Tyrosine283 Mutation of the Rat GnRH Receptor by Polymerase Chain Reaction

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Tyrosine283 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
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Gonadotropin-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.

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Introduction

Hypothalamus
GnRH
Pituitary
LH/FSH
Gonads
Gametes
Secondary sex characteristics
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Research 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.

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Research 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.

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Hind III
Bam HI
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Hind III
BamHI
bf1
GnRH receptors
dy1
bfBdy
bfHdy
dyBbf
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Hind III
BamHI
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Purpose
To mutate the tyrosine283 amino acid into leucine
and phenylalanine on the rat GnRH receptor by
polymerase chain reaction.
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Scheme
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
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Rat 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
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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
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1 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
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Conclusion

• Mutation was achieved.
• Confirmation through sequencing.
• Further research continues at the lab to measure
  the effects of this mutation.

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Acknowledgments

• 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

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