A Few Questions Regarding Genetic Engineering in Plants...

[Herron]

Hello,

 

I'm a student entering university soon and have a, frankly, rudimentary understanding of the intricacies genetic engineering of plants. Thus, I have a few questions regarding it.

 

1. This is probably the simplest of my questions; when injecting foreign DNA into plant cells, does it necessarily matter where you place the new gene into the plant's genome as long as the necessary "trigger" sequences are in the gene (that is, the sequences that tells the plant to produce the certain encoded enzyme)?

 

2. What generally dictates/limits how much of a certain compound is produced by an enzyme (other than availability of raw reactants) with regards to genetic sequences. Would it do any good to insert the same gene for a certain enzyme multiple times to increase the prevalence of a compound?

 

3. How does one "tell" restriction enzymes where to cut? I know that there is a 'header' sequence telling the enzyme where to cut, but how does one put that particular sequence in the restriction enzyme? And how many 'nucleotides' should one put in the 'header' sequence? Is it possible that the restriction enzyme cuts at the wrong spot/multiple spots?

 

 

Thanks!

[Mr Skeptic]

1. This is probably the simplest of my questions; when injecting foreign DNA into plant cells, does it necessarily matter where you place the new gene into the plant's genome as long as the necessary "trigger" sequences are in the gene (that is, the sequences that tells the plant to produce the certain encoded enzyme)?

 

I don't think so, but you at least have to make sure that it is not inserted in the middle of another gene and deactivates that one.

 

2. What generally dictates/limits how much of a certain compound is produced by an enzyme (other than availability of raw reactants) with regards to genetic sequences. Would it do any good to insert the same gene for a certain enzyme multiple times to increase the prevalence of a compound?

 

Some enzymes have a feedback loop regulating them. For example, the first enzyme in a chain might have the final product as an inhibitor. Some enzymes don't have this kind of regulation, of course, and are limited by the concentration of substrate.

 

3. How does one "tell" restriction enzymes where to cut? I know that there is a 'header' sequence telling the enzyme where to cut, but how does one put that particular sequence in the restriction enzyme? And how many 'nucleotides' should one put in the 'header' sequence? Is it possible that the restriction enzyme cuts at the wrong spot/multiple spots?

 

Don't have a clue on that one. It's possible for the enzyme to cut the wrong spot, but I don't know how likely.

[CharonY]

As sceptic said, plus:

 

1) If promoters are intact and you do not run into any frame-shifts you should be fine.

 

2)Also of course certain enzymes have higher activities than others, this is cannot usually be predicted by looking at the sequence, though. Unless, of course it has been experimentally elucidated before. Adding the sequence several times can increase enzyme concentration, and hence substrate conversion. This however depends on their regulation. Sometimes it is easier to alter the promoter (that is, exchanging it for a stronger one) to enhance enzyme production rather than adding a gene.

 

3)I am not sure what you mean. Restriction enzymes (at least type II) are intrinsically specific for their cutting sites. Normally you do not need to "tell" them anything. While there are efforts to artificially construct restriction enzymes afaik it is not yet possible to predict cutting sites merely by looking at the protein sequences. In other words, to cut at a specific site you do not construct a restriction enzyme, but look which existing enzyme might cut there. Essentially you are limited to the available cutting sequences (that is why most cloning vectors have multiple clonings sites=collection of restriction sites). And yes they may cut at the wrong sites but only under certain conditions. This is referred to as star activity and happens usually with the wrong buffer and high enzyme to substrate ratio.

Note this is only for type II restriction endonucleases, which are the only ones used for this purposes. Other restriction enzyme classes (I and III) do not cut specific to a sequence.