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gene promoter - promoter luciferase (Nov/27/2005 )

If one finds that a 5'-flanking region of a gene shows promoter activity with luciferase assays.....
how can one confirm if this identified promoter region is actually the promoter of this gene...

we have strong promoters like CMV ...what identifies them as CMV promoters.....since they can transcribe any gene behind them (is this really true?)

most promoter identification experiments use luciferase or some other reporter assays..what is the underlying hypothesis under these sort of experiments...
what make us think that if a 5'-flanking region can show luciferase activity it is the promoter of our particular gene

if u r studying a promoter of EUKARYOTIC gene A..how can it be experimentally 'proved' that it is the gene A promoter .....please suggest some experiments....thanks a lot

isn't it true that any damn region if picked from the genome can EXHIBIT reporter activity...then what experiments can be done to prove that this is the promoter of a particular gene
please suggest some experiments....thanks a lot

-Watson-

Clone it (the putative promoter region fused to the promoterless luciferase gene) in the opposite orientation from the CMV promoter? It's tough to say it's the promoter, though -- some genes have more than one promoter, active under different circumstances.

But, an untranslated region that shows promoter activity an is spaced appropriately upstream from a gene can be proposed as a promoter...

You could also mutate that region and show loss of expression of the gene it putatively promotes...

-HomeBrew-

QUOTE (HomeBrew @ Nov 27 2005, 04:54 PM)
Clone it (the putative promoter region fused to the promoterless luciferase gene) in the opposite orientation from the CMV promoter? It's tough to say it's the promoter, though -- some genes have more than one promoter, active under different circumstances.

But, an untranslated region that shows promoter activity an is spaced appropriately upstream from a gene can be proposed as a promoter...

You could also mutate that region and show loss of expression of the gene it putatively promotes...

thanks
abt what ur saying .....but this will only prove that the identified regulatory region is not exhibiting promoter activity in the opposite direction.....(am I right?..I am not sure)

Moreover...I am studying a human gene promoter ..but the promoter of the same gene of lower organisms has not been studied ..like mouse or rat. promoter...

i tell u that because i was thinking of some experiments so that I can disrupt the promoter in vivo or in cell culture (since I am studying human gene ) and show that inactivation of this promoter by gene disruption can totally suppress expression of my gene in a particular cell in which it did use to express it..can this be done?...please suggest some reading..or experiments...can it be done the Cre rcombinase ways or the tet-O ways
I found a paper in which they fuse the promoter of a gene to CRe-recombinase..waht exactly is the
purpose of doing such experiments
link of th e paper
Paper- Neural expression of alpha-internexin promoter in vitro and in vivo
http://www3.interscience.wiley.com/cgi-bin...92749/HTMLSTART

-Watson-

QUOTE (Watson @ Nov 27 2005, 05:11 PM)
QUOTE (HomeBrew @ Nov 27 2005, 04:54 PM)

Clone it (the putative promoter region fused to the promoterless luciferase gene) in the opposite orientation from the CMV promoter? It's tough to say it's the promoter, though -- some genes have more than one promoter, active under different circumstances.

But, an untranslated region that shows promoter activity an is spaced appropriately upstream from a gene can be proposed as a promoter...

You could also mutate that region and show loss of expression of the gene it putatively promotes...

thanks
abt what ur saying .....but this will only prove that the identified regulatory region is not exhibiting promoter activity in the opposite direction.....(am I right?..I am not sure)

Moreover...I am studying a human gene promoter ..but the promoter of the same gene of lower organisms has not been studied ..like mouse or rat. promoter...

i tell u that because i was thinking of some experiments so that I can disrupt the promoter in vivo or in cell culture (since I am studying human gene ) and show that inactivation of this promoter by gene disruption can totally suppress expression of my gene in a particular cell in which it did use to express it..can this be done?...please suggest some reading..or experiments...can it be done the Cre rcombinase ways or the tet-O ways
I found a paper in which they fuse the promoter of a gene to CRe-recombinase..waht exactly is the
purpose of doing such experiments
link of th e paper
Paper- Neural expression of alpha-internexin promoter in vitro and in vivo
http://www3.interscience.wiley.com/cgi-bin...92749/HTMLSTART


does anyone have any more suggestions for this..please it will be greatly appreciated ..thanks

-Watson-