Donna Melinda Earl Ashton and Dr. William S. Bradshaw, Zoology

The malignant transformation of chicken embryo fibroblasts (CEF) following infection with the Rous sarcoma virus is mediated through the oncogene v-src. One of the first responses of infected cells is the transcriptional activation of a set of immediate-early genes. Among these are CEF-5 (homologue of EGRI, early growth response, in mammals) and serum induced kinase (snk).

These two genes have been cloned, and their nucleic acid and amino acid sequences determined. However, further research is needed on both these genes to establish their transcription initiation (cap) sites and to sequence and functionally characterize their promoters. The promoter is the upstream regulatory region of the gene.

The necessary DNA and RNA needed for these experiments was isolated and provided by Daniel L. Simmons and William S. Bradshaw. As the name implies, the CEF-5 nucleic acids were isolated from chicken embryo fibroblasts, while the snk nucleic acids were isolated from mouse tissue. The nucleic acid sequences were determined through the Sanger dideoxy sequencing method and the resulting auto-radiographs.

Existing autoradiographs and the sequences obtained from them were reviewed in order to determine any inconsistencies. A few regions of the promoter were determined to be uncertain. Three new primers were created to be used in the Sanger dideoxy sequencing method to resolve these discrepancies. The sequences of these primers were 5′- GGAACCTCGGCCCACTT-3′, 5′-CGGTCTAAGTGGGCCGA-3′, and 5′- CCGCAGGAGCTCCATCGT-3′. The use of these primers in Sanger dideoxy sequencing produced autoradiographs that were useful in resolving any uncertainty. In addition, the sequence of approximately 140 new nucleotides further upstream in the promoter were determined (Fig. 1).

A procedure known as primer extension was used in an attempt to determine the cap site of snk. In this procedure the enzyme reverse transcriptase is used to synthesize a fragment of DNA from purified samples of RNA. This synthesized DNA along with the genomic DNA is then submitted to the Sanger dideoxy sequencing method. The two samples are then electrophoresed side-by-side to determine the cap site. In this procedure snk DNA was used along with CEF RNA. Since CEF is homologous to snk it was thought that the primer created for use with snk would also function with CEF. However, although formerly successful protocols were used, the desired results were not obtained. The same procedure using both snk DNA and RNA should allow for the determination of the cap site.

The reported findings along with the determination of the cap sites of CEF-5 and snk when combined with the other information already obtained will help give a clearer understanding of the activity of CEF5 and snk during normal cell division and in the development of malignancy.

References

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