gtagtgcatatacctatgtaatcggaacgcagagcactggctgccctgaa 1660 xm_007063 homosapiens gtagtgcttatacctatatagtccaaaggaagaatgacagctgccctgaa 1697 ******* ********* ** ** ** * *** *********** sequencetosubmitgenbank ttgcatatgtttgaagatatttcacccaacacagttaacatggctctgca 1710 xm_007063 homosapiens gtgaaggtgtttgaagatatttcagccaacacagttaacatggctctgca 1747 ** * ***************** ************************* sequencetosubmitgenbank gatcccgcagtacttcctcatcacctgcggcgaggtggttttctctgtca 1760 xm_007063 homosapiens aatcccgcagtattttcttctcacctgtggcgaagtggtcttctctgtca 1797 *********** ** ** ******* ***** ***** ********** sequencetosubmitgenbank caggactggagttctcatattctcaggccccctccaacatgaatcggtg 1810 xm_007063 homosapiens cgggattggaattctcatattctcaggctccttccaacatgaagtcggtg 1847 * *** **** ***************** ** ****************** sequencetosubmitgenbank cttcaggcgggatggctgctgacagtggct -------------------- 1840 xm_007063 homosapiens cttcaggcaggatggctgctgaccgtggctgttggcaacatcattgtgct 1987 ******** ************** ****** sequencetosubmitgenbank -------------------------------------------------- xm_007063 homosapiens catcgtggcaggggcaggccagttcagcaaacagtgggccgagtacattc 1947 sequencetosubmitgenbank -------------------------------------------------- xm_007063 homosapiens tatttgccgcgttgcttctggtcgtctgtgtaatttttgccatcatggct 1997 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens cggttctatacttacatcaacccagcggagatcgaagctcaatttgatga 2047 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens ggatgaaaagaaaaacagactggaaaagagtaacccatatttcatgtcag 2097 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens gggccaattcacagaaacagatgtgaaggtcaggaggcaagtggaggatg 2147 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens gactgggcccgcagatgccctgacctctgcccccaggtagcaggacactc 2197 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens cattggatggcccctgatgaggaagacttcagaattgggaactaaaccat 2247 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens gaatgctattttcttttttctttttcttttcttttttttttttttttttt 2297 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens ttttgagacagagttttgctcttgttgtccaggctggagtgcaatggcac 2347 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens gatctcagctcactgcaacctccgcctcccaggttcaagtaattctcctg 2397 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens cctcagcctcccgagtggctgggattagcggcatgcaccaccacgcccag 2447 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens ctatttttgtatttttagtagagatggggtttcaccatgttggccaggat 2497 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens ggtctcgatctcttgacctggtgatctgcccacctcggcctgccaaagtg 2547 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens ctgggattacaggcttgagctaccgcgcccggccgtgaacgctattttct 2597 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens aagcagccagcagtgaatctaaaactctggaagaagtcttctgtttgaaa 2647 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens ggcttatttaagccacacgtacacacactgtcttagagtactgtgagccc 2697 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens accccacattggtcatcttccctatcacacaaatgatgttattttggact 2747 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens agcttaattttgaaatggtaacaaagtttcctattccatactgttgattt 2797 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens ctaatactcttacgaaaactattctaaaggaggccaggagccaaggccaaa 2847 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens agtgaacgtacaggtttgaaatggctgtgataaggaccagctggtattaa 2897 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens ctgataactttacctttgggtttttgttattttgtttttctagtccctac 2947 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens ctgtgtttaaattatggataactcgaaagacagctcaggtgaaggccagt 2997 sequencetosubmitgenbank --------------------------------------------------- xm_007063 homosapiens aatgatttttttgaagtttcaatggtgtgaaataaatttctgttctta 3045 protein sequence of canine 5 ′ 3 ′ frame 2 catcttcttcatcgtggtcaatgagttctgtgaaagattttcctactatggaatgagagca ( seq id no : 8 ) i f f i v v n e f c e r f s y y g m r a ( seq id no : 13 ) ctcctgattctgtacttcagacggttcatcgggtgggacgataatctgtccacggccatc l l i l y f r r f i g w d d n l s t a i taccacacgtttgtggctctgtgctacctgacgccgatcctcggcggcactgatcgcagac