Title: Tyrosine283 Mutation of the Rat GnRH Receptor by Polymerase Chain Reaction
1Tyrosine283 Mutation of the Rat GnRH Receptor by
Polymerase Chain Reaction
- Maria Abreu1, Da Young Oh2, Dr. Jae Y.
Seong2, Dr. Hyuk B. Kwon2
1 Barry University, Miami Shores, FL 2 Hormone
Research Center Chonnam National University
Kwangju, Republic of Korea
2Gonadotropin-releasing hormone (GnRH)
- Decapeptide that is synthesized and secreted from
hypothalamic neurons. - Important in human reproduction and can possibly
be used in cancer therapy. - Research has been focused on finding distinct
types of GnRH receptors using mammalian and
non-mammalian models.
3Introduction
Hypothalamus
GnRH
Pituitary
LH/FSH
Gonads
Gametes
Secondary sex characteristics
4Research Background
Signaling Transduction Pathways
- Hormones (GnRH) bind to receptors.
- Second messengers are activated.
- Inositol triphosphate (IP) functions as a
signaling molecule by releasing intracellular
calcium ions and activating protein kinases.
5Research Background
- In order to determine how the GnRH receptors
work, they measured the IP production. - GnRH receptor genes were cut in three segments to
form chimeric receptors to determine which part
is responsible for IP production.
6Hind III
Bam HI
7Hind III
BamHI
bf1
GnRH receptors
dy1
bfBdy
bfHdy
dyBbf
8Hind III
BamHI
9Purpose
To mutate the tyrosine283 amino acid into leucine
and phenylalanine on the rat GnRH receptor by
polymerase chain reaction.
10Scheme
V I C W
T P Y Y GTC ATC
TGC TGG ACT CCC TAC TAC V
L G I W Y
W F GTC CTA GGA ATC
TGC TAC TGG TTF
Tyrosine TAC Leucine
TTA Phenylalanine TTC
5 CCC TAC TTA GTC 3
5 CCC TAC TTC GTC 3
Designed Primers
11Rat GnRH Receptor
L-R
VECTOR
VECTOR
L-F
PC-R
PC-F
Primers PC-F -----------universal forward
PC-R-----------universal reverse L-F----------le
ucine forward L-R----------leucine reverse
F-F----------phenylalanine forward F-R----------
phenylalanine reverse
12 Methods
Procedure to Incorporate Mutation
- Polymerase Chain Reaction (PCR)
- Gel Electrophoresis
- Restriction Enzyme Digestion
- DNA Ligation
- Amplification of Mutated DNA by using
competent cells. - Transformation
- Inoculation
- Plasmid MiniPrep
- Gel Electrophoresis
- DNA concentration
DNA sequencing
131 kb marker
100 bp marker
Figure 2. Full length of mutated Rat GnRH
receptor.
Figure 1. Amplification of GnRH receptor using
the designed primers.
L-R
vector
vector
L-F
PC-F
PC-R
Figure 4. Gel after inoculation of Y-L mutation.
Figure 3. Vector DNA bands and full length GnRH
mutated product.
1 2 3 4 5 6 7 8 9
1 kb marker
1 kb marker
14Conclusion
- Mutation was achieved.
- Confirmation through sequencing.
-
- Further research continues at the lab to measure
the effects of this mutation.
15Acknowledgments
- Hormone Research Center, S. Korea
- Da Young Oh
- Dr. Jae Seong
- Dr. Hyuk Kwon
- Barry University
- Dr. Peter Lin
- Dr. Ana Jimenez
- Dr. Flona Redway
- Sr. John Karen Frei, O.P., Ph. D.
- NIH FIC MIRT Grant
-
16