hi all,

 

I've question

we have human gene (with its promoter , TE , ....ect ) which code for a certain protein inserted in plasmed of bacteria , but what happen is there was no expressing for this gene !!! ... why????

 

-I think that may be mRNA degrated before translation by the action of nuclease enzymes. in other words, no capping or cleavage make the mRNA exposed to 3' exonuclease & 5' exonuclease.

 

- OR may be because there is a gene control the splicing for this gene which is not transferred to plasmed. I mean there is other gene may code for protein control splicing in specific way for exons that stop the translation or produce inactive protein. is it possible ?!

 

I hope someone help me in this question .

 

Thank you ,

 

Jumana

there is no 'human gene' as far as i know. we are not defined by a single gene but by much larger patterns in our genome and its not at all close to the pattern of a bacteria.

 

so, the 'human' gene is not expressed in bacteria because it doesn't exist. i can't find any reference to a gene named as such.

Apart from the poor phrasing, let me ask a question back at you. What would you need for a gene to be expressed in a given organism (in this case, bacterium).

Based on how the question is phrased I assume it is a theoretical or homework question. If it is really supposed to be a troubleshooting question, well the question itself needs some reworking as there is something definitely not right.

did you design the plasmid correctly? does expression of the protein have some deleterious effect?

It is a theoretical question.

What i was trying to say is we took a human gene and transferred to plasmed. We were expecting this gene gonna give its protein in bacteria, but it is not. I think the key thing here is the differences in gene expression between Eukaryote& prokaryote.

The key to this theoretical question lies in some of the information you provided.

 

The first thing to consider is the differences between eukaryotic and prokaryotic transcription. So you suppose that prokaryotic promoter sequences and transcription enhancer sequences are the same as eukaryotes? Will prokaryotic transcription enzymes recognise eukaryotic promoter regions?

 

Equally, what about the differences in post transcription processing between eukaryotes and prokaryotes? Do prokaryotes and eukaryotes process RNA into mRNA the same way? What are the differences between splicing for example.

 

Think about these questions and you'll arrive at your answers!

Almost too detailed, but important points. Although one could always wonder whether a EST was cloned or the native gene...

 

I'll move it into the homework section for now.

Human gene is not being expressed when it's inserted in bacterium plasmed. The reasons come from the differences in the gene expression between eukaryotic and prokaryotic.

 

- First difference is in the promoter structure and position. I think this surly will effect the transcription. Prokaryotic promoter has different structure from Eukaryotic one. This will affect the recognition of the transcription factors. The position is also different. Prokaryote promoter locates 10 to 35 nt.

 

- Second difference is in the RNA polymerase. In prokaryote there is only one type of RNA polymerase that does the transcription. While in eukaryotic there is three different RNA polymerases which means they are specialized for certain genes. Prokaryotic RNA will not recognize the special recognition signal in inserted human gene.

 

- Third reason is bacteria (prokaryotic) gene have only exons, with no entrons. Therefore there is no splicing process in bacteria. Human gene has entrons and must be spliced.

 

- Forth reason is some codons in bacteria code for different amino acid that human codons code for.

 

This is my assumptions.

First one is the major point although the last sentence

Prokaryote promoter locates 10 to 35 nt.

is incorrect. The -10 to -35 region is the sigma factor binding site which does not equal the promoter region.

 

The second is not a real issue. The mRNA of eukaryotes are also only transcribed by one polymerase species. The important bit is that each polymerase needs specific factors in order to interact with specific promoters.

 

The thirds is also a good point.

 

The fourth less so. The slight differences in some bacteria compared to animals may result in translation error but it does not necessary abolish the protein synthesis. However, the points mentioned above will be prohibitive for proper transcription so that translation would not occur in the first place.

 

If we assume that those problems are corrected there are even more problems during the protein synthesis parts.

And even if you could get the protein to be produced with the correct sequence (or even at all), it is unlikely to itself be properly processed (properly folded, glycosylated, etc.) nor have the correct pH, temperature, substrates, etc to function. Really better off using yeast or some other eukaryote.