y h t f v a l c y l t p i l g a l i a d tcctggctgggaaagttcaagacaatcgtgtcactctccattgtctacacaattggacag s w l g k f k t i v s l s i v y t i g q gcggtcactgcagtaagctcaattaatgacctcacagactataacaaagatggaactcct a v t a v s s i n d l t d y n k d g t p gacaatctgtccgtgcatgtggcactgtccatgattggcctggccctgatagctctggga d n l s v h v a l s m i g l a l i a l g actggaggaataaagccctgtgtgtctgcatttggtggagaccagtttgaagagggccag t g g i k p c v s a f g g d q f e e g q gaaaaacaaagaaacagattcttttccatcttttatttggccattaatgctggaagcttg e k q r n r f f s i f y l a i n a g s l atttccactattgtcactcccatgctcagagttcacgaatgtggaatttacagtcagaaa i s t i v t p m l r v h e c c i y s o k gcttgttacccactggcatttggggttcctgctgctctcatggccgtatctctgattgta a c y p l a f g v p a a l m a v s l i v tttgtcattggcagtggaatgtacaagaagtttcagccccagggtaatgtcatgggtaaa f v i g s g m y k k e q p q c n v m g k gttgtcaagtgcattggttttgccctcaaaaataggtttaggcaccggagtaagcagttt v v k c i g f a l k n r f r h r s k q f cccaagagggagcactggctggactgggctaaagagaaatacgatgagcggctcatctct p k r e h w l d w a k e k y d e r l i s caaattaagatggtcacaaaagtgatgttcttgtacatcccactcccaatgttctgggcc q i k m v t k v m f l y i p l p m f w a ctgtttgaccagcagggctccaggtggacactgcaagcaacagctatgagtgggaaaatt l f d q q g s r w t l q a t a m s g k i ggacttcttgaagttcagccagatcagatgcagactgtgaatgccatcttgattgtcgtc g l l e v q p d q m q t v n a i l i v v atggtccccatcatggatgccgtggtgtaccctctgattgcaaaatgtggcttcaatttc m v p i m d a v v y p l i a k c g f n f acctccttgaagaggatgacagttggaatgttcctggcttccatggccttcgtgatggcg t s l k r m t v g m f l a s m a f v m a gcgattgttcagctggaaattgataaaactcttccagtcttccccaaacaaaatgaagct a i v q l e i d k t l p v f p k q n e v caaatcaaagtactgaatataggaaatggtgccatgaatgtatcttttcctggagcggtg q i k v l n i g n g a m n v s f p g a v gtgacagttagccaaatgagtcaatcagatggatttatgacttttgatgtagacaaactg v t v s q m s q s d g e m t f d v d k l acaagtataaacatttcttccactggatcaccagtcattccagtgacttataactttgag t s i n i s s t g s p v i p v t y n f e cagggccatcgccatacccttctagtatgggcccccaataattaccgagtggtaaaggat q g h r h t l l v w a p n n y r v v k d ggccttaaccagaagccagaaaaaggagaaaatggaatcagatttataaatagtcttaat g l n q k p h k g e n g i r f i n s l n gagagcctcaacatcaccatgggcgacaaagtttatgtgaatgtcaccagtcacaatgcc e s l n i t m g d k v y v n v t s h n a agcgagtatcagttcttttctttgggcacaaaaaacattacaataagttcaacacaacag s e y q f f s l g t k n i t i s s t q q atctcacaaaattgtacaaaagttctccaatcatccaaccttgaatttggtagtgcatat i s q n c t k v l q s s n l e e g s a y acctatgtaatcggaacgcagagcactggctgccctgaattgcatatgtttgaagatatt t y v i g t q s t g c p e l h m f e d i tcacccaacacagttaacatggctctgcagatcccgcagtacttcctcatcacctgcggc s p n t v n m a l q i p q y f l i t c g gaggtggttttctctgtcacaggactggagttctcatattctcaggccccctccaacatg e v v f s v t g l e f s y s q a p s n m aagtcggtgcttcaggcgggatggctgctgacagtggcttgttggcaacatcattgtgct k s v l q a g w l l t v a c w q h h c a cattgtggcaggagcaggccagttcagtgaacagtgggctgaatacatcctatttgcggc h c g r s r p v q - t v g - i h p i c g attgcttctggttgtctgtgtaatatttgccatcatggcccggttttacacttacgtcaa i a s g c l c n i c h h g p v l h l r q tccagcagagattg s s r d 5 ′ 3 ′ frame 2 ( seq id no : 13 ) iffivvnefcerfsyygmrallilyfrrfigwddnlstaiyhtfvalcyltpilgaliad swlgkfktivslsivytigqavtavssindltdynkdgtpdnlsvhvalsmiglalialg tggikpcvsafggdqfeegqekqrnrffsifylatnagslistivtpmlrvhecgiysqk acyplafgvpapimavslivfvigsgmykkfqpqgnvmgkvvkcigfalknrfrhrskqf pkrehwldwakekyderlisqiknvtkvmflyiplrmfwalfdqqgsrwtlqatamsgki gllevqpdqmqtvnailivvmvpimdavvypliakcgfnftslkrmtvgmflasmafvma aivqletdktlpvfpkqnevqikvlnigngamnvsfpgavvtvsqmsqsdgfmtfdvdkl tsinisstgspvipvtynfeqghrhtllvwapnnyrvvkdglnqkpekgengirfinsln eslnitmgdkvyvnvtshnaseyqffslgtknitisstqqisqnctkvlqssnlefgsay tyvigtqstgcpelhmfedispntvnmalqipqyflitcgevvfsvtglefsysqapsnm ksvlqagwlltvacwqhhcahcgrsrpvq - tvg - ihpicgiasgclcnichhgpvlhlrq ssrd multiple alignment of amino - acid sequences sequence 1 : caninesubmitted 662 aa sequence 2 : xm_007063 homosapiens proteinseq 706 aa sequence 3 : ds0306ratproteinsequence 710 aa sequence 4 : nm_053079 