Supervisor: Prof. S K Goswami

Presented By:

AMAN SHARMA

2011EBT18

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)

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-coilCaveolin

binding

Calmodulin

binding

WD-40 repeat

Striatin

Zinedin

SG2NA

95% 85%

Benoist et al., 2006

Spliced Variants of SG2NA

83 kDa

82 kDa

78 kDa

38 kDa

87 kDa

35 kDa

E7 E8 E9

In 7/8

Figure 1: Strategy for the construction of expression vector 87kDa SG2NAs with puromycin as selection marker.

SG2NA

HinDIII

ClaI

Nco-I

Nco-I

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.

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.

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.

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..

.

4000bp3500bp

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,

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.

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

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

My Supervisor & Lab mates