A 10 base-pair sequence within Domain III of the GPEET procyclin promoter is essential for the autonomous replication of a plasmid in procyclic Trypanosoma brucei

Abstract

Very little is known about sequences that mediate nuclear DNA replication in Trypanosoma brucei. We have previously reported the construction and characterization of a panel of autonomously replicating plasmids and artificial linear mini-chromosomes for this organism [1,2]. The episome was obtained by ligating random DNA fragments from the T. brucei genome on to a plasmid that was otherwise incapable of stable replication in this organism. We call these inserted genomic fragments plasmid maintenance sequences (PMS). One member of this panel of episomes (pT13-11) and the corresponding artificial linear mini-chromosomes have been extensively characterized [1][2][3][4]. This plasmid, and by implication its many derivatives (such as pEV-luc; please see below), as well as the linear mini-chromosomes exist at a copy number of 1-2 per cell in procyclic T. brucei and demonstrate substantial mitotic stability in the absence of selection. We are using these autonomous replicons as model systems to delineate critical nuclear DNA replication control elements for this organism.The plasmid pEV-luc ( Figure 1) has been described previously [4]. In this episome, 433 base pairs (-408 to + 25 relative to the start site of transcription) of the GPEET procyclin promoter (also called PARP-A) drives the RNA polymerase I-mediated expression of firefly luciferase and neomycin phosphotransferase (G418 R ). We have previously demonstrated that the autonomous replication of pEV-luc is dependent on the PMS (as expected) and, surprisingly, on the segment bearing the GPEET-promoter [4]. The close association between elements controlling transcription and DNA replication and the possible interaction between factors assembling at these sites is intriguing and fuels our long-term interest in this area. We have sought to delineate the critical promoter-proximal replication control element(s) and determine the overlap (if any) between the two sets of regulatory sequences.The GPEET procyclin promoter has been extensively studied [5][6][7]. It comprises of 4 domains which map to positions -7 to -40 (Domain I), -57 to -72 (Domain II), -90 to -143 (Domain III) and -207 to -222 (Domain IV) with respect to the start point of transcription [5,7; also see Fig 1]. Ten base pair (bp) linker-substitutions within Domains I, II, III and IV reduce GPEETpromotermediated expression of a reporter gene to approximately <0.1%, 5-10%, 30-50%, and 60-70% of wild-type levels respectively [5,7]. Two other RNA Pol I promoters (RRNA and VSG) have been characterized in T. brucei but demonstrate little sequence conservation [8][9][10][11]. Nevertheless, transcription mediated by the GPEET, RRNA or VSG promoters in * Contributed equally to this study ** Corresponding author Tel: 973 720 3454 Fax: 973 720 2338 Email: patnaikp@wpunj.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typeset...