musmusculus proteinseq 709 aa sequence 5 : ay027496ovis 707 aa sequence 6 : u13707 oryctolaguscunic proteins 707 aa sequence 7 : ay029615 gallusgallus proteinseq 714 aa start of pairwise alignments aligning . . . sequences ( 1 : 2 ) aligned . score : 76 sequences ( 2 : 3 ) aligned . score : 84 sequences ( 3 : 4 ) aligned . score : 91 sequences ( 4 : 5 ) aligned . score : 80 sequences ( 1 : 3 ) aligned . score : 77 sequences ( 2 : 4 ) aligned . score : 83 sequences ( 3 : 5 ) aligned . score : 82 sequences ( 4 : 6 ) aligned . score : 76 sequences ( 1 : 4 ) aligned . score : 75 sequences ( 2 : 5 ) aligned . score : 82 sequences ( 3 : 6 ) aligned . score : 77 sequences ( 4 : 7 ) aligned . score : 63 sequences ( 1 : 5 ) aligned . score : 77 sequences ( 2 : 6 ) aligned . score : 80 sequences ( 1 : 6 ) aligned . score : 72 sequences ( 3 : 7 ) aligned . score : 64 sequences ( 5 : 6 ) aligned . score : 77 sequences ( 1 : 7 ) aligned . score : 60 sequences ( 2 : 7 ) aligned . score : 63 sequences ( 6 : 7 ) aligned . score : 61 sequences ( 5 : 7 ) aligned . score : 64 guide tree file created : [/ net / nfs0 / vol1 / prosuction / w3nobody / tmp / 936042 . 678539 - 441485 . dnd ] start of multiple alignment there are 6 groups aligning . . . group 1 : sequences : 2 score : 14016 group 2 : sequences : 2 score : 14858 group 3 : sequences : 4 score : 13893 group 4 : sequences : 5 score : 14022 group 5 : sequences : 6 score : 12718 group 6 : sequences : 7 score : 12338 alignment score 68091 clustal - alignment file created [/ net / nfs0 / vol1 / production / w3nobody / tmp / 936042 . 678539 - 441485 . aln ] your multiple sequence alignment : 936042 . 678539 - 441485 . aln clustal w ( 1 . 81 ) multiple sequence alignment ( seq id no : 14 ) xm_007063 homosapiens proteinseq --- mskshs ----- ffgyplsiffivvnefcerfsyygmraililyftnf 42 ( seq id no : 18 ) u13707 oryctolaguscunic proteins - mgmsksls ----- cfgyplsiffivvnefcerfsyygmrallilyfrnf 44 ( seq id no : 15 ) d50306ratproteinsequence - mgmsksrg ----- cfgyplsiffivvnefcerfsyygmrallvlyfrnf 44 ( seq id no : 16 ) nm_053079 musmusculus proteinseq - mgmsksrg ----- cfgyplsiffivvnefcerfsyygmrallvlyfrnf 44 ( seq id no : 17 ) ay027496ovis - mgmsvpks ----- cfgyplsiffivvnefcerfsyygmrallilyfqrf 44 ( seq id no : 13 ) caninesubmitted --------------------- iffivvnefcerfsyygmrallilyfrrf 29 ( seq id no : 19 ) ay029615 gallusgallus proteinseq maakskskgrsvpncfgyplsiffivinefcerfsyygmravlvlyfkyf 50 xm_007063 homosapiens proteinseq iswddnlstaiyhtfvalcyltpilgaliadswlgkfktivslsivytig 92 u13707 oryctolaguscunic proteins igwddnlstviyhtfvalcyltpilgaliadawlgkfktivwlsivytig 94 d50306ratproteinsequence lgwdddlstaiyhtfvalcyltpilgaliadswlgkfktivslsivytig 94 nm_053079 musmusculus proteinseq lgwddnlstaiyhtfvalcyltpilgaliadswlgkfktivslsivytig 94 ay027496ovis lgwndnlgtatyhtfvalcyltpilgaliadswlgkfktivslsivytig 94 caninesubmitted igwddnlstaiyhtfvalcyltpilgaliadswlgkfktivslsivytig 79 ay029615 gallusgallus proteinseq lrwddnfstatyhtfvalcyltpilgaliadswlgkfktivslsivytig 100 : *:*::.*. *********************:********* ******** xm_007063 homosapiens proteinseq qavtsvsstndltdhnhdgtpdslpvhvvlsliglalialgtggikpcvs 142 u13707 oryctolaguscunic proteins qavtslssvneltdnnhdgtpdslpvhvavcmiglllialgtggikpcvs 144 d50306ratproteinsequence qavtsvssindltdhdhdgspnnlplhvalsmiglalialgtggikpcvs 144 nm_053079 musmusculus proteinseq qavisvssindltdhdhngspdslpvhvalsmvglalialgtggikpcvs 144 ay027496ovis qvvtavssindltdfnhdgtpnnisvhvalsmiglvlialgtggikpcvs 144 caninesubmitted qavtavsstndltdynkdgtpdnlsvhcalsmiglalialgtggikpcvs 129 ay029615 gallusgallus proteinseq qavmavssindmtdqnrdgnpdniavhialsmyglilialgtggikpcvs 150 *. * ::**:*::** :::*.*:.:.:.:*:.:. : ** ************** xm_007063 homosapiens proteinseq afggdqfeegqekqrnrffstfylainagsllstiitpmlrvqqcgihsk 192 u13707 oryctolaguscunic proteins afggdqfeegqekqrnrffstfylainagsllstiitpmlrvqqcgihsk 194 d50306ratproteinsequence afggdqfeegqekqrnrffsifylainagsllstiitpilrvqqcgihsq 194 nm_053079 musmusculus proteinseq afggdqfeegqekqrnrffsifylainggsllstiitpilrvqqcgihsq 194 ay027496ovis afggdqfeegqekqrnrffsifylainagsllstiitpmlrvqvcgihsk 194 caninesubmitted afggdqfeegqekqrnrffstfylainagslistivtpmlrvhecgiysq 179 ay029615 gallusgallus proteinseq afggdqfeehqekqrsrffsifylsinagslistiitpilraqecgihsr 200 ********* *****.