32 - AIIMS novemeber 2008 biochemistry mcqs - 1

Q1. what is the source of energy for light production in the firefly ?” 

1. ATP 
2. GTP 
3. phosho creatine 
4. NADH 

The answer is ATP…. 


Let me start the discussion with this question …..“DO YOU THINK THAT THE QUESTION IS RELEVANT TO US ?” 

If you think this question is irrelevant , then go through the following discussion & then you ll understand why these AIIMS people want us to know about the metabolic machinery of this humble organism. 

The firefly (Photinus pyralis) contains an enzyme called LUCIFERASE. This enzyme acts on its substrate – luciferin in the presence of ATP and calcium. The reaction produces visible light. This reaction has been exploited by the scientific community for research on 

1. antibiotic susceptibility testing 
2. pyro sequencing 
3. studies of metabolic pathways 
4. tumorigenesis 
5. transcriptional activity 
6. others 


ANTI BIOTIC SUSCEPTIBILITY TESTING 

Eg : MDR TB 

MDR TB is characterized by the presence of resistance to the two drugs 
Isoniazide …….. due to mutations of genes Kat G & INH A 
Rifampicin……..due the mutations in the gene rpo B gene 

To the mycobacteria cultured from the patient`s clinical samples we add adeno virus carrying the FFLUX gene (which codes for luciferase). Within a few minutes the mycobacteria exhibit bioluminescence i.e they emit light. Now add the antibiotic , say INH. If the mycobacteria are sensitive , they soon become non viable & their metabolic machinery is paralysed and hence ATP is depleted. In the absence of ATP , the light production is reduced. In fact the degree of reduction of the bio luminescence can be taken as an indicator of the potency of the drug . 
If the strains are resistant , there is NO reduction in light production 



PYRO SEQUENCING 


This is a type of DNA sequencing. 
Take the template DNA which is to be sequenced. To this we add luciferase, luciferin, calcium , DNA polymerase, ATP sulfurylase 
Then we add the deoxy nucleotides (A, C , G , T) one by one and wash them off. 
For instance let the d-NTP in x position of the template be A. When we add A, G , C there will be no reaction. But when we add T , base pairing occurs , releasing a PYRO PHOSPHATE, which is acted upon by ATP sulfurylase to produce ATP. 
When there is ATP , luciferase is activated and there is luminescence. So we know that the template contains the d NTP which is complementary to the one that we added.in the same way the entire NA can be sequenced 




STUDYING METABOLIC PATHWAYS 

Coenorhabditis elegans (a nematode) was the FIRST MULTICELLULAR ORGANISM FOR WHICH THE ENTIRE GENOME WAS SEQUENCED. 

An interesting aspect is that its cuticular layer is transparent. We can add the luciferase gene to its genome with the help of adeno or lenti virus. So whenever its metabolic machinery is activated an ATP is produced, light is emitted. 
Likewise whenever a metabolic poison is given the ‘light is switched off’ 
The degree of bio luminescence is proportional to the amount of ATP present 



STUDYING TRANSCRIPTIONAL ACTIVITIES 

To study the transcriptional activity of a gene , incorporate the luciferase gene close to the index gene `s promoter 
Whenever there is transcription, luciferase is also activated and light is emitted 


TUMORIGENESIS MODELS 

The tumor cells tagged with luciferase gene become luminescent and thereby their growth and susceptibility to chemo therapeutic agents can be studied in vivo within mouse models 


OTHERS 

Luciferase gene is also used in forensic medicine - detect blood and in studying specific cell lines