1. Supervisor: Prof. S K Goswami
Presented By:
AMAN SHARMA
2011EBT18

3. SG2NA is a molecular scaffold of WD-40 repeat superfamily
 It was originally reported in 1994 as a nuclear autoantigen
(Landberg and Tan., 1994).
 It shows a Cell Cycle regulated expression (Muro etal.,
1995)
 It is ubiquitously expressed with enriched expression in
brain (Castets , F. et al., 2000)..
 It is a cytosolic and membrane-bound protein (Benoist M.
etal., 2006)

4. It is a member of Striatin family with multiple protein-protein
interaction domains, namely:
 Caveolin binding domain
 Coiled-coil motif
 Calmodulin binding domain
 Multiple WD-40 repeats
Coiled-coil
Caveolin
binding
Calmodulin
binding
WD-40 repeat
Striatin
Zinedin
SG2NA
95% 85%
Benoistet al.,2006

5. Spliced Variants of SG2NA
83 kDa
82 kDa
78 kDa
38 kDa
87 kDa
35 kDa
E7 E8 E9
In 7/8

9. Figure 1: Strategy for the construction of expression vector 87kDa SG2NAs with puromycin as selection marker.
SG2NA
HinDIII
ClaI
Nco-I
Nco-I

10. 6000 bp
3000 bp
1000 bp
myc 87 kDa SG2NA
fragment (6.5kbp)
Puro fragment (965bp)
Figure 2: Cloning of puromycin expression casette in SG2NA-pcDNA 3.1 vector
backbone. Upper): SG2NA expression vectors were PCR amplified using forward primer
spanning the SV40 polyadenylation site and the reverse primer spanning the f1 origin. The
amplified product was devoid of neomycin expression casette. (Lower) Restriction digestion
of GFP-V-RS plasmid with HindIII & ClaI. Upon digestion with HindIII and ClaI, 965 bp
fragment containing SV40 promoter and the puromycin ORF was released.

11. Figure 3. Screening of recombinant plasmids with expression cassettes for
SG2NA and puromycin. The candidate recombinant plasmids were amplified
using primers spanning 15 th exon of 87 kDa & a fragment internal to
puromycin acetylase of the recombinants. The candiate colnes were turn out to
be incorrect & were not pursued for further study.

12. Figure 4. Screening of recombinant plasmids with expression cassettes for
SG2NA and puromycin. . The candidate recombinant plasmids were amplified
using primers spanning 15 th exon of 87 kDa & a fragment internal to puromycin
acetylase of the recombinants. The candidate recombinant plasmids were digested
with restriction enzyme Nco-I to further confirm the identity of clones, which
turned out to be incorrect as they were ligated in reverse direction.

13. Positive
Positive
Figure 5. Confirmation of recombinant plasmids with expression cassettes for
SG2NA and puromycin. The candidate recombinant plasmids were amplified using
primers spanning 15 th exon of 87 kDa & a fragment internal to puromycin
acetylase of the recombinants. Two plasmids got amplified & are circled..
.

14. 4000bp
3500bp
Fig 6: Confirmation of the recombinant plasmids: The candidate
recombinant plasmids were digested with restriction enzyme Nco-I to further
confirm the identity of clones, both the correct plasmids are shown encircled,

15. CONCLUSION
 Got the desired clone
 Neomycin selection marker causes
epigenetic changes (Dutta.et.al, 2012)
 Introduction of the neomycin-resistant
gene is fraught with variability in gene
expression.

16. Acknowledgement
Special thanks to
1.Prof. S.K Goswami ( supervisor)
2.Shweta Pandey (mentor)
3.Nikhat saleem
4.Gautam Tanti
5.Buddhi Jain
6.Jahangir Alam

17. REFERENCES
 Baillat, G., Gaillard, S., Castets, F., and Monneron, A. (2002).
Interactions of phocein with nucleoside-diphosphate kinase, Eps15,
and Dynamin I. J. Biol. Chem. 277, 18961–18966.
 Bartoli, M., Ternaux, J.P., Forni, C., Portalier, P., Salin, P., Amalric,
M., and Monneron, A. (1999). Down-regulation of striatin, a neuronal
calmodulin-binding protein, impairs rat locomotor activity. J.
Neurobiol. 40, 234–243.
 Benoist, M., Gaillard, S., and Castets, F. (2006). The striatin family:
a new signaling platform in dendritic spines. J. Physiol. Paris 99,
146–153.
 Burns, T.F., and El-Deiry, W.S. (1999). The p53 pathway and
apoptosis. J. Cell. Physiol. 181, 231–239.
 Castets, F., Bartoli, M., Barnier, J.V., Baillat, G., Salin, P., Moqrich,
A., Bourgeois, J.P., Denizot, F., Rougon, G., Calothy, G., et al.
(1996). A novel calmodulin-binding protein, belonging to the WD-
repeat family, is localized in dendrites of a subset of CNS neurons.
J. Cell Biol. 134, 1051–1062.
 Dutta,P¤, Tanti, GK., Sharma,S., . Goswami, SK., Komath1, SS.,
Mayo, MW. Joel W. Hockensmith2*, Muthuswami1,R.* Global
Epigenetic Changes Induced by SWI2/SNF2 Inhibitors Characterize
Neomycin-Resistant Mammalian Cells. Plos ONE. November 2012 |
Volume 7, Issue 11, e49822

18. My Supervisor & Lab mates