
	1999 Summer Scholars Program

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Plasmid Construction in the Role of NFkB in Human T-Cell Leukemia Virus Type 1

By Katherine Kosman
Mentors: Dr. Lee Ratner, Mike Robek
Departments of Molecular Microbiology, Pathology, Washington University School of Medicine, St. Louis, MO

Human T-Cell Leukemia Virus Type 1 (HTLV- 1 ), detected in the 1970s as the first retrovirus in humans, can cause two major diseases: Adult T-cell leukemia/lymphoma, a hematopoietic malignancy, and Tropical Spastic Paraparesis/HTLV-1 associated myelopathy. As of 1995, an estimated 10-20 million people worldwide were infected by HTLV-1. Adult T-Cell Leukemia (ATL), the focus of this project, was first recognized in Japan as the first human cancer caused by a retrovirus. After a latency period of 20-30 years, ATL development can cause lymph node enlargement, hepatomelagy, splenomegaly, and skin lesions. Shortly after the recognition of ATL, the virus HIV was found to be associated with AIDS. Research in HTLV-1 and ATL has therefore not only benefited leukemia patients, but has consequently given valuable insight into the study of HIV and AIDS as well.

The nuclear phosphoprotein Tax is the viral transactivator of transcription of HTLV-1. Tax transgenic mice (possessing granzyme B promoter-driven expression of Tax in activated T cells and NK cells) are used as a model for ATL. These Tax transgenic mice develop leukemia, splenomegaly, and peripheral tumors. It is believed that the NFkB family of transcription factors plays an important role in the onset of leukemia in the Tax transgenic mice. In normal cells NFkB is present in the cytoplasm bound to the inhibitor IkB. However, the viral transactivator Tax activates a kinase that attaches a phosphate group to IkB at serine 32 and serine 36. This activity leads to a degradation of IkB and forces IkB to lose its ability to bind to NFkB and to hold NFkB in the cytoplasm. Tax thus promotes increased and disregulated NFkB activity resulting in cancer. Researchers are consequently searching for a method to control IkB and NFkB activity. The goal of this summer research opportunity in Dr. Lee Ratner's laboratory under the direction of Mike Robek was to make a plasmid by genetic cloning that will counteract Tax's effect on NFkB. This plasmid will contain a 44 bp N-terminal deletion mutant of IkB [IkBD(1-44)] under the control of the granzyme B promoter. This IkBD(1-44) resists phosphorylation and degradation thus prohibiting the release and activation of NFkB. To construct the plasmid, pcTAX (JGZBPT) was digested with PstI and XmaI to remove the Tax open reading frame and was run on an agarose gel. The large vector fragment cut by both enzymes was gel purified using Qiaex II protocol. Meanwhile IkBD(1 - 44) was amplified by PCR, run on an agarose gel, and purlfied. To confirm that the purification of both the vector fragment and the PCR product was successful, both the vector and the PCR product were run on a gel. The IkBD(1-44) PCR product was digested with XmaI and NsiI, and the vector fragment was ligated with the IkBD(1-44) PCR product. Ten (10) colonies were collected from the transformation. The plasmids were then screened to identify positives. A digest with PstI demonstrated success in plasmids one (1) through five (5). Plasmids two (2) and three (3) were determined correct after a digest with PstI and NcoI. These two plasmids were transformed and run in a large scale DNA prep to isolate the plasmid DNA. The concentration of DNA from plasmid 2 was 6.2 ug/ul and from plasmid 3 was 5.6 ug/ul. Both plasmids were cut with PstI and NcoI to confirm correct restriction digest patterns and were then analyzed by automatic sequencing.

This summer project is the beginning of a large project in Dr. Lee Ratner's laboratory. The plasmid will be digested with EcoRI and NheI and the resulting 2.8 kb fragment will be used to make transgenic mice that will be utilized to evaluate the role of NFkB in tumorgenesis in Tax transgenic mice. The fundamental question is whether or not the cross of a Tax mouse with a mouse with IkBD(1-44) and no NFkB activity will produce an offspring lacking tumors. Therefore this project will provide valuable insight into the role of NFkB in Tax transgenic mice and could potentially be applied as a therapeutic approach for leukemia patients.

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