L. Daniel Holsinger and Dr. Laura Bridgewater, Zoology

Abstract: The Human col11 2 gene is responsible for the production of type XI collagen, one of the three components of the collagen triple helix. Disruption of this gene results in varied problems including dwarfism, osteoarthritis and possibly cleft palate. In order to understand more about this gene, a series of experiments were carried out to identify proteins involved in its expression in cartilage-producing Chondrocytes. More specifically, based on what was learned in previous research, efforts were made to isolate the DNA sequences that encode those proteins acting as activators of three known enhancer elements in Chondrocyte cells. Using the protein identification system known as a Yeast-One Hybrid Screen (clonetech), plasmids were constructed using four copies of a known enhancer element. The new reporter plasmids were integrated into the genome of a host yeast strain and used as bait to attract DNA encoding the protein used to activate the specific enhancer element. Based on the technology of hybridizing a binding-domain and an activation-domain that acts as a carrier for cDNA molecules, specific sequences in theory could be sought after, isolated and be translated into functional proteins. The results of this study will ultimately be coordinated with studies of the rest of the gene. These results in turn will provide insight into control and expression pathways of col11 2. Such information will provide potential sources of gene therapy to individuals with collagen gene dysfunction.

Introduction

This study is a continuation of past research that was conducted to identify the precise location of protein binding-sites in enhancer elements of the col11a2 gene. The techniques involved in the analysis are fairly new ones.

The enhancers of interest are located in the first intron in the middle region. The previously identified enhancers were analyzed to identify where activator proteins were binding within the known enhancer element. The binding sites were identified by performing a series of point mutations three base-pairs long every six base pairs. The mutations that changed the binding sites of the activator proteins drastically reduced activity when tested in vitro. Thus the binding sites of the activator proteins for the enhancer elements were identified.

Knowing where the activator proteins were binding, the main focus of our research became classification of the proteins. To identify the proteins whose binding sites had thus been discovered, a protein identification screen was used. The basic methodology was as follows: Activator proteins binding to the enhancer element can not be isolated as proteins. Therefore, a series of steps were followed to find and isolate the cDNA’s that then could be translated into functional proteins. Reporter plasmids with the target enhancer elements were constructed and integrated into the yeast genome. A cDNA library from human condrocytes was amplified and screened. (This is made possible because the library is constructed so that the encoded proteins are synthesized as fusions with strong transcription activation domains.) Finally, yeast cells harboring the reporter gene were transformed with the cDNA library and using a special selection strategy, cDNA’s of interest were identified.

In theory, the cDNA would then be isolated from the selected cells and further characterization to confirm that the encoded protein binds the DNA sequence of interest would be carried out.

Materials and Methods

The amplification of the cDNA library was the first step in the project. A complete human chondrocyte cDNA library was ordered from ClontechTM. Amplification involved a several day process of tittering and then growing several hundred thousand colonies on agar plates. The bacteria were harvested and after a brief period in a shaking incubator the cells were lysed and the DNA was recovered using a DNA recovery kit for large quantities of DNA (Qiagen Megaprep). An optical density reading was taken for the DNA and then it was stored for use later in the experiment.

To conduct the assay, we prepared new yeast reporter strains having the sequence of a specific target element upstream of the reporter gene. Target elements BC, DE and FG were constructed through a multiple cloning process. Following PCR amplification, these were inserted upstream of the reporter gene promoter. Next, we transformed the target-reporter construct into yeast cells and, by marker gene selection, obtained recombinants with genomically integrated reporters to make a new target-reporter strain. Integration was straight-forward because the reporter vectors provided in the kit permitted high-frequency, site-specific recombination. In our case, dual reporter genes, useful for more stringent library screening, were generated by sequentially integrating the HIS3 and lacZ reporters into the same yeast genome at different loci ( HIS3 and URA3, respectively).

Screening of the library to find and isolate the activator proteins did not take place because of the amount of time required for the first steps in this experiment. The next steps in this experiment however involve screening the human chondrocyte library for a gene encoding the DNA-binding proteins of interest. We will transform the target-reporter strain with an AD library of fusions between the target-independent AD and potentially target-specific DNA-BPs. Transformants would then be plated on selective medium. If an AD/library hybrid protein interacts with the target element, HIS3 reporter gene expression will be activated, allowing colony growth on minimal medium lacking histidine, but containing the concentration of 3-AT needed to inhibit background HIS3 expression. A beta-galactosidase assay will be performed to verify the DNA-protein interaction and help eliminate any false positive clones. Finally, AD/library plasmids will be isolated from the His + transformants and DNA binding should be confirmed through gel shift assays.

Implications of this experiment will be broad but very useful. First of all, identified proteins would be tested to see what, if any, role they play with sox-9 in chondrocyte cells. Finding all of the activator proteins of the gene will help us to recognize and possibly locate other enhancer elements in the gene. This will ultimately lead to helping us identify remaining activator proteins. The bottom line is that with current gene therapy techniques, social issues aside, this study will eventually lead to a potential cure (or prevention) for some acute genetic problems in the human race. How would it be to be free from worrying about arthritis and other skeletal abnormalities?

This ground-breaking research will not only do that but lead to insight on the general mechanisms of gene expression and correction which will be invaluable knowledge in the quest for genetic cures on all levels.