********:**.***:***:**::*. : ***: : xm_007063 homosapiens proteinseq qacyplafgvpaalmavaltvfvlgsgmykkfkpqgnimgkvakcigfai 242 u13707 oryctolaguscunic proteins qacyplafgipailmavslivfiigsgmykkfkpqgnilskvvkcicfai 244 d50306ratproteinsequence qacyplafgvpaalmavalivfvlgsgmykkfqpgnimgkvakcigfai 244 nm_053079 musmusculus proteinseq qacyplafgvpaalmavalivfvlgsgmykkfqpqgnimgkvakcigfai 244 ay027496ovis qacyplafgvpaalmavslivevigsgmykkfqpgnimskvarcigfai 244 caninesubmitted kacyplafgvpaalmavslivfvigsgmykkfqpqgnvmgkvvkcigfal 229 ay029615 gallusgallus proteinseq qqcyplafgvpaalmavslwfiagsgmykkvqpqgnimvrvckcigfai 250 : *******:** ****:*:**: *******. :****:: :* :** **: xm_007063 homosapiens proteinseq knrfrhrskafpkrehwldwakekyderlisqikmvtrvmflyiplpmfw 292 u13707 oryctolaguscunic proteins knrfrhrskqfpkrahwldwakekyderliaqikmvtrvlflyiplpmfw 294 d50306ratproteinsequence knrfrhrskafpkrehwldwakekyderlisqikmvtkvmflyiplpmfw 294 nm_053079 musmusculus proteinseq knrfrhrskaypkrehwldwakekyderlisqikmvtkvmflfiplpmfw 294 ay027496ovis knrishrskkfpkrehwldwasekyderlisqikmvtrvmflyiplpmfw 294 caninesubmitted knrfrhrskqfpkrehwldwakekyderlisqikmvtkvmflyiplpmfw 279 ay029615 gallusgallus proteinseq knrfrhrskeypkrehwldwasekydkrliaqtkmvlkvlflyiplpmfw 300 ***: **** :*** ******. ****:***:* *** :*:**:******* xm_007063 homosapiens proteinseq alfdqqgsrwtlqattmsgktgaleiqpdqmqtvnailivimpvpifdavl 342 u13707 oryctolaguscunic proteins alfdqqgsrwtlqattmsgrigileiqpdqmqtvntiliiilvpimdavv 344 d50306ratproteinsequence alfdqqgsrwtlqattmtgkigtteiqpdqmqtvnailivimvpivdavv 344 nm_053079 musmusculus proteinseg glfdqqgsrwtlqattmngkiganeiqpdqmqtvnailnvnngpnvdavv 344 ay027496ovis alfdqqgsrwtlqattmsgkigiieiqpdqmqtvnailivvmvpivdavv 344 caninesubmitted alfdqqgsrwtlqatansgkigllevqpdqmqtvnailivvmvpimdavv 329 ay029615 gallusgallus proteinseq alfdqqgsrwtlqattmdgdfgamqiqpdqmqtvnpiliiimvpvvdavi 350 . **************:* * :* ::*********. ** : * . ***: xm_007063 homosapiens proteinseq ypltakcgfnftslkkmavgmvlasmafvvaaivqveidktlpvfpkgne 392 u13707 oryctolaguscunic proteins ypliakcglnftslkkmtigmflasmafvaaailqveidktlpvfpkane 394 d50306ratproteinsequence ypliakcgfnftslkkmtvgmflasmafvvaaivqveidktlpvfpsgnq 394 nm_053079 musmusculus proteinseq yrstakcgfnftslkkmtvgmflasmafvvaaivqvwidktlpvepggnq 394 ay027496ovis ypliakcglnftslkkmtvgmflasmafvaaaivqvdidktlpvfpkgne 394 caninesubmitted ypliakcgfnftslkrmtvgmflasmafvmaaivqleidktlpvfpkqne 379 ay029615 gallusgallus proteinseq ypliqkckinftplrritvgmflaglafvaaallqvqidktlpvfpaagq 400 * * ** :***.*:::::**.**. :*** **::*::********* . : xm_007063 homosapiens proteinseq vqtkvlnignntmnislpg -- emvtlgpmsqtnafmtfdvnkltriniss 440 u13707 oryctolaguscunic proteins vqtkvlnvgsenmttslpg -- qtvtlnqmsqtnefmtfnedtltsinits 442 d50306ratproteinsequence vqtkvlntgnndmavyfpg -- knvtvaqmsqtdtfmtfdvdqltsinviss 442 nm_053079 musmusculus proteinseq vqtkvlnignnnmtvhfpg -- nsvtlaqmstdtfmtfdidkltsiniss 442 ay027496ovis vqikvlnignnsmtvsfpg -- ttvtcdqmsqtngfltfnvdnls - iniss 441 caninesubmitted vqtkvlntgngannvsfpg -- avvtvsqmsqsdgfmtfdvdkltsiniss 427 ay029615 gallusgallus proteinseq aqiktinlgdsnanvtflpnlqnvtvlpmestg - yrmfessqlksvmvnf 449 .***::*:*. : : ** *..:. : *: . *. : :. xm_007063 homosapiens proteinseq pgsp - vtavtddfkqgqrhtllvwapnhyqvvk - dglnqkpekgengirf 488 u13707 oryctolaguscunic proteins - gsq - vtmitpsleagqrhtllvwapnnyrvvn - dgltqksdkgengirf 489 d50306ratproteinsequence pgspgvttvahefepghrhtllvwgpnlyrvvk - dglnqkpekgengirf 491 nm_053079 musmusculus proteinseq sgspgvttvahdfeqghrhnllvwepsqyrvvk - dgpnqkpekgrngirf 491 ay027496ovis tgtp - vtpvthnfesghrhtllvwapsnyqvvk - dglnqkpekgrngirf 489 caninesubmitted tgsp - vipvtynfeqghrhtllvwapnnyrvvk - dglnqkpekgengirf 475 ay029615 gallusgallus proteinseq gsesrsentdsissnthtvttknaaagivsslrsdnftskpeegknlvrf 499 . : . : . : .. :. *. ..