Progress Report 1
Identification of transcription factors that recognize the ATTCATTCATGA site
Fatih KOCABAS
In the one of the recent studies in the Stathopoulos laboratory, it was found that the ind enhancer, which affects dorsal and lateral patterning in Drosophila (Stathopoulos A., and Levine M., 2005), has a novel silencer element, and it also contains various activator and repressor elements. After that, following step should be to find related proteins which bind to this enhancer element by doing one hybrid screens which is the motivator part of my project.
In my project, there are five main steps to reach my goal; to identify transcription factors that recognize the 5’-ATTCATTCATGA -3’ sequence found in the A-box:
To Construct a DNA Reporter Vector
To Generate a cDNA Library
To Construct an AD Fusion Library
To Screen by Cotransformation
Analysis of positive interactions and conformation of DNA-binding activity.
In our project, our sequence is 5’-ATTCATTCATGA -3’. To find transcription factors which bind to this sequence we designed 2 different oligonucleotides and one mutant oligonucleotide that contains 5’-ATTAATTAATTA -3’ sequence with three different nucleotides.
First of all, before coming to Surf, my superviser Phoebe Tzou designed these sequences for me to start my project on time:
5’ GCA GCG CAT TCA TTC ATG AGG CCA AGA GCT 3’
3’ CTT CGC GTA AGT AAG TAC TCC GGT TCT CGA 5’
And
5’ GGA TGT ATT CAT TCA TGA AGT GTC TGA GCT 3’
3’ CCT ACA TAA GTA AGT ACT TCA CAG ACT CGA 5’
And mutant oligo:
5’ GGA TGT ATT AAT TAA TTA AGT GTC TGA GCT 3’
3’ CCT ACA TAA TTA ATT AAT TCA CAG ACT CGA 5’
After preparation of oligonucleotides from designed sequences, pHIS2 vector is used to clone the oligonucleotides into E.coli. pHIS2 is a reporter vector used in yeast one-hybrid screens to identify and characterize DNA-binding proteins because it can be maintained in both yeast and bacteria. After digestion of pHIS2 with SacI restriction enzyme and digested pHIS2 vector is ligated with three oligonucleotides, then pHIS2 vector is transformed easily into E.coli (DH5α).
During these studies, we also collected early Drosophila embryos (wild type) which are 0-4 hour’s embryos. After transformation, we selected 8 colonies from three oligos and made PCR to test colonies whether they contain pHIS2 vector with our sequences. We found some good results but to be sure we made more experiments such as mini prep preparation from culture of colonies and we again did PCR reaction by using reverse primers such as
5’ TAT CAT ATG GTC CAG AAA CC 3’
And
5’ AGT CAC CAA TGC ACT CAA CG 3’
As a result, we obtained very good 2 colonies from first oligo, 3 good colonies from second oligo and 7 good colonies from mutant oligo. From PCR reactions we also saw that second reverse primer (5’ AGT CAC CAA TGC ACT CAA CG 3’) worked better than first primer (5’ TAT CAT ATG GTC CAG AAA CC 3’).
After insertion of desired sequence into cloning site of pHIS2, we made more Plasmid mini prep to send plasmid to sequencing and to be sure about absence of any mutation in the plasmid and inserted sequence. While waiting the sequencing results, we collected more embryos and started to isolate total RNA from early embryos. After isolation of RNA, I run a sample of total RNA at a RNA gel and I saw two bands at the top and a smear at the bottom so that I continued to make cDNA by using random primer as defined and supplied with BD Matchmaker Library Construction & Screening Kit. For cDNA preparation, I used protocol given in this kit and then I amplified ds cDNA by Long Distance PCR (LD-PCR). After obtaining enough amount of cDNA, to prepare library, I purified it by using BD CHROMA SPIN TE-400 Column given by kit and I stored it at -200C until in vivo recombination with pGADT7-Rec2.
I have already generated a cDNA and reporter vector (pHIS2) is also ready. In the following weeks, after transformation of reporter vector with selected yeast strain (Y187), I will determine the optimum 3-AT concentration needed to suppress background growth on SD/-His/-Trp plates. Then, I will start to construct an AD Fusion Library explained in the BD Matchmaker Library Construction & Screening kit. By the way, I will perform necessary control experiment; it is again explained nicely in that kit. After library construction, it is time to start one-hybrid screening by plating transformants on SD/-His/-Leu/-Trp+ optimal [3-AT] plates.
With the advance of biotechnology, like in my project, to make one-hybrid screens become so easy. Finally, with the advice of my mentor Dr. Stathopoulos and help of my supervisor Dr. Phoebe this project will be the best experience in my career.
REFERENCES:
Stathopoulos A., and Levine M. (2005). Localized repressors delineate the neurogenic ectoderm in the early Drosophila embryo. Developmental Biology, 280, 482-493.
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