Date of Award

5-1-1993

Document Type

Thesis

Degree Name

Master of Arts (MA)

Department

Biology

First Advisor

Dr. A. Thomas Weber

Abstract

Previous studies in Dictyostelium discoideum on the transposable element Tdd-1, have identified two transcription units of opposite polarity. One is a major, developmentally regulated 4.5 kb transcript believed to encode proteins involved in the retrotransposition of this element, and the other, 1.4 kb in size, is a heat-shock inducible RNA. Based on the sequence of the 4.5 kb transcript and the encoded amino acid sequence, it has been proposed that Tdd-1 is a retrotransposon. This implies that the 4.5 kb mRNA is copied by reverse transcriptase, resulting in DNA copies that insert into new sites. The purpose of this study was to identify similar retrotransposons in two other Dictyostelium strains, D. discoideum V-12 and D. mucoroides DM-7, and to estimate retrotransposon copy number. To identify the Tdd-1 gene in D. discoideum strain NC-4, polymerase chain reaction (PCR) primers were designed using the published nucleotide sequence in the known highly conserved amino acid regions of reverse transcriptase. The product of this amplification was a 370-bp fragment, which was then cloned. The identity of the fragment as part of Tdd-1 was confirmed by restriction mapping and DNA sequencing. To estimate gene copy number, Southern blots were analyzed using a probe made from the cloned fragment. In all Southern blot analysis NC-4 had the highest copy number, followed by D. discoideum V-12 and D. mucoroides DM-7, respectively. Although the band patterns on Southern blots were not reproducible, D. discoideum NC-4 always had more bands than D. discoideum V-12, which had more bands than D. mucoroides DM-7. This was consistent with PCR results where NC-4 always yielded the greatest concentration of amplified product of the expected size. A probe was used to identify clones with complementary sequences to the Tdd-1 fragment in a genomic library of D. mucoroides. Digoxigenin (dig)-labeling of the cloned probe was successfully accomplished by employing the PCR. Identification of genomic clones from the lambda library was accomplished using these dig-labeled probes.

Comments

A Thesis Presented to the Department of Biology and the Faculty of the Graduate College University of Nebraska In Partial Fulfillment of the Requirements for the Degree Master of Arts University of Nebraska at Omaha. Copyright 1993 Gail A. Henderson.

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