*.::*. * :** xm_007063 homosapiens proteinseq vntfnelttitmsgkvyanissynastyqffpsgikgftisste - ippqc 537 u13707 oryctolaguscunic proteins vntysqptnvtmsgkvyehiasynaseyqfftsgvkgftvssag - iseqc 538 d50306ratproteinseguence vstlnemittkmsgkvyenvtshsasnyqffpsgqkdytintte - iapnc 540 nm_053079 musmusculus proteinseq vntlnemvtnkmsgkvyekftshnasgykflpsgekqytintta - vaptc 540 ay027496ovis vnafgesfgvtmdgevynnvsghnaseylffssgvksftinspe - isqqc 538 caninesubmitted inslneslnitmgdkvyvnvtshnaseyqffslgtknitisstqqisqnc 525 ay029615 gallusgallus proteinseq vnnlpqtvnitmgdttfgileetsisnyspfsggrtydivitag - stnc 547 :. : . .*.. . : . . * * :. * . : :. . * xm_007063 homosapiens proteinseq qpnfntfylefgsaytytvq - rkndscpevkvfedisantvnmalqipqy 586 u13707 oryctolaguscunic proteins rrdfespylefgsaytylit - sqatgcpqvtefedippntmnmawqipqy 587 d50306ratproteinsequence ssdfkssnldfgsaytyvtrsrasdgclevkefedippntvnmalqipqy 590 nm_053079 musmusculus proteinseq ltdfkssnldfgsaytyvir - rasdgclevkefedippntvnmalqipqy 589 ay027496ovis ekqfktsylefgsaftyvis - rksdgcpekifedispntvsmalqipqy 587 caninesubmitted tkvlqssnlefgsaytyvig - tqstgcpelhmfedispntvnmalqipqy 574 ay029615 gallusgallus proteinseq kp -- tseklgyggayttvtn - ecsgdctqlryiediqpntvhmawqipqy 594 : * :*. *:* :: . : :*** . **: ** ***** xm_007063 homosapiens proteinseq flltcgevvfsvtglefsysqapsnmksvlqagwlltvavgniivlivag 636 u13707 oryctolaguscunic proteins flitsgevvfsitglefsysqapsnmksvlqdrwlltvavgniivlivag 637 d50306ratproteinsequence flltcgevvfsvtglefsysqapsnmksvlqagwlltvaigniivlivae 640 nm_053079 musmusculus proteinseq flltcgevvfsvtglefsysqapsnmksvlqagwlltvagniivlivag 639 ay027496ovis flltcgevvfsitglefsysqapsnmksvlqagwlltvavgniivlivag 637 caninesubmitted flitcgevvfsvtglefsysqapsnmksvlqagwlltvacwqhhcahhcahcgr 624 ay029615 gallusgallus proteinseq ftltccevvfsvtglefsysqapsnmksvlqagwlltvagniivlivag 644 *::*. ******:******************* ****** : . xm_007063 homosapiens proteinseq agqfskqwaeyilfaalllvvcvifaimarfytyinpaeieaqfdedekk 686 u13707 oryctolaguscunic proteins agqinkqwaeyilfaalllvvcvifaimarfytyvnpaeieaqfeedekk 687 d50306ratproteinsequence aghfdkqwaeyvlfaslllvvciifaimarfytyinpaeieaqfdedekk 690 nm_053079 musmusculus proteinseq aghfpkqwaeyilfaslllvvcvifaimarfytyinpaeieaqfdedekk 689 ay027496ovis agqfpkqwaeyvlfaalllvvciifaimarfytyvnpaeieaqfdeddke 687 caninesubmitted srpvq - tvg ----------- ihpicgiasgclcnichhgpvlhlrqssrd 662 ay029615 gallusgallus proteinseq asklseqwaeyvlfaallfavciifavmayfytytdpneveaqldeeekk 694 : . . : * . : : :: :..:. xm_007063 homosapiens proteinseq nrleksnpyfmsgansqkqm 706 u13707 oryctolaguscunic proteins knpekndlypsvapvsqtqm 707 d50306ratproteinsequence kgvgkenpysslepvsqtnm 710 nm_053079 musmusculus proteinseq kgigkenpysslepvsqtqm 709 ay027496ovis ddleksnpyakldfvsqtqm 707 caninesubmitted -------------------- ay029615 gallusgallus proteinseq kqikqdpdlhgkeseavsqm 714 alignment of amino - acid sequences for canine and human sequence format is pearson sequence 1 : xm_007063 homosapiens proteinseq 706 aa sequence 2 : caninesubmittedclone37 662 aa start of pairwise alignments aligning . . . sequences ( 1 : 2 ) aligned . score : 76 guide tree file created : [/ net / nfs0 / vol1 / production / w3nobody / tmp / 789481 . 229198 - 238519 . dnd ] start of multiple alignment there are 1 groups aligning . . . group 1 : sequences : 2 score : 12826 alignment score 3129 clustal - alignment file created [/ net / nfs0 / vol1 / production / w3nobody / tmp / 789481 . 229198 - 238519 . aln ] your multiple sequence alignment : 789481 . 229198 - 238519 . aln clustal w ( 1 . 81 ) multiple sequence alignment ( seq id no : 14 ) xm_007063 homosapiens proteinseq mskshsffgyplsiffivvnefcerfsyygmraililyftnfiswddnls 50 ( seq id no : 13 ) caninesubmittedclone37 ------------- iffivvnefcerfsyygmrallilyfrrfigwddnls 37 ********************:***** .**. ****** xm_007063 homosapiens proteinseq taiyhtfvalcyltpilgaliadswlgkfktivslsivytigqavtsvss 100 caninesubmittedclone37 taiyhtfvalcyltpilgaliadswlgkfktivslsivytigqavtavss 87 *********************************************:*** xm_007063 homosapiens proteinseq indltdhnhdgtpdslpvhvvksliglialgtggikpcvsafggdqfe 150 caninesubmittedclone37 indltdynkdgtpdnlsvhvalsmiglalialgtggikpcvsfggdqfe 137 ******:*:*****.