We have developed methods to transiently and selectably transform the human-infective protist Trichomonas vaginalis. This parasite, a common cause of vaginitis worldwide, is one of the earlier branching eukaryotes studied to date. We have introduced three heterologous genes into T. vaginalis by electroporation and have used the 5 and 3 untranslated regions of the endogenous gene ␣-succinyl CoA synthetase B (␣-SCSB) to drive transcription of these genes. Transient expression of two reporter proteins, chloramphenicol acetyltransferase (CAT) or luciferase, was detected when electroporating in the presence of 50 g closedcircular construct. Optimal levels of expression were observed using Ϸ2.5 ؋ 10 8 T. vaginalis cells and 350 volts, 960 Fd for electroporation; however, other conditions also led to significant reporter gene expression. A time course following the expression of CAT in T. vaginalis transient transformants revealed the highest level of expression 8-21 hr postelectroporation and showed that CAT activity is undetectable using TLC by 99 hr postelectroporation. The system we established to obtain selectable transformants uses the neomycin phosphotransferase (neo) gene as the selectable marker. Cells electroporated with 20 g of the NEO construct were plated in the presence of 50 g͞ml paromomycin and incubated in an anaerobic chamber. The paromomycin-resistant colonies that formed within 3-5 days were cultivated in the presence of drug and DNA was isolated for analyses. The NEO construct was shown to be maintained episomally, as a closed-circle, at between 10-30 copies per cell. The ability to transiently and selectably transform T. vaginalis should greatly enhance research on this important human parasite.
To identify regulatory elements that play a role in transcription initiation in ancient eukaryotes, we have analyzed the upstream regions of protein-cing genes from Trichomonas vaginalis, one of the most ancient eukaryotes studied to date. Characterization of seven protein-coding genes from this protist invariably revealed the presence of a highly conserved DNA sequence motif immediately u pstm of the coding region. This 13-nt motif was shown to surround and contain precise sites for transcription initiation. No typical TATA boxes, positioned at 25-30 nt upstream of the transcription start sites of these genes, were found. The start-site regions from all seven T. vaginaijs genes impart strong specific initiation of transcription in a mammalian in vitro transcription assay. This consensus promoter element in an ancient eukaryote is similar, both structurally and functionally, to initiator elements found in promoters of higher eukaryotes.Transcriptional regulation of protein-coding genes has been studied extensively in higher eukaryotes, leading to the identification of conserved DNA sequence elements responsible for the accurate initiation of mRNA synthesis by RNA polymerase II. At least two types of core promoter elements exist, TATA boxes and initiator (Inr) elements. The TATA box, a conserved element found in the promoter regions of many genes, contains the sequence motif TATAAA (1, 2). This TATA motif is usually located 25-30 bp 5' of the site of transcription initiation and plays a critical role in start-site selection and recruitment of the general transcription machinery to the promoter (1, 3). A second less-abundant class of promoters lack a TATA box but contain an element called an Inr, which surrounds the RNA start site and appears to carry out the same functions as TATA (4-8). The remaining core promoters contain both TATA and Inr elements or neither.The evolutionary origins of the structural components of eukaryotic core promoters are not known. TATA-like promoter sequences have been reported in archaebacteria (9). In addition, the location of the TATA box relative to the transcription start site is similar to the location of a critical element found at -35 in eubacterial promoters, but it is not clear whether this similarity is relevant to the origin of the TATA box. The DNA-binding domain of the TATA-binding protein has sequence similarity with prokaryotic o factors; however, the similarity is restricted to the region of the a factors thought to interact with the -10 element of bacterial promoters (3, 4).One reason for our lack of knowledge about the evolutionary origin of eukaryotic transcriptional control elements and transcription initiation mechanisms is that, to date, the study of eukaryotic transcription has been confined largely to animals, plants, and fungi. As illustrated by the phylogenetic tree shown in Fig.
We have examined transcription in an early diverging eukaryote by analyzing the effect of the fungus-derived toxin alpha-amanitin on the transcription of protein-coding genes of the protist Trichomonas vaginalis. In contrast to that typical in eukaryotes, the RNA polymerase that transcribes T. vaginalis protein-coding genes is relatively resistant to alpha-amanitin (50% inhibition = 250 microg alpha-amanitin/ml). We have also characterized the gene encoding the largest subunit of RNA polymerase II, the subunit that binds alpha-amanitin. This protein is 41% identical to the mouse RNA polymerase II. Sequence analysis of the 50-amino-acid region thought to bind alpha-amanitin shows that this region of the trichomonad RNA polymerase II lacks many of the conserved amino acids present in the putative binding site, in agreement with the observed insensitivity to this inhibitor. Similar to other RNA polymerase IIs analyzed from ancient eukaryotes, the T. vaginalis RNA polymerase II lacks the typical heptapeptide (Tyr-Ser-Pro-Thr-Ser-Pro-Ser) repeat carboxyl-terminal domain (CTD) that is a hallmark of higher eukaryotic RNA polymerase IIs. The trichomonad enzyme, however, does contain a short modified CTD that is rich in the amino acid residues that compose the repeat. These data suggest that T. vaginalis protein-coding genes are transcribed by a RNA polymerase II that is relatively insensitive to alpha-amanitin and that differs from typical eukaryotic RNA polymerase IIs as it lacks a heptapeptide repeated CTD.
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