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Gene expression

Genetics as it applies to evolution, molecular biology, and medical aspects.

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Gene expression

Postby Linn » Mon May 08, 2006 1:45 am

Expression of UGA-Containing Mycoplasma Genes in Bacillus subtilis. ...

Can someone or (lilkim?)please explain what this means?
I have read about it but i do not know about genetics and need
a explanation :lol: for dummies
Thanx in advance
~Lynne
"How far you go in life depends on your being tender with the young, compassionate with the aged, sympathetic with the striving and tolerant of the weak and strong. Because someday in life you will have been all of these".

~ George washington Carver
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Postby LilKim » Mon May 08, 2006 4:08 am

Hey Linn!

Unfortunately... i'm not familiar with "expression of UGA-Containig Mycoplasma Genes in bacillus subtilis"

and..to make matters worse i'm CRAMMING for my final (it's a take home we get it tuesday and it's due on friday)....

.. so I don't expect to have a lot of time to 'research' it over the next few days.

Hopefully, somone else will know how to answer your questions. (and, if not .. I'll try to figure it out towards the end of the week).

Sorry about that!
- Kim
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Postby Linn » Mon May 08, 2006 2:13 pm

Hey! Good Luck on your finals,
hope you dont get a migraine like I used to! :(
In the mean time I am going to try to figure it out wikipedia has an easy definition of gene

Expression:http://en.wikipedia.org/wiki/Gene_expression

I just want to make sure I understand.
~Lynne
"How far you go in life depends on your being tender with the young, compassionate with the aged, sympathetic with the striving and tolerant of the weak and strong. Because someday in life you will have been all of these".

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Postby LilKim » Tue May 09, 2006 3:04 pm

Hey Linn!

Ok... i'm procrastinating at the moment and did a quick pub med search for this question.

I found an interesting introduction that kinda explains the UGA codon thing...

So, generally in most eukaryotic organisms mRNA translation will terminate at UGA sequences (UGA is a stop codon). However, Mycoplasma doesn't "stop" translation at UGA.. instead it inserts a trytophan in the growing amino acid sequence and the nacent peptide will continue to grow until different stop codon is encountered (UAA or UAG .. those are 2 other stop codons)

Apparently this is an interesting feature of mycoplasm and it makes it difficult to perform classical genetic studies. For instance, (use your imagination) normally if a scientist scientist found a new mouse gene that made the pups glow fluorescent pink for the first 20 days of their lives... the scientist may opt to investigate this gene by inserting a copy of the pink gene into anorther organisms (such as e.coli or yeast) and then waiting to see if the new colonies started to glow-pink. And, if/when the colonies started glowing.. the scientist could feel confident in saying that the yeast/e.coli glow because of the gene that they identified in the mouse.

Conversely, if a scientist identified a new mycoplasma gene (containing UGA codons) causing pink fluorescence ... He would not be able to further investigate if the protein was-in-fact causing pink-fluorescent color by inserting the gene into e.coli or yeast. Because both yeast and e.coli read "UGA" as a stop.. instead of a tryptophan. As a result the "Pink" protien would be truncated and non-functional.

BUT!!!

B. subtillis is a known "model-organism" which many scientist work with. And i guess subtilis also inserts tryptophan instead of stopping at UGA sequences. Therefore, if the scientist inserted the mycoplasma (UGA containing)"Pink" gene sequence into subtilis... subtilis theoretically should be able to accurately translate the full legnth fluorescent "pink" protiens (Simplyy because Mycoplasma and subtilis utilize the same amino acid codons). Therefore, B. Subtilis is one of the best model organisms to express mycoplasma derived genes.

.... Anyways, i hope that this isn't too complicated. Please let me know if it is, and i'll "bring-it-down-a-notch". I based this explanation on the introduction of the paper below (i cut and pasted the intro. and reference):