*.***. **:************************** xm_007063 homosapiens proteinseq egqekqrnrffsifylainagsllstiitpmlrvqqcgihskqacplaf 200 caninesubmittedclone37 egqekqrnrffsifylainagslistivtpmlrvhecgiysqkacyplaf 187 ***********************:***:******::***:*::******* xm_007063 homosapiens proteinseq gvpaalmavalivfvlgsgmykkfkpqgnimgkvakcigfaiknrfrhrs 250 caninesubmittedclone37 gvpaalmavslivfvigsgmykkfqpgnvmgkvvkcigfalknrfrhrs 237 *********:*****:********:****:****. ******:******** xm_007063 homosapiens proteinseq ksfpkrehwldwakekyderlisqikmvtrvmflyiplpmfwalfdqqgs 300 caninesubmittedclone37 kqfpkrewldwakekyderlisqikmvtkvmflyiplpmfwalfdqqgs 287 * ****************************:******************** xm_007063 homosapiens proteinseq rwtlqattmsgkigaleiqpdqmqtvnailivimvpifdavlypliakcg 350 caninesubmittedclone37 rwtlqatamsgkigllevqpdqmqtvnailivvmvpimdavvypliakcg 337 *******:****** **:**************:****:***:******** xm_007063 homosapiens proteinseq fnftslkkmavgmvladmafvvaaivqveidktlpvfpkgnevqikvlni 400 caninesubmittedclone37 fnftslkrmtvgmflasmafavmaaivqleidktlpvfpkqnevqikvlni 387 *******:*:***. *******:*****:*********** ********** xm_007063 homosapiens proteinseq gnntmnislpgemvtlgpmsqtnafmtfdvnkltrinisspgspvtavtd 450 caninesubmittedclone37 gngamnvsfpgavvtvsqmsqsdgfmtfdvdkltsinisstgspvipvty 437 **. :**:*:** :**:. ***::. ******:*** *****,**** . ** xm_007063 homosapiens proteinseq dfkqgqrhtllvwapnhyqvvkdglnqkpekgengirfvntfnelititm 500 caninesubmittedclone37 nfeqghrhtllvwapnnyrvvkdglnqkpekgengirfinslneslnitm 487 :*:**:**********:*:*******************:*::** :. *** xm_007063 homosapiens proteinseq sgkvyanissynastyqffpsgikgftisst - eippqcqpnfntfylefg 549 caninesubmittedclone37 gdkvyvnvtshnaseyqffslgtknitisstqqisqnctkvlqssnlefg 537 ..***. *::*:*** ****. * *. :***** :*. :* ::: **** xm_007063 homosapiens proteinseq saytyivqrkndscpevkvfedisantvnmalqipqyflltcgevvfsvt 599 caninesubmittedclone37 saytyvigtqstgcpelhmfedispnyvnmalqipqyflitcgevvfsvt 587 *****:: :. .***:::*****. **************;********** xm_007063 homosapiens proteinseq glefsysqapsnmksvlqagwlltvavgniivlivagagqfskqwaeyil 649 caninesubmittedclone37 glefsysqapsnmksvlqagwlltvacwqhhcahcgrsrpvq - tvg ---- 632 ************************** : . : .. . xm_007063 homosapiens proteinseq faalllvvcvifaimarfytyinpaeieaqfdedekknrlenksnpyfmsg 699 caninesubmittedclone37 ------- ihpicgiasglcnichhgpvlhlrqssrd ------------- 662 : * . * : * :: :..:. xm_007063 homosapiens proteinseq ansqkqm 706 caninesubmittedclone37 ------- after analyzing the protein sequence and performing alignment with other species , the underlined , italicized was removed for submission to genbank . sequence to submit to genbank ( seq id no : 7 ) catcttcttcatcgtggtcaatgagttctgtgaaagattttcctactatggaatgagagcactcctgattctgtacttcagacgg ttcatcgggtgggacgataatctgtccacggccatctaccacacgtttgtggctctgtgctacctgacgccgatcctcggc gcactgatcgcagactcctggctgggaaagttcaagacaatcgtgtcactctccattgtctacacaattggacaggcggtc actgcagtaagctcaattaatgacctcacagactataacaaagatggaactcctgacaatctgtccgtgcatgtggcactgt ccatgattggcctggccctgatagctctgggaactggaggaataaagccctgtgtgtctgcatttggtggagaccagtttg aagagggccaggaaaaacaaagaaacagattcttttccatcttttatttggccattaatgctggaagcttgatttccactattg tcactcccatgctcagagttcacgaatgtggaatttacagtcagaaagcttgttacccactggcatttggggttcctgctgct ctcatggccgtatctctgattgtatttgtcattggcagtggaatgtacaagaagtttcagccccagggtaatgtcatgggtaa agttgtcaagtgcattggttttgccctcaaaaataggtttaggcaccggagtaagcagtttcccaagagggagcactggct ggactgggctaaagagaaatacgatgagcggctcatctctcaaattaagatggtcacaaaagtgatgttcttgtacatccc actcccaatgttctgggccctgtttgaccagcagggctccaggtggacactgcaagcaacagctatgagtgggaaaattg gacttcttgaagttcagccagatcagatgcagactgtgaatgccatcttgattgtcgtcatggtccccatcatggatgccgt ggtgtaccctctgattgcaaaatgtggcttcaatttcacctccttgaagaggatgacagttggaatgttcctggcttccatgg ccttcgtgatggcggcgattgttcagctggaaattgataaaactcttccagtcttccccaaacaaaatgaagtccaaatcaa agtactgaatataggaaatggtgccatgaatgtatcttttcctggagcggtggtgacagttagccaaatgagtcaatcagat