The cloning and expression of SNAP-25a and b in zebrafishMaia Lavarias*, Dr. Wendy Boehmler

Department of Biology, York College of Pennsylvania, York, PA 17403

INTRODUCTION

• Synaptosome-associated protein of 25 kDa (SNAP-25) is important in the attachment of SNARE proteins to tagmin (Ca+ sensor) in the neuron. SNARE proteins are critical for the docking of transport vesicles to the membrane so that they can fuse and release their neurotransmitter into the synaptic cleft.

• There are two splice variants of SNAP-25 in humans; a and b. • Botulism is an illness caused by a toxin produced by

Clostridium botulinum bacteria. The symptoms include vomiting, nausea, blurred vision, and flaccid paralysis leading to death.

• The botulinum toxin (BoNT) functions by cleaving the SNAP-25 proteins in neurons and therefore preventing the docking of transport vesicles and the release of neurotransmitters.

• BoNT has the potential to be used as a bioweapon, and therefore identifying inhibitors to the toxin would be beneficial.

• Zebrafish have been used increasingly as model systems for human disease and drug discovery.

 

OBJECTIVES

1) To identify and sequence the SNAP-25 gene in zebrafish.

2) To determine the SNAP-25 gene’s expression in various adult zebrafish tissues and different developmental time points in zebrafish embryos.

METHODS RESULTS

SUMMARY

FUTURE EXPERIMENTS

REFERENCESDelgado-Martines, I. et al. Differential abilities of SNAP-25 homologs to support neuronal function. Journal of Neuroscience 2007, 27(35): 9380-91.

Schiavo, G. et al. Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasma membrane t-SNARE, SNAP-25, can explain docked vesicles at neurotoxin-treated synapse. Proct. Natl. Acad. Sci 1997, (44):997-1001.

Schiavo, G. et al. Botulinum neurotoxins serotypes A and E cleave SNAP-25 at distinct COOH-terminal peptide bonds. FEBS Letters 1993, 335 (1):99-103.

Woods, I. et al. The zebrafish gene map defines ancestral vertebrate chromosomes. Genome Res 2005 (15):1307-1314.

http://www.med.umich.edu/opm/newspage/2006/nphp.htm

Figure 2. Miniprep result of SNAP-25a and b. The SNAP-25a and b genes were transformed into a pDrive vector and cloned in E. coli. The 500 bp ladder is represented by Lad, A represents SNAP-25a, and B1 and B2 represent two different bacterial colonies that were transformed with the vector containing SNAP-25b.

Figure 1. RT-PCR analysis of SNAP-25a and b . The SNAP-25a and b genes were amplified using adult zebrafish brain cDNA. The 500 bp ladder is represented with Lad, while the SNAP-25 a and b genes are represented by A and B, respectively.

http://www.pnas.org/content/104/8/2697/F4.expansion.html

• A search of the NCBI database for the human SNAP-25 gene was completed.

• A BLAST search recovered two zebrafish homologs of the SNAP-25a and b splice variants

• PCR products were ligated into a pDrive vector and transformed into E. coli.

• Plasmids containing SNAP-25a and b DNA were isolated from the bacteria and sequenced.

Figure 3. RT-PCR analysis of SNAP-25a gene from various adult zebrafish tissues. The SNAP-25a gene was amplified from adult brain (B), eye (E), heart (H), gut (G), and muscle (M) cDNA and 24, 48, and 72 hpf zebrafish embryos. L represents the 500 bp ladder.

Figure 4. RT-PCR analysis of SNAP-25b gene from various adult zebrafish tissues. The SNAP-25a gene was amplified from adult brain (B), eye (E), heart (H), gut (G), and muscle (M) cDNA and 24, 48, and 72 hpf zebrafish embryos.

Figure 5. Alignment of zebrafish, mouse, and human SNAP-25. Zebrafish, mouse, and human SNAP-25 amino acid sequences were aligned using CLUSTALW. Identical amino acids are shown using an asterisk. A colon identifies conserved substitutions, and a period is for less conserved substitutions. Dashes allow for optimal alignment for amino acid insertions or deletions. Cleavage sites of BoNT/A and BoNT/E are marked with arrows. The four cysteine residues highlighted in red are conserved residues which associate the protein with the plasma membrane. The bolded regions after the cleavage sites are sequences necessary for inducing the BoNT/A and E toxins to attach and cleave the protein.

• In zebrafish, SNAP-25a and b are separate genes, not splice variants.

• Expression of both SNAP-25a and b genes were found in the brain, eye, heart, and muscle tissues.

• Amino acid alignment and syntenic analysis supports our identification of SNAP-25a and b in zebrafish.

Representation of SNAP-25a and b and the primers created. The primers used encompassed the predicted ATG start codon and stop codon in both genes.

• Use in situ hybridization to determine the location of SNAP-25a and b expression in embryos at various developmental stages

• Determine function of SNAP-25a and b using morpholino knockdown.

Table 1: Markers and Genes Syntenic with SNAP-25a and b Genes in Zebrafish and

Humans