Brown BIOL1220:Notebook/SynBio in Theory and Practice/Genes/Synaptotagmin and VAMP

Ashley Kim BIOL 1220: Synthetic Biology 4 February 2009 Primer Design: Synaptotagmin I and VAMP

Synaptotagmin and VAMP: Gene Backgrounds

1. Synaptic transmission between neurons (cells of the nervous system) can be chemical and involve the release of neurotransmitter filled vesicles from the axon terminals of pre-synaptic neurons to the dendrites of post-synaptic neurons. (The Neuron Doctrine states that neurons communicate through contact, not continuity, indicating the need for signals between cells. Chemical synapses actually include a 20-50nm wide synaptic cleft.)

2. NT release occurs at the active sites of pre-synaptic neuronal membranes. When an AP arrives at the axon hillock of a pre-synaptic neuron, depolarization causes the voltage-gated Ca2+ channels at the active zone to open allowing Ca2+ (at lower concentrations inside the cell than outside the cell) to rush in.

3. Synaptotagmin, a protein observed on synaptic vesicles, is thought to be a Ca2+ sensor that triggers vesicle fusion to the pre-synaptic membrane and exocytosis releasing NT into the synaptic cleft.

4. This vesicle fusion occurs through the interaction of v-SNARE and t-SNARE proteins on the vesicle and “target” pre-synaptic membrane (respectively). VAMP is a family of v-SNARE proteins.

Unaligned Sequences:

Synaptotagmin I:

>sp syn I mgdqevsqaa vpnesaaapp apteaaadgg ggggiggafa nagtnmknki sdvmdkiplp twavvaiaiv aglillcccf cickkccckk rkkkegkkgl kgavdlksvq mlgnsykekp dvddldnggd eegdtdsvks eiklgklqfs ldydfqegkl nvgvmqasel pgmdfsgtsd pyvkvylmpd kkkkyetkvh rktlnpvfne tftfkvpyse vssktlvfai ydfdrfsrhd iigevkvkls qvdlgsvvee wrdlqsaevp ggegkselgd icfslryvpt agkltvvile aknlkkmdvg glsdpyvkis lymnnkrmkk kkttikkrtl npyynesfgf evpfeqiqkv tlvvtvvdyd rmgssepigk vvlgcnatga glrhwsdmla sprrpiaqwh tlqepeegk

>lv syn I

maeqevsqaa vpnesaagpp aptegaadgg ggggaggala nmgnniknkv gdlvdkiplp twaviaiaiv aglillcccf cickkccckk rkkkegkkgl kgavdlksvq mlgnsykeki qpdvddlddg gdeegdtdsv kseiklgklq fsldydfqeg klnvgvmqas elpgmdfsgt sdpyvkvylm pdkkkkyetk vhrktlnpvf netftfkvpy sevssktlvf aiydfdrfsr hdiigevkvk lsqvdlgsvv eewrdlqsae vpggegksel gdicfslryv ptagkltvvi leaknlkkmd vgglsdpyvk islymnnkrm kkkkttikkr tlnpyynesf gfevpfeqiq kvtlvvtvvd ydrmgssepi gkvvlgcnat gaglrhwsdm lasprrpiaq whtlqepeen k

VAMP

>sp vamp a

maappppqpa psnkrlqqtq aqvdevvdim rvnvdkvler dqalsvlddr adalqqgasq fetnagklkr kywwknckmm iilaiiiivi liiiivaivq sqkk

>lv vamp

maaqptqptp aptnkrlqqt qaqvdevvdi mrvnvdkvle rdqalsvldd radalqhgas qfetnagklk rkywwknckm miilaviiiv iliiiivaiv qstkk

>sp vamp b qqtqaqvdev vdimrvnvdk vlerdqalsv lddradalqq gasqfetnag klkrkywwkn ckmm

>sp vamp c

thnttthssn yanryttnkr lqqtqaqvde vvdimrvnvd kvlerdqals vlddradalq qgasqfetna gklkrkywwk nckmmiilai iiiviliiii vaivqsqkk

