Biology help needed! JC1...

[junyang]

Scenario:

You are about to receive the “Mad Scientist Award” from HCJC for your pioneering work on the “Sick Cow Syndrome”, which costs dairies worldwide billions of dollars in terms of losses.



In the course of your research, you managed to isolate the cDNA sequence of the gene that you believe is responsible for the disease. Note that the cDNA sequence reverse-transcribed from mRNA – the first cDNA strand is complementary to the mRNA strand. The first cDNA strand is then used as a template in DNA replication to obtain the double-stranded cDNA



Armed with your knowledge of Bioinformatics, you sought to learn more about the gene.



The nucleotide sequence (non-transcribing strand) of the cDNA is as follows:

1 tgcattcgag gctgccagca agcaggtcct cgcagccccg ccatggccct gtggacacgc
61 ctgcggcccc tgctggccct gctggcgctc tggccccccc ccccggcccg cgccttcgtc
121 aaccagcatc tgtgtggctc ccacctggtg gaggcgctgt acctggtgtg cggagagcgc
181 ggcttcttct acacgcccaa ggcccgccgg gaggtggagg gcccgcaggt gggggcgctg
241 gagctggccg gaggcccggg cgcgggcggc ctggaggggc ccccgcagaa gcgtggcatc
301 gtggagcagt gctgtgccag cgtctgctcg ctctaccagc tggagaacta ctgtaactag
361 gcctgccccg acacaataaa cccttacgag caag

To begin with, you decide to translate the nucleotide sequence into meaningful polypeptide sequence. Using the ExPASy Molecular Biology Server (http://us.expasy.org/), translate the nucleotide sequence into polypeptide sequence using the translate tool from the Tools and Software Package.



You have six possible reading frames for the gene. Which is the correct reading frame (i.e. one that will yield a meaningful sequence of amino acids)?

 

[Wai]

hmm....never study this before when i was in JC...

anyway... :rbounce: for u...

i know there is at least one Biology teacher around, he should be able to help u

 

[Poledra]

:what: And that's JC1 work?

What the hell?

Lucky I'm not even taking science..

You want me to ask Year 2 Bio students from my school?

 

[mervlam]

Took a short course on Life Science last semester (and I'm from the College of Engineering, not some Biological Sci student ). I try to see if I can solve or else make some sense out of it. :what:

 

[junyang]

sigh.. i hate my jc.. :angry:

 

[mervlam]

Originally posted by junyang
sigh.. i hate my jc.. :angry:

take it easy. Life is not so good in Univ as well. I'm an Electrical student and I still have to take subjects from Life Science, Materials, Mechancial and Civil Engineering.

Take a deep breath and try again.

 

[mervlam]

you translated the nucleotides to polypeptides already?

 

[Prismatic]

Originally posted by mervlam
take it easy. Life is not so good in Univ as well. I'm an Electrical student and I still have to take subjects from Life Science, Materials, Mechancial and Civil Engineering.

Take a deep breath and try again.

Haha.... I'm in civil engineering and 1/3 of my subjects are mathematics subjects which I have never seen being applied on my engineering subjects.

 

[denizenx]

Originally posted by mervlam
you translated the nucleotides to polypeptides already?

I think that's the question itself...

is there any coincidence that the numbers all start with multiple of 6?

 

[mervlam]

Originally posted by denizenx
I think that's the question itself...

is there any coincidence that the numbers all start with multiple of 6?

i realised after translating the nucleotides to polypeptides, I don't have enough knowledge to choose which is the correct one from the six possible. :bsmilie:

Sorry junyang, can't help on these

 

[dehuis]

eh.. can't help much, return my genetics liao. but you can try yeocolin. he is a year 2 medic... should be closer to his area.

 

[junyang]

ahh nvm i guessed its the last one

few more to go :| i think i'm going to sleep. haha

2. The start codon would determine the start of translation (i.e. the start of the coding region). Identify the nucleotides for the start codon in the correct reading frame.

3. Your research confirms that the polypeptide obtained corresponds to that translated from the correct reading frame. With the amino acid sequence obtained, determine the molecular weight and pI of the protein using the Peptide Mass tool.