ggatttatgacttttgatgtagacaaactgacaagtataaacatttcttccactggatcaccagtcattccagtgacttataact ttgagcagggccatcgccatacccttctagtatgggcccccaataattaccgagtggtaaaggatggccttaaccagaag ccagaaaaaggagaaaatggaatcagatttataaatagtcttaatgagagcctcaacatcaccatgggcgacaaagtttat gtgaatgtcaccagtcacaatgccagcgagtatcagttcttttctttgggcacaaaaaacattacaataagttcaacacaac agatctcacaaaattgtacaaaagttctccaatcatccaaccttgaatttggtagtgcatatacctatgtaatcggaacgcag agcactggctgccctgaattgcatatgtttgaagatatttcacccaacacagttaacatggctctgcagatcccgcagtactt cctcatcacctgcggcgaggtggttttctctgtcacaggactggagttctcatattctcaggccccctccaacatgaagtcg gtgcttcaggcgggatggctgctgacagtggct canine pept1 nucleotide sequence ( seq id no : 20 ) atgggcatgtccaagtcatatggttgctttggttaccccttgagcatcttcttcatcgtggtcaatgagttctgtgaaagatttt cctactatggaatgagagcactcctgattctgtacttcagacggttcatcgggtgggacgataatctgtccacggccatcta ccacacgtttgtggctctgtgctacctgacgccgatcctcggcgcgcactgatcagactcctggctgggaaagttcaaga caatcgtgtcactctcattgtctacacaattggacaggcggtcactgcagtaggctcaattaatgacctcacagactatgg caaagatggaactcctgacaatctgtccgtgcatgtggcactgtccatgattggcctggccctgatagctctgggaactgg aggaataaagccctgtgtgtctgcatttggtggagaccagtttgaagagggccaggaaaaacaaagaaacagattctttt ccatcttttatttggccattaatgctggaagcttgatttccactattgtcactcccatgcttcacgaatgtggaatttac agtcagaaagcttgttacccactggcatttggggttcctgctgctctcatggccgtatctctgattgtatttgtcattggcagt ggaatgtacaagaagtttcagccccagggtaatgtcatgggtaaagttgtcaagtgcattggttttgccctcaaaaataggt ttaggcaccggagtaagcagtttcccaagagggagcactggctggactgggctaaagagaaatacgatgagcggctca tctctcaaattaagatggtcacaaaagtgatgttcttgtacatcccactcccaatgttctgggccctgtttgaccagcagggc tccaggtggacactgcaagcaacagctatgagtgggaaaattggacttcttgaagttcagccagatcagatgcagactgt gaatgccatcttgattgtcgtcatggtccccatcatggatgccgtggtgtaccctctgattgcaaaatgtggcttcaatttca cctccttgaagaggatgacagttggaatgttcctggcttccatggccttcgtgatggcggcgattgttcagctggaaattga taaaactcttccagtcttccccaaacaaaatgaagtccaaatcaaagtactgaatataggaaatggtgccatgaatgtatctt ttcctggagcggtggtgacagttagccaaatgagtcaatcagatggatttatgacttttgatgtagacaaactgacaagtat aaacatttcttccactggatcaccagtcattccagtgacttataactttgagcagggccatcgccatacccttctagtatggg cccccaataattaccgagtggtaaaggatggccttaaccagaagccagaaaaaggagaaaatggaatcagatttataaat agtcttaatgagagcctcaacatcaccatgggcgacaaagtttatgtgaatgtcaccagtcacaatgccagcgagtatcag ttcttttctttgggcacaaaaaacattacaataagttcaacacaacagatctcacaaaattgtacaaaagttctccaatcatcc aaccttgaatttggtagtgcatatacctatgtaatcggaacgcagagcactggctgccctgaattgcatatgtttgaagatat ttcacccaacacagttaacatggctctgcagatcccgcagtacttcctcatcacctgcggcgaggtggttttctctgtcaca ggactggagttctcatattctcaggccccctccaacatgaagtcggtgcttcaggcgggatggctgctgacagtggctgtt ggcaacatcattgtgctcattgtggcaggagcaggccagttcagtgaacagtgggctgaatacatcctatttgcggcattg cttctggttgtctgtgtaatatttgccatggcccggttttacacttacgtcaatccagcagagattgaagctcagtttgacg acgatgagaaaaagaacctggaaaagatgaatgtatattccacggtaactccggtctcacagacacagatg canine pept1 amino acid sequence ( seq id no : 21 ) mgmsksygcfgyplsiffivvnefcerfsyygmrallilyfrrfigwddnls taiyhtfvalcyltpilgaliadswlgkfktivslsivytigqavtavssindl tdynkdgtpdnlsvhvalsmiglalialgtggikpcvsafggdqfeegqek qrnrffsifylainagslistivtpmlrvhecgiysqkacyplafgvpaalma vslivfvigsgmykkfqpqgnvmgkvvkcigfalknfrhrskqfpkreh wldwakekyderlisqikmvtkvmflyiplpmfwalfdqqgsrwtlqata msgkigllevqpdqmqtvnailivvmvpimdavvypliakcgfnftslkrm tvgmflasmafvmaaivqleidktlpvfpkqnevqikvlngngamnvsfp gavvtvsqmsqsdgfmtfdvdkltsinisstgspvipvtynfeqghrhtllv wapnnyrvvkdglnqkpekgengirfinslneslnitmgdkvyvnvtshn aseyqffslgtknitisstqqisqnctkvlqssnlefgsaytyvigtqstgcpe lhmfedispntvnmalqipqyflitcgevvfsvtglefsysqapsnmksvlq agwllltvavgniivlivagagqfseqwaeyilfaalllvvcvifaimarfyt yvnpaeieaqfdddekknlekmnvystvtpvsqtqm all publications , patents and patent documents are incorporated by reference herein , as though individually incorporated by reference . the invention has been described with reference to various specific and preferred embodiments and techniques . however , it should be understood that many variations and modifications may be made while remaining within the scope of the invention . asp asp asn leu ser thr