Aligned Sequences

Synaptotagmin I

CLUSTAL W (1.83) multiple sequence alignment

sp MGDQEVSQAAVPNESAAAPPAPTEAAADGGGGGGIGGAFANAGTNMKNKISDVMDKIPLP lv MAEQEVSQAAVPNESAAGPPAPTEGAADGGGGGGAGGALANMGNNIKNKVGDLVDKIPLP

               *.:**************.******.********* ***:** *.*:***:.*::******

sp TWAVVAIAIVAGLILLCCCFCICKKCCCKKRKKKEGKKGLKGAVDLKSVQMLGNSYKE-- lv TWAVIAIAIVAGLILLCCCFCICKKCCCKKRKKKEGKKGLKGAVDLKSVQMLGNSYKEKI

               ****:*****************************************************  

sp KPDVDDLDNGGDEEGDTDSVKSEIKLGKLQFSLDYDFQEGKLNVGVMQASELPGMDFSGT lv QPDVDDLDDGGDEEGDTDSVKSEIKLGKLQFSLDYDFQEGKLNVGVMQASELPGMDFSGT

               :*******:***************************************************

sp SDPYVKVYLMPDKKKKYETKVHRKTLNPVFNETFTFKVPYSEVSSKTLVFAIYDFDRFSR lv SDPYVKVYLMPDKKKKYETKVHRKTLNPVFNETFTFKVPYSEVSSKTLVFAIYDFDRFSR

               ************************************************************

sp HDIIGEVKVKLSQVDLGSVVEEWRDLQSAEVPGGEGKSELGDICFSLRYVPTAGKLTVVI lv HDIIGEVKVKLSQVDLGSVVEEWRDLQSAEVPGGEGKSELGDICFSLRYVPTAGKLTVVI

               ************************************************************

sp LEAKNLKKMDVGGLSDPYVKISLYMNNKRMKKKKTTIKKRTLNPYYNESFGFEVPFEQIQ lv LEAKNLKKMDVGGLSDPYVKISLYMNNKRMKKKKTTIKKRTLNPYYNESFGFEVPFEQIQ

               ************************************************************

sp KVTLVVTVVDYDRMGSSEPIGKVVLGCNATGAGLRHWSDMLASPRRPIAQWHTLQEPEEG lv KVTLVVTVVDYDRMGSSEPIGKVVLGCNATGAGLRHWSDMLASPRRPIAQWHTLQEPEEN

               ***********************************************************.

sp K lv K

               *

VAMP

CLUSTAL W (1.83) multiple sequence alignment

a -----MAAPPPPQPAPSNKRLQQTQAQVDEVVDIMRVNVDKVLERDQALSVLDDRADALQ lv ----MAAQPTQPTPAPTNKRLQQTQAQVDEVVDIMRVNVDKVLERDQALSVLDDRADALQ b ---------------------QQTQAQVDEVVDIMRVNVDKVLERDQALSVLDDRADALQ c THNTTTHSSNYANRYTTNKRLQQTQAQVDEVVDIMRVNVDKVLERDQALSVLDDRADALQ

                                    ***************************************

a QGASQFETNAGKLKRKYWWKNCKMMIILAIIIIVILIIIIVAIVQSQKK lv HGASQFETNAGKLKRKYWWKNCKMMIILAVIIIVILIIIIVAIVQSTKK b QGASQFETNAGKLKRKYWWKNCKMM------------------------ c QGASQFETNAGKLKRKYWWKNCKMMIILAIIIIVILIIIIVAIVQSQKK

               :************************

Primer Design (See highlighted regions)

Synaptotagmin I

Primer 1: CKKCCCKK TGC/T AAA/G AAA/G TGC/T TGC/T TGC/T AAA/G AA

Primer 2: DKKKKYE TA C/TTT C/TTT C/TTT C/TTT G/ATC

VAMP

Primer 1: QQTQAQV CAA/G CAA/G ACA/C/G/T CAA/G GCA/C/G/T CAA/G GT

Primer 2: WWKNCKMM CAT CAT C/TTT A/GCA A/GTT C/TTT CCA CCA