• CHARACTERISTICS OF RH PROTIEN
INTRODUCTION:
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Rhesus (Rh) factor is an inherited protein found on the surface of red
blood cells.
• It is the second most important blood group system
• Rh blood group system consists of 49 defined blood group antigens
• clinical importance of Rh-antigens
• 1- role in transfusion
• 2-role in autoimuune disease
• 3-hemolytic disease in newborns
• 4-abnormalitys in Rh null people
AIM:TO DETERMINE THE ROLE OF RH PROTEIN
IN TRANSFER ON NH4+ (AMMONIUM)
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To check if the Mep protein and Rh protein have the same function
we must check if there is a similarity in the sequence via the use of
BLAST( basic local alignment search tool)If I want to know the
function of a certain gene encoding protein I can go to method of
alignment using BLAST.
BLAST is a database that search for stretches of similarity in the
sequence between genes
There are several BLAST that we can use (N,P,X)
AMT,MEP have role in ammonium transportation.
From this alignment we concluded that Rh,MEP,AMT act as one
family of certain function but each in different organism.
• So, do Rh acts as ammonium transporter?
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ARTICLE OF MARINI IN
1997
Until article of Marini in 1997,some topological
characteristics of Rh protein were determined:
• Rh protein spans the membrane 12 times which is a
property of most transporters, its was proposed
that they are responsible for the transfer of
ammonium from the outside to the inside of the
cell.
• To validate if Rh proteins transport ammonium or
not some experiments were done.
YEAST SACCHAROMYCES
CEREVISIAE
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It is a unicellular eukaryotic organism,belongs to fungi
It needs carbon and nitrogen source to live(ammonium)
Used for fermentation
Used as model organism to study human diseases.
MEP PROTIEN:
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Mep genes (mep1, mep2,mep3,) are genes found in the yeast
saccharomyces cerevisiae ,they are redundant genes that code for
the proteins mep1, mep2 and mep3 respectively, all of these proteins
have the same function of transferring amonia in and out of the cell.
MEP PROTEIN FUNCTION
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The mep protein is responsible to transfer ammonium into the yeast
to provide a nitrogen source for it to live.
At high concentration (>5mM) ammonium can enter to the yeast by
diffusion, but at low concentration (<5mM) the Mep protein helps
uptake ammonium so the yeast can live in such conditions.
DELETING THE MEP GENES
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deleting ONEof the Mep genes (MEP delta )can be compensated by
the other Mep gene ( example the deletion of Mep1 is compensated by
Mep2 and Mep3)
A triple Mep delta yeast has all the Mep genes deleted,where it can’t
live in a medium containing [ammonium]<5mM
SECOND ARTICLE OF
MARINI
Aim is to determine wheather Rh protein transport ammonium or not.
To show if RhAG will compensate function of triple MEP delta gene in yeast in
less than 5mM of ammonium,3 vectors were designed:
• Vector 1:empty(no gene cloned)
• Vector 2:expression vector containing RhAG gene expressed under the control
of methionine promoter
• Vector 3:RhGK gene was expressed under control of methionine promoter.
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They transformed those vectors into the yeast with the previously mentioned
conditions.
• Then western blotting was applied .
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These
results were obtained
RESULTS OF THE
EXPERIMENT:
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The wild type transformed with empty vector expressed no protein
• Also in case of MEP delta within empty vector no protein was
expressed
• However,MEP delta transformed with vector containing RhAG ,a band
of protein was detected .so cloning in this case succeeded ,yeast is
able to express RhAG at the protein level.
So RhAG protein (same for RhGK) complement a yeast mutant
deficient in ammonium transport.
★ Also RhGK functions slightly more efficiently than RhAG
★
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SPECIFICITY OF RH
PROTEIN:
To make sure that the function of the Rh protein is to transport ammonium only.
1-we insert the Rh genes into gap1 delta cells that can’t uptake amino acids.
result of 1:after putting them on a medium containing amino acids the cells died since they
can’t uptake amino acids.
2-Also we insert the Rh gene into trk1 delta that can’t uptake potassium.
result of 2- After placing them in a meduim containing postassium the cells died since they
cant uptake potassium
★ SO THE TRANSPORT FUNCTION RESTORED BY
RHAG AND RHGK IN YEAST APPEARS
AMMONIOUM SPECIFIC BECAUSE :
● THE RH PROTEINS COULD NOT REPAIR THE
GROWTH DEFECTS OF THE GAP1 DELTA AND
AGP1 DELTA DOUBLE MUTANT DEFICIENT IN
AA TRANSPORT ,NOR THOSE OF THE TRK1
DELTA AND TRK2 DELTA DOUBLE MUTANT
DEFICIENT IN POTASSIUM TRANSPORT.
AIM: TO DETERMINE THE FUNCTION OF
RH PROTEIN IN AMMONIUM EXPORT.
When yeast is grown in a medium containing high concentration of
ammonium
accumulation of ammonium leading to toxicity
● There should be a balance ,yeast should utilize certain concentration
of ammonium , where excess should be excreted out.
● So MEP proteins have the ability to export ammonium to avoid
accumulation into toxic levels
● But do RhAG and RhGK have role in export of ammonium?
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They grow wild type and triple MEP delta in a medium containing methyl
ammonium at 200mM (form of ammonium but when it accumulates inside cell,it
cause toxicity)
Results of this experiment:
Usually triple MEP delta cells die at this high [ammonium] since it is toxic to it
,yet RhAG and RhGK containing cells were able to grow since they were able to
express excess of ammonium out of the cells.
SO:
1. RhAG and RhGK proteis confer resistance to methylammonium to yeast cells.
2. RhAG and RhGK promote excretion of ammonium.
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the End
Done by:Rawan saleh
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