ala ile tyr his thr phe val ala leu cys asp gly thr pro asp asn leu ser val his val ala leu ser met ile ile ser thr ile val thr pro met leu arg val his glu cys gly ile tyr ser gln lys ala cys tyr pro leu ala phe gly val pro ala ala ile gly phe ala leu lys asn arg phe arg his arg ser lys gln phe arg leu ile ser gln ile lys met val thr lys val met phe leu tyr ile pro leu pro met phe trp ala leu phe asp gln gln gly ser arg trp thr leu gln ala thr ala met ser gly lys ile gly leu leu glu ile pro val thr tyr asn phe glu gln gly his arg his thr leu leu val trp ala pro asn asn tyr arg val val lys asp gly leu asn gln glu ser leu asn ile thr met gly asp lys val tyr val asn val thr ser his asn ala ser glu tyr gln phe phe ser leu gly thr lys asn leu gln ser ser asn leu glu phe gly ser ala tyr thr tyr val ile gly thr gln ser thr gly cys pro glu leu his met phe glu asp ile ser pro asn thr val asn met ala leu gln ile pro gln tyr phe leu tyr ser gln ala pro ser asn met lys ser val leu gln ala gly trp ser arg pro val gln thr val gly ile his pro ile cys gly ile ala ile val val asn glu phe cys glu arg phe ser tyr tyr gly met arg leu ile ala leu gly thr gly gly ile lys pro cys val ser ala phe ile ile thr pro met leu arg val gln gln cys gly ile his ser lys lys pro gln gly asn ile met gly lys val ala lys cys ile gly phe ser gln ile lys met val thr arg val met phe leu tyr ile pro leu pro met phe trp ala leu phe asp gln gln gly ser arg trp thr leu gln ala thr thr met ser gly lys ile gly ala leu glu ile gln pro ile phe asp ala val leu tyr pro leu ile ala lys cys gly phe asn gly pro met ser gln thr asn ala phe met thr phe asp val asn lys thr asp asp phe lys gln gly gln arg his thr leu leu val trp ala pro asn his tyr gln val val lys asp gly leu asn gln lys pro glu tyr leu glu phe gly ser ala tyr thr tyr ile val gln arg lys asn asp ser cys pro glu val lys val phe glu asp ile ser ala asn thr val asn met ala leu gln ile pro gln tyr phe leu leu thr cys gly pro ser asn met lys ser val leu gln ala gly trp leu leu thr val ser lys gln trp ala glu tyr ile leu phe ala ala leu leu leu val leu glu lys ser asn pro tyr phe met ser gly ala asn ser gln lys met gly met ser lys ser arg gly cys phe gly tyr pro leu ser ile met arg ala leu leu val leu tyr phe arg asn phe leu gly trp asp asp asp leu ser thr ala ile tyr his thr phe val ala leu cys tyr phe lys thr ile val ser leu ser ile val tyr thr ile gly gln ala ser thr ile ile thr pro ile leu arg val gln gln cys gly ile his lys phe gln pro gln gly asn ile met gly lys val ala lys cys ile pro leu pro met phe trp ala leu phe asp gln gln gly ser arg trp thr leu pro val phe pro ser gly asn gln val gln ile lys val leu asn ile gly asn asn asp met ala val tyr phe pro gly lys asn val val trp gly pro asn leu tyr arg val val lys asp gly leu asn gln lys pro glu lys gly glu asn gly ile arg phe val ser thr leu asn ser his ser ala ser asn tyr gln phe phe pro ser gly gln lys asp tyr thr ile asn thr thr glu ile ala pro asn cys ser ser asp phe lys ser ser asn leu asp phe gly ser ala tyr thr tyr val ile arg ser arg ala ser asp gly cys leu glu val lys glu phe glu asp ile pro pro asn thr val asn met ala leu gln ile pro gln tyr phe leu tyr ser gln ala pro ser asn met lys ser val leu gln ala gly trp ala gly his phe asp lys gln trp ala glu tyr val leu phe ala ser met gly met ser lys ser arg gly cys phe gly tyr pro leu ser ile met arg ala leu leu val leu tyr phe arg asn phe leu gly trp asp asp asn leu ser thr ala ile tyr his thr phe val ala leu cys tyr phe lys thr ile val ser leu ser ile val tyr thr ile gly gln ala ser thr ile ile thr pro ile leu arg val gln gln cys gly ile his lys phe gln pro gln gly asn ile met gly lys val ala lys cys ile gly phe ala ile lys asn arg phe arg his arg ser lys ala tyr pro gly pro asn val asp ala val val tyr arg ser ile ala lys cys gly thr thr val ala his asp phe glu gln gly his arg his asn leu leu val trp glu pro ser gln tyr arg val val lys asp gly pro asn gln lys pro glu lys gly glu asn gly 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