SURF Project: August Progress Report (2)
Student name: Fatih Kocabas
Mentor name: Angelike Stathopoulos
I have reviewed this paper with the student and approve it as the progress report for August for this SURF project.
Mentor’s Signature Date
Identification of Transcription Factors That Recognize the ATTCATTCATGA Site Affecting Dorsalateral Patterning in Drosophila
In our laboratory, several studies are going on the ind enhancer and especially a newly defined silencer element found in it. In this study, we also study this silencer element – ATTCATTCATGA- called A-box (Stathopoulos A., and Levine M., 2005) found within the module B of ind enhancer. I mostly study together with my supervisor Pheobe, a postdoc in our laboratory. I almost everyday meet my mentor.
Purpose of my project is to identify and isolate transcription factor(s) bind to this conserved element by using one-hybrid assays. We used One-Hybrid assays because it enables us to study transcription factor(s) that bind(s) to a target element that activates transcription from minimal promoter as shown in figure 1. In this method, DNA-binding proteins like transcription factors are expressed as hybrid proteins, in other words fusions to GAL4 activation domain (AD) by cloning the cDNA into pGADT7-Rec2 vector (Figure 2). This vector is a low-copy number vector, it is Sma1 linearized and it has special ends to mediate homologues recombination with cDNA in yeast cells by using cells’ recombinases. In our study, we cloned one of target sequence into pHIS2 vector, as described in BD Matchmaker™ One-Hybrid Manual, it acts as a reporter vector contains the nutritional reporter gene HIS3. Binding of hybrid protein to the target sequence activates transcription of HIS3, makes possible to grow yeast strain Y187, a His auxotroph, on minimal medium lacking histidine.
To make experiments rapid, we use cotransformation method to construct AD fusion library. Moreover, to find optimum 3-AT concentration to suppress leaky HIS3 expression in reporter plasmid pHIS2 with target sequence, we also use control vector pGAD-Rec2-53 (Figure 3) and cotransform with reporter pHIS2 vector and plate on a more stringent medium SD/-His/-Leu/-Trp than SD/-His/-Trp which we previously tried, but it was failed.
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Figure 1:
One-hybrid System, library protein from cDNA will form a hybrid protein with GAL4 Activation Domain protein. Then, this hybrid protein will activate transcription of HIS3 so that cells with this reporter vector containing target sequence will grow on minimal media lacking histidine whereas other with mutant target sequence will not.
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Figure 2: pGADT7-Rec2 Vector, The BD Matchmaker One-Hybrid Library Construction & Screening Kit contains the Sma I-linearized form of this vector, the form used for recombination-mediated cloning in yeast.
Because yeast cells grow slower in minimal medium lacking some essential amino acids than complete media, we waited for a week to determine optimum 3-AT concentration in library search to decrease number of positive false results.
We prepared SD/-His/-Leu/-Trp plates containing different concentration of 3-AT ranging from 0-5-10-20-40-60 mM. Although some difficulties seen at yeast media to find optimal 3-AT concentration for library studies, at last we decided optimal 3-AT concentration as 5 mM because we found that 5 mM was enough to suppress leaky histidine production on SD/-His/-Leu/-Trp agar plates. Moreover, positive control tests showed that with the activation of p53+GAL4 hybrid protein produced by p53HIS2 vector, yeast cells can form 2-3 mm of colonies on SD/-His/-Leu/-Trp up to 20mM 3-AT concentration. Therefore, to make conditions more stringent and to look at stronger interactions with our sequence, we chose 7 mM because cotransformed yeast cells with p53HIS2 Control vector (figure3) and pGAD-Rec2-53 control vector (figure4) can easily form colonies at 10 mM 3-AT concentration. However, there is a lit bit difference at this cotransformation from our cotransformations (pHIS2/target sequence + pGAD-Rec2-53 control vector), p53HIS2 Control vector contains 3x p53 DNA elements so there are 3 times more activation than one DNA element. We cloned into pHIS2 only one DNA element, therefore 7 mM will be affective as much as 20 mM, so we could see high affinity bindings to our target element ATTCATTCATGA.
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Figure 3: p53HIS2 Control vector Figure 4: pGAD-Rec2-53 control vector
In this week, we will construct AD fusion library in one step; cotransformation of yeast strain Y187 with ds cDNA, pGADT7-Rec2 and DNA target/pHIS2 reporter plasmid. After obtaining positive interactions, we will perform yeast plasmid isolation and analyze cDNA insert by sequencing. Then, I will compare the sequence with that of other proteins in GenBank, EMBL, or other databases.
On the other hand, due to some problems related with auxotroph yeast strain and its growing on minimal media, we decided to perform LacZ expression experiments to see LacZ induction by the corresponding library binding protein (Li J.J., and Herskowitz I., 1993). Therefore, we designed new oligonucleoties to clone into pJL368 vector containing lacZ. In this system, we will activate transcription of LacZ instead of HIS3. Moreover, pJL368 vector contains Ura2 gene, so it will enable us to select transformants on minimal media lacking uracil amino acid by using a new yeast strain which is ura-, so it can not grow on media lacking essential amino acid uracil. By looking color of colonies formed, as described in figure 5, we will decide which colony contain cDNA insert expressing binding transcription protein. Similarly with other study, an expression library of hybrid proteins will be formed by using Sma1 linearized pGADT7-Rec2 vector and cDNA. These hybrid proteins will recognize the binding site and will act as transcriptional activators of the reporter gene and they will turn cells blue in a β-Galactosidase assay.
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Figure 5: Diagram for one-hybrid screen for identifying proteins that recognize a binding sequence of interest by using LacZ activation and β-Galactosidase assay. Reporter genes containing either wild-type (top, blue colony formation) or mutant (bottom) binding sites in their promoter regions are used to test the sequence specificity of the LacZ induction.
Finally, from colonies that grow on medium that lacks histidine due to reporter activation, we will isolate the cDNA insert. We expect to find 1 or 2 transcription factors that bind ATTCATTCATGA with high affinity. Moreover, we will conform that by β-Galactosidase assay. Results of this work are important because no other repressor functioning in dorsal regions of the Drosophila early embryo has been characterized to date.
References:
Li JJ and Herskowitz I (1993). Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science 262(5141):1870-4
Stathopoulos A. and Levine M. (2005). Localized repressors delineate the neurogenic ectoderm in the early Drosophila embryo. Developmental Biology, 280, 482-493.
BD Matchmaker™ Library Construction & Screening Kits User Manual. Retrieved August 1, 2005 from http://www.clontech.com/clontech/techinfo/manuals/PDF/PT3529-1.pdf
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