4. You then decide to identify the gene. You are going to compare the gene sequence with existing sequences in the National Centre for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/). Choose the BLAST function and perform a standard nucleotide-nucleotide BLAST. From your nucleotide BLAST results, identify the gene involved in the “Sick Cow Syndrome

5. One possible therapy for the sick cows would be to inject the native, “non-mutant” protein purified from close vertebrate relatives. You would want the protein to be as conserved as possible (i.e. with as little variations as possible) between the species to minimise the risk of rejection. From your nucleotide BLAST results, which “relative” would be the most ideal donor?

6. Being a young entrepreneur, you decide to make you first millions through cloning and expression of the proper cow’s gene. First of all, you observed that there is a highly conserved stretch of five amino acids at the beginning of the polypeptide sequence: FVNQH (N-Phe-Val-Asn-Gln-His-C). This would be useful in your primer synthesis for PCR cloning of the gene.
Which of the following would be an appropriate primer sequence? Note PCR primers are DNA primers
a 5’ TTTGTTAACCAGCAC 3’

b 5’ UUUGUUAACCAGCAC 3’

c 5’CACGACCAAUUGUUU 3’

d 5’CACGACCAATTGTTT 3’

 

[Wai]

Originally posted by Poledra
:what: And that's JC1 work?

What the hell?

Lucky I'm not even taking science..

You want me to ask Year 2 Bio students from my school?

That's why i dropped bio in the end when i was in JC...and there my hope of becoming a doctor was gone :cry: :cry:

A lvl vs O lvl biology is quite big

just compare number of terms u need to remember for a simple plant cell.....

 

[Poledra]

Originally posted by Wai
That's why i dropped bio in the end when i was in JC...and there my hope of becoming a doctor was gone :cry: :cry:

A lvl vs O lvl biology is quite big

just compare number of terms u need to remember for a simple plant cell.....

Haha..
I don't even have Sec 4 Bio. Not smart enough like some people to take it.

 

[mpenza]

kinda miss the study/school time.... definitely more free time compared to working.

 

[hongsien]

Here are the six possible frames: DNA always has a start and an end (start and stop codon= three aminoacid sequence, start codon is always ATG which is translated into methionine ATG and stop codon: TAG, TAA, or TGA)

5'3' Frame 1
C I R G C Q Q A G P R S P A Met A L W T R L R P L L A L L A L W P P P P A R A F V N Q H L C G S H L V E A L Y L V C G E R G F F Y T P K A R R E V E G P Q V G A L E L A G G P G A G G L E G P P Q K R G I V E Q C C A S V C S L Y Q L E N Y C N Stop A C P D T I N P Y E Q


5'3' Frame 2
A F E A A S K Q V L A A P P W P C G H A C G P C W P C W R S G P P P R P A P S S T S I C V A P T W W R R C T W C A E S A A S S T R P R P A G R W R A R R W G R W S W P E A R A R A A W R G P R R S V A S W S S A V P A S A R S T S W R T T V T R P A P T Q Stop T L T S K


5'3' Frame 3
H S R L P A S R S S Q P R H G P V D T P A A P A G P A G A L A P P P G P R L R Q P A S V W L P P G G G A V P G V R R A R L L L H A Q G P P G G G G P A G G G A G A G R R P G R G R P G G A P A E A W H R G A V L C Q R L L A L P A G E L L Stop L G L P R H N K P L R A


3'5' Frame 1
L A R K G L L C R G R P S Y S S S P A G R A S R R W H S T A P R C H A S A G A P P G R P R P G L R P A P A P P P A G P P P P G G P W A C R R S R A L R T P G T A P P P G G S H T D A G Stop R R R G P G G G A R A P A G P A G A A G V S T G P W R G C E D L L A G S L E C


3'5' Frame 2
L L V R V Y C V G A G L V T V V L Q L V E R A D A G T A L L H D A T L L R G P L Q A A R A R A S G Q L Q R P H L R A L H L P A G L G R V E E A A L S A H Q V Q R L H Q V G A T Q Met L V D E G A G R G G G P E R Q Q G Q Q G P Q A C P Q G H G G A A R T C L L A A S N A


3'5' Frame 3
C S Stop G F I V S G Q A Stop L Q Stop F S S W Stop S E Q T L A Q H C S T Met P R F C G G P S R P P A P G P P A S S S A P T C G P S T S R R A L G V Stop K K P R S P H T R Y S A S T R W E P H R C W L T K A R A G G G G Q S A S R A S R G R R R V H R A Met A G L R G P A C W Q P R Met

Above there is only one which makes sense.......so which one? Make a choice.......think it is quite obvious now, isn't it?

