restriction enzyme gene in eucaryotes

[Dak]

hi everyone

 

am currently wrighting my bacholorate dissertation, and was wondering if i could pick your brains on a subject?

 

basiccally, i was wondering if anyone knows whether the prokaryotic genes encoding restriction enzymes will be properly expressed by eukaryotes, in particular humans

 

i cannot find any published data on this subject, so if anyone has any idea -- anecdotal or otherwize -- i would very much appreciate it

 

thanx all

[Bluenoise]

Just a running thought here.

 

Well microbes that produce REN's have thier REN sites methylated to protect themselves right? So wouldn't producing functional REN's in eukaryotes lacking these protection cut-up thier own DNA? Assuming the REN is capable of moving accross the nuclear membrane.

 

There should be no reason why the ORF cannot be properly transcribed and translated. I guess It boils down to

1) if the protein produced will be functional (my guess yes).

2) What interaction said REN if functional will have with a eukaryotic cell.

I think this second question is where the problem lies that either the cell will actively degrade the REN or that the REN will degrade your hosts DNA. So you'd either get no RENS, or a cell that dies once properly modified.

[rakuenso]

hi everyone

 

am currently wrighting my bacholorate dissertation' date=' and was wondering if i could pick your brains on a subject?

 

basiccally, i was wondering if anyone knows whether the prokaryotic genes encoding restriction enzymes will be properly expressed by eukaryotes, in particular humans

 

i cannot find any published data on this subject, so if anyone has any idea -- anecdotal or otherwize -- i would very much appreciate it

 

thanx all[/quote']

 

Even if it was possible, I'm not sure exactly as how you can express it feasibly since it would require you to directly insert it into the gametes

[Skye]

But that's quite possible, a good description:

 

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/TransgenicAnimals.html

[rakuenso]

Even if it was technically possible, crazy right wings and religious groups wouldnt allow it for the heck of it. But interesting read...

[Dak]

hi there, cheers all for your replys

 

the idea that im investigating in my dissertation is the potential use(s) of restriction enzymes to combat hiv infection, so to answre the issues that you rased:

 

the gene would not be inserted into the gamete, but into the genome of the cd4+ cells (helper-T-cells, macrophages, dendrites) using a HIV vector

 

the host genome would be protected from restriction by either: using a restriction enzyme that cannot re-enter the nucleus after being trannslated (which could restrict the HIV genes as they travel through the cytoplasm); using a restriction enzyme that is regulated by a HIV-tat promotor, ie upon hiv infection would create a restriction enzyme to completely distroy the cell; or introdusing both the restriction enzyme gene and the methylation enzyme gene aswell,

 

There should be no reason why the ORF cannot be properly transcribed and translated. I guess It boils down to

1) if the protein produced will be functional (my guess yes).

2) What interaction said REN if functional will have with a eukaryotic cell.

I think this second question is where the problem lies that either the cell will actively degrade the REN or that the REN will degrade your hosts DNA. So you'd either get no RENS, or a cell that dies once properly modified.

 

if it would be translated, then does anyone know whether or not the cell would degrade the res enzm? i cant find any published info on this anywhere (unsurprisingly).

 

and what does ORF mean?

 

cheers again for all your replys

[Dak]

oh right, open reading frame. makes scence now

[Bluenoise]

I think the main question you should ask is why do Eukaryotes not have restriction enzymes in the first place? They seem like a very valuable defense so there might be a good reason behind their absense. I'll ask molecular biology proff of mine and see what she has to say.

 

Here's something you should think of. HIV is a retero virus, so it doesn't contain DNA. If the reverse transcription happens in a manner where the DNA might get into the nucleus before the REN can get to it then you might need a REN that has to be able to access the neucleus to remove it. Which would also require methylase activity to protect the host. hmm

[Dak]

"hmm" indeed... alot of annoying hurdles to sort out in this idea

 

hiv reverse-transcribes in the cytoplasm before transporting a DNA copy of its genome into the nucleus, so cytoplasmic ResNms could cut it up in the cytoplasm and leave the host genome intact, as long as the ResNms do not gain entry into the nucleus.

 

i believe that eukaryotes probably ditched the bacterial Res-Mod system as we use methylations for other purposes -- cytosine methylation is used to deactivate genes and adenosine methylation performs an unknown function in eukaryotes, but it is thought to maybe be involved in binding long loops of dna to the nuclear membrane during dna synthesis (any imput on the role of A methylation would be appreciated), and C and A are the only two bases that can be methylased. i guess gene regulation is a more important function than cellular immunity, as we already have an immune system to protect us, and methylations cannot easaly perform both functions simultaniously.

[Bluenoise]

If the reason we don't have restriction enzymes is because methylation is reserved for higher purposes, then it'd make sense that RENs are able to enter the nuclues. If they couldn't methylation wouldn't matter either way. So either evolution didn't create REN's that are not able to acess Eukaryotic DNA, or having REN'S not active towards the Host's Chromosome lowers their usefullness to the point where they wouldn't be maintained in an Eukaryotic population. If they can't cut out of the nucleus, some viral DNA might still slip by and infect it at which point it'd be safe from restriction enzymes. Maybe the metabolic load of maintaining enough REN to be usefull might also be too high, we have other imune defenses already.

Yeah lots of questions.

[nahid]

hi everyone

 

am currently wrighting my bacholorate dissertation' date=' and was wondering if i could pick your brains on a subject?

 

basiccally, i was wondering if anyone knows whether the prokaryotic genes encoding restriction enzymes will be properly expressed by eukaryotes, in particular humans

 

i cannot find any published data on this subject, so if anyone has any idea -- anecdotal or otherwize -- i would very much appreciate it

 

thanx all[/quote']

Hi,

I have valuable information about your idea.PLZ give me your email adress.

sincerely

[mattbimbo]

hi dak, you may find it interesting to consider RNAinterference. and VDJ recombination.

[Dak]

Weee, it's my first ever post

 

Thanks guys, but I've handed my dissertation in now.

[Nashyboyo]

Just out of interest

Which restriction enzyme was it ? Why do you want to put it into a eukaryotic cell ? There are restriction enzyme properties in many proteins present in the eukaryotic nucleus!

[Dak]

it was for my dissertation, disscussing the possible anti-viral uses of restrictases.

[BioWizard]

For your purposes, you might be interested to look into interfering RNA (RNAi) instead of restriction enzymes, which as everyone has pointed out, might be a bit troublesome.