Active Genes & Protein Expression: Why Can't Active Genes Give LacZ Expression?

[sotellme]

all i learned 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!

 

[iansmith]

By active genes, you prof meant that about 80% of the genes have functions that essantial for the for the organism to grow. Therefore these genes are virtually always turned on. When you create a gene fusion by using random techniques, like the one you described, the fusion migth have an effect on the protein properties and therefore inhibiting the grow of or killing the organism. Also some genes are only turned on under specific condtions such as low iron, decreased temperature, presence of certain sugars, etc.

It is hard to define what genes would give permit the expression of the genes in the experiment you described. These genes will give a positive reaction with lacZ because this enzyme can fulfill its role even when the with significant amount of amino acid are attach to it. The expression is usually recorded using IPTG, a molecules that mimic lactose.