Changing Course

So after many attempts and re-workings, new primer sets and fresh enzymes, plates with colonies and negative cultures, we decided that the LacZ fusion was not going to work and devoting any more time to it did not make sense.  We still needed a mechanism for visualizing Rad23 expression at our fourth chromosome heterochromatin site.  A visual marker, like the white gene I described earlier, is the easiest way to monitor gene expression, but wasn’t ideal because we weren't sure it would function as a fusion protein with Rad23, and because it is a fly gene we worried it might have its own effects on heterochromatin formation, if only minor.  One of my major concerns about this situation has been the lack of the knowledge of the Rad23 protein.  There has not yet been work done on the Drosophila Rad23 protein.  There are high quality predictions of the Rad23 coding sequence, which we used when designing our attempts to generate the Rad23-LacZ reporter.  To get around this, I decided to design a reporter that would not require an accurate Rad23 prediction to work.  Instead of Rad23 fused to a reporter protein, I made use of an internal ribosome entry site construct in conjunction with a fluorescent marker mVenus. 

Summary of transcription and translation:  From NIH Stem Cell report.  

©Terese Winslow.

A bit of background/refresher; for an mRNA message that was transcribed from a gene to lead to protein production, the mRNA must be translated.  The concept of transcription versus translation can be a bit tricky.  I always remember it by thinking of a conversation, if you were making a copy of a conversation, in the same language, but using your own vernacular, you’d be transcribing.  This is much the same as an RNA transcript being generated from DNA in the genome, RNA and DNA are essentially the same language, both nucleic acid based, but they have their own special vernacular, in RNA you have Uracil instead of Thymine found in DNA.  In contrast, if you were copying a conversation from English to French, that would be a translation, changing it from one language to another.  In generating protein, this is essentially what happens, the RNA nucleic acid language must be translated into the protein amino acid language, the two are very different, but you can only make a protein if you have the correct mRNA to translate.  Translation is done by ribosomes, first they bind to mRNA by recognizing a ribosome entry site.  Then ribosomes use the mRNA template to make a protein by joining amino acids together in a specific order.  Some mRNA transcripts have more than one ribosome entry site, so more than one separate protein can be generated from the same transcript.

The Rad23-mVenus construct.  The bent arrow indicates the transcription start site (TSS), the asterisk indicates the start codon (beginning of protein coding sequence).  UTR: untranslated region, IRES: internal ribosome entry site, SV40T: SV40 terminator sequence.

This is what I chose to exploit for our new construct.  We included Rad23 with an upstream repeat sequence known as 1360 and its 5’ untranslated sequence through the stop codon, then we added an internal ribosome entry site (IRES) and the mVenus coding sequences, as well as a strong transcriptional terminator (SV40T), which is necessary to ensure good transcript levels.  This construct was used with the MiMIC vector system and injected into embryos.  Next up, we’ll talk about generating flies with your desired sequences, the techniques and genetics involved.

You can’t always get what you want…

Our goals for this project include understanding how a typical gene found on the Drosophila melanogaster fourth chromosome has adapted to allow expression in the context of heterochromatin.  Generally, we consider heterochromatic regions of chromosomes to be hostile towards gene expression, maintaining a compact state that prevents necessary transcription factors and/or transcriptional machinery to access genes.  We have selected the Rad23 gene, located with in a stereotypically heterochromatic region on the fourth chromosome, but known to be expressed.  This gene should allow us to answer questions regarding how a gene adapts to heterochromatin.  We want to know if we move Rad23 to another heterochromatic region on the fourth, will it behave as it does in its normal location?  In order to do this, we have to make a version of Rad23 that we can analyze.  To do this, we planned to link Rad23 to the enzyme LacZ (also known as beta-galactosidase).  In enzymatic assays using X-gal, LacZ produces a product that can be visualized and quantitated.  This technique is commonly used in molecular biology research, and Drosophila researchers have used it in the past (including our collaborators: Lu BY, Ma J, and Eissenberg JC.  Development 1998).

Cloning strategy for creating the Rad23-LacZ fusion.

For this technique to work, we attempted to create a protein fusion between Rad23 and LacZ based on the predicted protein structure of Rad23.  Using standard molecular cloning techniques, we attempted to bring together the DNA of Rad23 with the DNA of LacZ, and a linker region that would form a flexible hinge between the two proteins, in an effort to ensure that the LacZ protein would retain its enzymatic function.  This is where we ran into problems.  The LacZ DNA cloning did not go well, but we were able to grow it in our desired vector if we only grew our bacteria with the DNA on plates and not in liquid culture.  However, once we attempted to add the Rad23 to the LacZ DNA, nothing would grow.  We tried different primer sets, different restriction enzymes.  We checked the ligations to ensure that the DNA pieces were being linked together.  Everything was working as it should, but once the DNA was put in the bacteria so we could grow it up and work with it, the bacteria wouldn’t grow. 

Ligations of 5'UTR and Rad23
with LacZ and the 3'UTR
*Indicate successful ligation products
of expected sizes.

So why didn’t our strategy work?  It’s hard to know exactly.  There are a couple possibilities.  It could be that LacZ was toxic to the cells we are using.  This seems unlikely, at least on it’s own, as LacZ is commonly used in this way.  However in the context of our particular DNA vector, it is possible that the LacZ gene was not stable or caused some other problems.  When we started having problems we inquired of other labs to see if they had experience with this sort of construct and were told they no longer used LacZ in the way we were trying to because they’d encountered similar problems.  After a lot of work trying to create the Rad23-LacZ fusion we decided it was time to cut our losses and find a different system to mark Rad23.