 

[hongsien]

The answer for question one was actually given in question two! See, you don't need to be smart, just read carefully, and if you listened carefull during the class you will know which software to use at http://us.expasy.org

Molecular Biology is just a tool to do things in biology nowadays, it is like learning HTML, there is no magic in it, as long as you know the rules of the game you will find your answer! So, please don't see it as something difficult. Just doing all these steps is just applying the rules, but to make sense of it and to know which tools to use depends on your experience.

Hong Sien

 

[hongsien]

I am sad to see question six though: making money seems to only thing that is on the mind, why must people always emphasize on making money?Is finding a cure not important anymore? Personally I find this a wrong motivation to do research.................

Hong Sien

 

[plsoong]

hi,

translate them to amino acids

then look for the longest open reading frame (ORF)

how?
the longest ORF is usually the correct amino acid sequence that encodes a functional protein

so look for the start codon (Met) and count the number of a.a. sequence until the stop codon.

the longest one in either frames 1 - 6 will the correct one

alternatively if u're lazy or have so much sequences until counting is impossible and tedious,

just plonk your a.a. sequence into www.ncbi.nlm.nih.gov (the ORF FINDER) and look for the longest shaded frame..click on it and it will give u the relevant sequences for it.

then highlight the complete sequence and do a BLASTp on the database on www.ncbi.nlm.nih.gov and check if its your correct protein.

All the best mate....

anyway, anything related to bio, (like tuition required ;p or any questions can PM me)

cheers

 

[aosis]

Originally posted by hongsien
Here are the six possible frames: DNA always has a start and an end (start and stop codon= three aminoacid sequence, start codon is always ATG which is translated into methionine ATG and stop codon: TAG, TAA, or TGA)

5'3' Frame 1
C I R G C Q Q A G P R S P A Met A L W T R L R P L L A L L A L W P P P P A R A F V N Q H L C G S H L V E A L Y L V C G E R G F F Y T P K A R R E V E G P Q V G A L E L A G G P G A G G L E G P P Q K R G I V E Q C C A S V C S L Y Q L E N Y C N Stop A C P D T I N P Y E Q


5'3' Frame 2
A F E A A S K Q V L A A P P W P C G H A C G P C W P C W R S G P P P R P A P S S T S I C V A P T W W R R C T W C A E S A A S S T R P R P A G R W R A R R W G R W S W P E A R A R A A W R G P R R S V A S W S S A V P A S A R S T S W R T T V T R P A P T Q Stop T L T S K


5'3' Frame 3
H S R L P A S R S S Q P R H G P V D T P A A P A G P A G A L A P P P G P R L R Q P A S V W L P P G G G A V P G V R R A R L L L H A Q G P P G G G G P A G G G A G A G R R P G R G R P G G A P A E A W H R G A V L C Q R L L A L P A G E L L Stop L G L P R H N K P L R A


3'5' Frame 1
L A R K G L L C R G R P S Y S S S P A G R A S R R W H S T A P R C H A S A G A P P G R P R P G L R P A P A P P P A G P P P P G G P W A C R R S R A L R T P G T A P P P G G S H T D A G Stop R R R G P G G G A R A P A G P A G A A G V S T G P W R G C E D L L A G S L E C


3'5' Frame 2
L L V R V Y C V G A G L V T V V L Q L V E R A D A G T A L L H D A T L L R G P L Q A A R A R A S G Q L Q R P H L R A L H L P A G L G R V E E A A L S A H Q V Q R L H Q V G A T Q Met L V D E G A G R G G G P E R Q Q G Q Q G P Q A C P Q G H G G A A R T C L L A A S N A


3'5' Frame 3
C S Stop G F I V S G Q A Stop L Q Stop F S S W Stop S E Q T L A Q H C S T Met P R F C G G P S R P P A P G P P A S S S A P T C G P S T S R R A L G V Stop K K P R S P H T R Y S A S T R W E P H R C W L T K A R A G G G G Q S A S R A S R G R R R V H R A Met A G L R G P A C W Q P R Met

Above there is only one which makes sense.......so which one? Make a choice.......think it is quite obvious now, isn't it?

I don't know how true is it. I never learn it anyway. Up for u !