Expression of UGA-Containing Mycoplasma Genes in Bacillus subtilis

T. R. Kannan and Joel B. Baseman*
Department of Microbiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7758


Received 3 September 1999/Accepted 16 February 2000

The Mollicutes represent a class of unique cell wall-less procaryotes that includes members of the genus Mycoplasma, a distinct subgroup pathogenic for humans and animals (3, 12). Gene structure and function analyses of mycoplasmas and other genera taxonomically categorized under the Mollicutes are limited by the lack of classical genetic systems and the inability to express cloned genes in native Mycoplasma hosts (7). A fundamental limitation of Mycoplasma gene expression is the unusual codon usage pattern displayed by mycoplasmas (24). Like mitochondria, Mycoplasma species utilize the UGA codon to encode tryptophan (9) rather than to serve as a stop codon. In general, Mycoplasma genes containing one or more UGA codons will not be expressed in commonly used expression systems that strictly adhere to the universal genetic code, due to premature termination. An exception is the very limited readthrough of the UGA stop codon in Escherichia coli and Salmonella enterica serovar Typhimurium (18, 19). Interestingly, although UGA also functions as a termination codon in Bacillus subtilis, the efficiency of UGA readthrough in this gram-positive bacterium is high due to the presence of a tRNA that reads the UGA termination codon as tryptophan (13). Therefore, UGA readthrough in B. subtilis is substantially leakier than that in members of the family Enterobacteriaceae (11). Furthermore, B. subtilis mutants with mutations in the structural gene (prfB) for release factor 2 (RF2) demonstrated increased readthrough levels by overcoming UGA-mediated termination (11). These observations enabled us to use B. subtilis as a host to express specific UGA-containing Mycoplasma genes.


... please let me know if my explanation is "gibberish"

have a sparkling day!
- KIM
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Postby Linn » Tue May 09, 2006 7:18 pm

Thank you so much for your help. It really helped lot. :D
I cant figure out why they would want to mess with mycoplasma,
e-coli etc :?

There are some new mycoplasma diseases cropping up ans its resistant to antibiotics.
in horticulture it is attacking legume species/peanuts.
_
And here is some interesting use of B. sublitis (A really interesting bacteria)

https://rpas.web-p.cisti.nrc.ca/RPAS/RPViewDoc?

They even use it in food.

Thanx again
~Lynne
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Postby canalon » Tue May 09, 2006 7:28 pm

Messing with all this I can tell you why. Bacteria tend to reproduce fast and with little demand. And they have a simplified but mostly compatible genetic system with the rest of the living world (a good argument in favor of evolution...) so you can use to isolate and study little part of more complex organisms/cells.
In the case of Mycoplasma, it is because those bugs are demanding and usually growing very slowly, so if you transfer the genes that interest you in another bacteria you will be able to isolate proteins etc. much faster.
Patrick

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Postby Linn » Tue May 09, 2006 7:43 pm

In the case of Mycoplasma, it is because those bugs are demanding and usually growing very slowly, so if you transfer the genes that interest you in another bacteria you will be able to isolate proteins etc. much faster.


So then, if mycoplasma genes are transfered in to B. sublitis, What changes (mutations) could occur with it (sublitis) ?
"How far you go in life depends on your being tender with the young, compassionate with the aged, sympathetic with the striving and tolerant of the weak and strong. Because someday in life you will have been all of these".

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Postby canalon » Tue May 09, 2006 8:41 pm

You do not except mutation.
You just want your B. subtilis to produce the mycobacterium gene the way you want (in bigger amount, only at certain time...)
Kim paper just state that there is a minor difference between the genetic code of the mycoplasma, and most of the rest of the living world. But B. subtilis sort of make sometimes the same mistake, allowing the production of complete Mycoplamic proteins instead of the truncated form that you would get with other expression system.
Patrick

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Postby Linn » Tue May 09, 2006 11:19 pm

Patrick,
Oh so by "you do not acept mutation' do you mean it is controlled?
So B sublitis is another word for using it called the (binding protein)
that is used?

I am wondering because I have come across a few articles of B sublitis used in products and allergic reactions.

and I am researching the
micoplasma, one Mycoplasma fermentans.

I was hoping no hybrids between the two exsist. :?:

Thank you mucho for the help. :)

~Lynne
"How far you go in life depends on your being tender with the young, compassionate with the aged, sympathetic with the striving and tolerant of the weak and strong. Because someday in life you will have been all of these".

~ George washington Carver
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Postby canalon » Tue May 09, 2006 11:37 pm

Soory for the type I meant "expect". You d onot expect a mutation when you are transfering genes because it would then chamge your produce, and this would not help you understand the role of the said gene.

B. subtilis is Gram positive bacteria, just like E. coli is a Gram negative one. Both are used as a model for those 2 big classes of bacteria because they are not demanding (in term of culture medium) and rather inocuous. In fact B. subtilis is even more inocuous than E. coli and can be used in human food.

Since it is a complete bacteria it can be used as a small factory to express genes (and the produce of a gene is a protein).

It can cause allergic reaction (but it is probably not very frequent considering that it belongs to our intestinal flora) as anything of its size and so full of protein.

Hybrids between B. subtilis and Mycoplasma are not likely, and would probably be very unstable anyway. But it is common practice in molecular biology to transfer genes between organisms to understand their role and regulation. But in this case Kim paper just says that B. subtilis is a better choice than E. coli to study mycoplasma genes because of this defective stop codon that they share in some extent.
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