active genes and expression problems! - (Sep/11/2004 )
all i learnt about protein expression till now is gone, may i say, just because an explanation from my professor...i just hope that he was not right about this, since it turns my "world" up side down!
the aim was to investigate the different expression degrees of the yeast genes. we used S. cerevisiae. we transformed the yeasts with a transposon carrying a lacZ gene. by homolog recombination the transposon with the lacZ gene was inserted into the different yeast genes. we selected for blue colonies. we got different degree of the blue colour. it showed that we got less blue colonies than expected. i asked my professor and he explained;
-> because 80% of the yeast genome codes for active genes ( open reading frames without non sense stop codons) if the transposon was inserted into the active genes then lacZ can't be expressed even if the insertion was in frame with the active genes!
yes, this was what he told me. from my knowledge i thought it does not make any different which genes a DNA segment is inserted into. as long as the fusion is in frame between the different genes, then expression would be! now he said that it does not include active genes. WHY CAN'T ACTIVE GENES GIVE LACZ EXPRESSION?
BTW what kind of genes gave the expression of lacZ we got in this experiment? how can they give the expression of the lacZ???
thanks so much for reading and help!
in my oppinion it has to do something with essential genes.
if your lacZ inserts into a vital gene of yeast, even if it's in frame it will destroy the original proteinsequence. so a vital protein is missing in this clone, so it won't grow at all, so no expression of lacZ, and you won't see the blue colony.
if lacZ "hits" a non-essential gene AND is in an exon (not spiced out) AND is in frame AND the gene the insertion occured is active under the conditions you grow your yeast, the yeast will grow and be blue - it may have some defects though.
i'm not a yeast researcher though, maybe I'm wrong.
it means alot to me that you replied! i have some more questions and i really hope that you can keep helping me out. it would help me alot.
this is not about yeast genes, but in generally about expression:
in the case where we get expresssed lacZ , should the insertion of lacZ be infront of the other's gene start codon or behind it? if lacZ is behind it, then the translation will give a product with both sequence of the other gene and lacZ. would not this interfere with lacZ function? and in this case would this interfere with the function of the other gene?
wait so much for your replies!
thanks a bunch!
the insertion lacZ should be "after" (downstream) of the gene's start codon, since the lacZ gene you use usually shound't have a promotor region of it's own - you're out to study gene-regulation of yeast genes, not the regulation of lacZ in yeast, aren't you?
and yes, the fusion of lacZ with other genes may interfer with function of both genes, but then again, it may not, in which case you get "blues".