Cloning of Zebrafish b4 Splice Variants. Cloning of first strand RACEready cDNA encoding b4 subunits

1
HYPOTHESIS Differential expression of b4
subunit splice variants suggests that b4a and b4b
serve very specific functions in different
neuronal subtypes. We hypothesize that b4 subunit
alternative splicing has been conserved
throughout chordate evolution. To test this
hypothesis we will attempt to clone the b4a and
b4b subunits from zebrafish (Danio rerio).
Zebrafish serve as an excellent model system for
the study of nervous system development and
genetics.
b4 Subunit Genomic Exon Distribution is Similar
Between Zebrafish and Humans
METHODS
Cloning of Zebrafish b4 Splice Variants. Cloning
of first strand RACE-ready cDNA encoding b4
subunits was performed by A. Srinivasan (Harvard
University). b4 subunit cDNAs were amplified
using 5 and 3 SMART- RACE PCR (Clontech) and
subcloned into pCR2.1 TA cloning plasmid
(Invitrogen) using traditional cloning methods.
Sequences were analyzed using BLAST (National
Center for Biotechnology Information) and
DNASTAR. Full length constructs and b4 subunit
probes were generated from the splice variants
found. Probes were subcloned into pBluescript II
SK() in order to generate mRNA using T3/T7
mMessage mMachine kit (Ambion).
Restriction Digest of Zebrafish b4a and b4b
probe DNA
1Kb StuI EcoR1 SE 100 bp XmnI EcoR1 XE
StuI
339bp
75bp
b4a
b4b
9
9
CONCLUSIONS
Exon 1 of the b4 Subunit is Alternatively Spliced

• Alternative splicing of the voltage-dependent
  Ca2 channel b4 subunit is conserved in
  Zebrafish. The genomic pattern of Exon 1
  splicing is nearly identical to that found in the
  human genome.
• The Zebrafish b4 subunit also undergoes
  alternative splicing in Exon 5.

Exon 1
Exon 2

• 15 (b4a) vs 49 (b4b) amino acids
• hb4a is closely related to ZFb4a and human b3
• ZFb4b differs from hb4b at its 5 end

h?4a
MYDNLYLHGIEDSEAGSAD
Differential Labeling in Cerebellar Cortex
ZF
MYDNLYLHGFEDSEAGSAD
MYDDSYVPGFEDSEAGSAD
h?3
B
A
FUTURE DIRECTIONS
b4a A Domain
MSSSSYAKNGTADGPHSPTSQVARGTTTRRSRLKRSDGSTTSTSFILRQG
SAD
AID
MSRHIYIRNKIGEGIQAPIFQVNRGTYSRRSRLKRSDGSTTSTSFILRQG
SAD

• Cloned splice variant probes of the Zebrafish b4
  subunit (b4a and b4b) will be used for in-situ
  hybridization to determine their temporal and
  differential distribution in Zebrafish embryos.
• Zebrafish b4 splice variant constructs will be
  used in electrophysiological studies to
  characterize the specific effects of alternative
  splicing on calcium channel gating.

Sequences used to make peptides for polyclonal
antibody production.
b2a SH3 and GK Domains
b4a-Ab punctate labeling in molecular layer and
granular layers (synapses) - note Purkinje fiber
ghosts
A
Exon 1 splice site
Exon 5 splice site
B
b4b-Ab labeling of Purkinje cell bodies and
Bergman glial cells
Funding Support contributed by Merck-Merial and
National Institutes of Health RO1 NS42600