The MRC Group in Periodontal Physiology - The Ligament: a newsletter, Winter 1995/96

In this Issue:

PhD Training in the MRC Group

In this issue we highlight our PhD students. The combination of the multidisciplinary backgrounds of the Group’s principal investigators and the focus on high quality research in periodontal tissues provides a unique environment for PhD studies. To date, 23 students have graduated from the Group with PhDs and six more are in progress. Of these, 22 have clinical training and 15 have, in addition, a clinical specialty. Twelve of our PhD graduates have held or currently hold dental faculty appointments, ten in Canada. Six of the seven current Canadian faculty members hold research grants. Our earliest PhD graduates were periodontists, Doug Pettigrew and Tim Gould, who were subsequently appointed in the dental faculties at the Universities of Alberta and British Columbia, respectively. Another early graduate was Ed Yen, who became head of the Orthodontics Department at Winnipeg before moving to UBC as Dean. Chris McCulloch, Howie Tenenbaum and Jeff Dixon, all periodontists, continued with post-doctoral studies following completion of PhD theses with our Group. Chris and Howie are now members of our Group while Jeff is in the Physiology Department at UWO and has close ties with their dental faculty. Another periodontist, Australian Chris Overall, did post-doctoral work with nobel laureate Michael Smith and is now a dental faculty member at UBC. Other PhD graduates from the Group with clinical qualifications from abroad are Cathy Birek (Romania), Jamila Farsi (Saudi Arabia), Lesley Ellies (Australia), Reynaldo Todescan (Brazil), Jin- kun Chen, Qi Zhang, and Hesheng Yu (China). Cathy did post-doctoral work and an Oral Pathology specialist programme in Toronto and is now on faculty in Winnipeg, while Jamila is running an orthodontics programme in Saudi Arabia. Lesley, who with Reynaldo completed specialties in prosthodontics in our faculty, is in California involved in research on glycoprotein expression in lymphocytes, while Reynaldo is conducting clinical trials on the Endopore system. Jin-kun, who came from Connecticut with an Oral Pathology specialty, is now in the Pedodontics Department at the University of Texas at San Antonio. Qi completed post-doctoral studies at the NIH and is currently in the DDS programme at Toronto. Hesheng is doing post-doctoral research on chondrocytes at Mt. Sinai. Two of our earlier graduates, Hardy Limeback and Emil Svoboda, completed clinical training in Toronto following their PhDs. Hardy is now heading Preventive Dentistry in our faculty.

Currently, we have Kiran Kulkarni with a specialty in pedodontics from our faculty finishing his PhD thesis, while Alexa Herbertson (orthodontics) and Mike Glogauer (periodontics) are well into combined clinical specialty/PhD programmes. Carmelo Domenicucci and Jun (Jack) Li are in the combined DDS/PhD programme.

Many of the non-clinical PhDs are also continuing academic pursuits. Marty Petkovich cloned retinoic acid receptors in his post-doctoral work with Chambon in Marseilles and is now on faculty at Queens. Jeff Wrana cloned the TGF-b receptors with Massagué at the Sloan Kettering Institute and is now on staff at Sick Children’s Hospital. Agi Grigoriadis studied fos gene ‘knockouts’ in transgenic mice with Wagner in Austria and is continuing post-doctoral studies on bone development in Paul Sharpe’s laboratory in England. Susan Mandel chose medicine and specialized in orthopaedic surgery. Kay Panchmatia and Kui-Lai Lee have joined pharmaceutical companies. Kursad Turksen did post-doctoral work with Elaine Fuchs in Chicago and is continuing his research at the Loeb Institute in Ottawa. Shen-Ling Xia is a research associate at UCLA, doing biophysics.

Laboratory Techniques: Differential Display - Polymerase Chain Reaction

The human genome contains over 100,000 genes, each one coding for specific RNA and/or a protein product. At any given time there are in all cells only a limited number of these genes expressed. Different tissues at different stages of growth and development express genes in a highly specific manner. In addition, during disease progression various genes are expressed inappropriately, resulting in abnormalities that define specific pathologies. These expressed genes can be identified and isolated by a number of methods, including subtractive-hybridization, differential cDNA cloning, and the newly described technique, differential display-polymerase chain reaction (DD- PCR). Subtractive-hybridization and differential cDNA cloning require large amounts of mRNA derived from cell or tissue samples, and are very laborious and time consuming, often taking many months from start to finish. However DD-PCR requires only small amounts of cell or tissue samples, and can be completed within weeks.

DD-PCR requires RNA derived from control (normal) and sample (diseased) cells or tissues; if temporal changes/alterations are sought then RNA from different stages of normal or disease may be compared. The samples of RNA being examined are converted into cDNA by reverse transcription using an oligo-dT primer with two penultimate specific bases to the 3’ end:

5’ TTTTTTTTTTTTMN 3’

(where M = A, G, or C; N = any nucleotide)
.

Upon conversion to cDNA the PCR amplification is done with arbitrary 5’ primer (8-10mers) using random sequences, and the same 3’ primer (shown above). In order to define most of the genes expressed in these cells, different sets of random primers may be used. After ~30 amplification cycles using radioactive S-35 labeled nucleotides the expressed cDNA fragments are displayed using DNA sequencing gels. Differentially expressed cDNAs are easily visualized following autoradiography. The X-ray films can be used directly as a template to identify bands in the gels that are differentially expressed. The DNA from these gels slices is recovered by boiling, and reamplified by PCR using the original primers. The amplified DNAs are characterized by sub-cloning, sequencing and data-base searches. To verify the differential expression status of each of these sub-clones, Northern-Blot assays are performed using RNA isolated from the original sample sources.

As an example, we have isolated three ETS 1 transcription factor target genes by using RNA from a control cell line and a cell line that expresses high levels of ETS 1. Recently we have developed ETS 2 transgenic mice that have bone abnormalities such as osteomalacia, dome shaped heads, and calavaria that do not close completely. We are currently culturing normal and transgenic calavaria cells and we will compare the RNA expression pattern differences by DD-PCR to identify genes that cause this disorder, and which may prove to be useful targets for understanding bone development.

Charles Lekic: Periodontal Ligament Cells

Predrag (Charles) Lekic joined our group in 1994 as a research associate. Prior to moving to Toronto, he was a practicing pedodontist and held a full professorship in the Department of Pedodontics and Preventive Dentistry at the University of Belgrade. He received his PhD in oral biology and completed a postdoctoral fellowship in immunology at the University of Belgrade. He has spent study and research leaves in Sweden and Denmark and has received many awards during his career in Belgrade. Most recently, he was awarded a research grant from the Faculty of Dentistry to study optimization of cell storage media and methods to maintain viability of periodontal ligament cells of avulsed human permanent teeth. Currently, Dr. Lekic’s research interest is in the regulation of connective tissue cells in periodontium, specifically in relation to periodontal regeneration and prevention of periodontal disease. Along with Drs. McCulloch and Sodek, he has five publications in press, and has presented papers at the AADR/CADR and the Canadian Musculoskeletal Conference. In addition to supervising graduate students in research, Dr. Lekic also lectures to graduate students in periodontal pathology and connective tissue physiology.

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