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

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In summary, the conversation discusses an experiment involving protein expression in yeast using a transposon carrying a lacZ gene. The results showed a lower degree of blue color than expected, which was explained by the professor as a result of the transposon being inserted into active genes that cannot express the lacZ gene. The conversation also touches on the factors that can affect gene expression and the role of IPTG in the experiment.
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sotellme
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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!
 
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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.
 
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First of all, I understand how frustrating it can be when new information contradicts what we have previously learned. However, it's important to remember that science is constantly evolving and what we think we know now may change in the future. It's also important to trust your professor's knowledge and expertise in the field.

To answer your question, active genes cannot give LacZ expression because they are constantly being transcribed and translated into functional proteins. The LacZ gene, on the other hand, is not a functional gene and does not have a specific function in the yeast cells. Therefore, even if the LacZ gene is inserted into an active gene, it will not be expressed because the yeast cells have no use for it.

The genes that gave expression of LacZ in your experiment are likely non-functional genes or genes that are not actively being transcribed and translated. These genes may have been in a state of low activity or not needed by the yeast cells, allowing for the LacZ gene to be expressed.

I hope this helps clarify the concept for you. Keep learning and questioning, and don't be afraid to ask your professor for further explanation. Good luck with your studies!
 

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

1. Why can't active genes give LacZ expression?

The LacZ gene is responsible for encoding beta-galactosidase, an enzyme that breaks down lactose into glucose and galactose. This enzyme is essential for the metabolism of lactose in bacteria, such as E. coli. However, active genes do not necessarily mean that they are producing the LacZ enzyme. The expression of a gene is a complex process that involves multiple steps, including transcription, translation, and post-translational modifications. If any of these steps are disrupted, the gene may not produce the desired product, in this case, the LacZ enzyme.

2. What factors affect the expression of LacZ?

The expression of LacZ can be affected by various factors, including the presence of inducers or repressors, the activity of regulatory proteins, the availability of necessary enzymes and cofactors, and the physical state of the DNA. For example, the LacZ gene is only expressed in the presence of lactose or its analogs, which act as inducers. On the other hand, glucose acts as a repressor, preventing the expression of the LacZ gene. Additionally, mutations in the regulatory elements or the gene itself can also affect its expression.

3. Can active genes give LacZ expression in all organisms?

No, active genes cannot give LacZ expression in all organisms. The LacZ gene is primarily found in bacteria, and its expression is tightly regulated by the lac operon. This operon is not present in all organisms, and therefore, the LacZ gene may not be expressed in those organisms. Additionally, different organisms may have different regulatory mechanisms for gene expression, so even if the LacZ gene is present, it may not be expressed in the same way as in bacteria.

4. How is LacZ expression measured in experiments?

LacZ expression can be measured using a technique called a beta-galactosidase assay. In this assay, a colorimetric substrate, such as X-gal, is added to the cells or tissue expressing LacZ. The LacZ enzyme cleaves the substrate, resulting in the production of a blue product. The intensity of the blue color can then be quantified using a spectrophotometer, providing a measure of the level of LacZ expression.

5. Can LacZ expression be used as a marker for active genes?

Yes, LacZ expression can be used as a marker for active genes, but it is not always a reliable marker. As mentioned earlier, the expression of a gene is a complex process, and many factors can affect it. Therefore, LacZ expression may not always reflect the activity of a gene accurately. Additionally, LacZ is only one of many genes that can be expressed and may not be representative of the overall gene expression in a cell or organism.

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