No abstract

Iron has been shown to regulate transcription in the protozoan pathogen Trichomonas vaginalis. In this study, a DNA transfection system was developed to monitor ap65-1 promoter activity in response to changing iron supply. In conjunction with electrophoretic mobility shift assay, iron-induced transcription of the ap65-1 gene was shown to be regulated by multiple closely spaced DNA elements spanning an iron-responsive region (؊110/؊54), including an iron-responsive DNA element ( ؊98 AGATAACGA ؊90 ), which overlaps with a 3-MYB-like protein binding sequence ( ؊95 TAACGATAT ؊87 ), and three nearby T-rich sequences ( ؊110 ATTTTT ؊105 , ؊78 AT-TATT ؊73 , and ؊59 ATTTTT ؊54 ). 5-and 3-flanking sequences of the iron-responsive region were shown to regulate basal transcription. A distal DNA regulatory region was shown to enhance both basal and iron-induced transcription. These findings delineate the DNA regulatory elements and nuclear proteins involving in ironinduced transcription of the ap65-1 gene, which provide useful tools for the future study of transcriptional regulation in T. vaginalis.Human infection by the protozoan pathogen Trichomonas vaginalis causes one of the most common sexually transmitted diseases throughout the world (1). Although this protozoan infection usually manifests itself as self-limiting in males, it can impose serious health problems for female patients especially during pregnancy, and it is also implicated as a risk factor for cervical cancer and as a predisposition to human immunodeficiency virus contagion (2-3). As one of the deepest branches of the eukaryotic lineage, this organism exhibits interesting features that deviate from higher eukaryotes and represents an important model system in phylogenetic studies (4). With the recent advent of gene transfer techniques for T. vaginalis (5), in-depth molecular and cellular research can be carried out in this organism.T. vaginalis trophozoites colonize the epithelial surface of the human urogenital tract in which they obtain nutrients, multiply, and face a constant challenge from host immune surveillance. Iron, which is an essential nutrient for almost every organism, is particularly important for T. vaginalis as it regulates growth rate, metabolic activities, and the expression of certain virulence phenotypes such as cytoadherence and resistance to complement lysis (6 -11). At the molecular level, iron has been shown to up-regulate the expression of a number of cellular proteins including a group of putative adhesin molecules, the identities of which are still controversial (8,9,(12)(13)(14)(15)(16)(17)(18)(19). Iron has also been shown to regulate the phosphorylation level of a major surface immunogen P270, which may be responsible for immune evasion (20). These observations suggest that iron is a key modulator in the versatile cellular activities in T. vaginalis. Because the expression of some of the putative adhesin proteins can be inhibited by actinomycin D (8), ironinduced gene expression may be regulated at the transcription level.The k...

The promoter elements that regulate transcription initiation in Giardia lamblia are poorly understood. In this report, the promoter of the Giardia ran gene was studied using a luciferase expression plasmid pRANluc؉ to monitor transcription efficiency. An AT-rich sequence spanning ؊51/؊20 relative to the translation start site of the ran gene was identified and was found to be required for efficient luciferase expression by deletion and mutation mapping of pRANluc؉. The ؊51/؊20 sequence was also sufficient for promoter activity as revealed from studies on a 32-base pair synthetic promoter derived from this region. Deletion mapping of the synthetic promoter revealed two minimal promoter elements, ؊51/؊42 and ؊30/؊20, sufficient for 6-and 30-fold luciferase expression above background, respectively. The transcription start sites on luc؉ messenger RNA were determined by the position of the synthetic promoter in the luciferase expression plasmids as shown by primer extension experiments. Results from electrophoretic mobility shift assays revealed multiple DNAprotein complexes upon binding of nuclear proteins with either DNA strand but not the double-stranded DNA derived from the ran promoter. Our results delineate the first promoter sequence of the Giardia gene (ran), which provides an excellent model for future studies on transcription regulation in this protozoan parasite.As a very common intestinal parasite of humans and one of the earliest diverging eukaryotic cells, Giardia lamblia is an important pathogen for biological studies (1, 2). The parasite adapts to drastic changes in host and outside environments by differentiating between actively dividing trophozoites and dormant cysts in response to extracellular stimuli (3, 4). Trophozoites emerge from cysts in the upper intestine and proliferate to cause gastrointestinal disease. Giardiasis usually manifests as self-limiting diarrhea; however, it can also lead to malabsorption and even death in children. Trophozoites differentiate into cysts in the lower intestine and are excreted to the environment.G. lamblia exhibits both prokaryotic and eukaryotic features (1, 2). Its genome is small and compact (genome size of ϳ1.2ϫ10 7 bp) 1 (1, 2). It is very interesting to study how the organism regulates its gene expression efficiently. Although intron sequences have not been reported in Giardia genes to date (5), a number of genes that code for putative components of the spliceosomal machinery have been found. The short 5Ј-untranslated sequence (as short as 1 nt) and the absence of 5Ј-cap structure of bulk Giardia messenger RNA raise the possibility that G. lamblia may exploit distinct mechanisms for translation initiation different from common strategies used by other eukaryotes (1,7,8). Recent developments using gene transfer technologies and on-going genome sequencing projects involving this organism will provide us additional opportunities to study the molecular and cellular mechanisms operating in this protozoan parasite (9 -13).Very little is known about the mechan...

The transcription efficiency of an adhesion protein gene, ap65-1, in Trichomonas vaginalis varies with changes in the iron supply and with the growth stage. In the present study, two Myb recognition elements, MRE-1/ MRE-2r and MRE-2f, were found to play antagonistic roles in regulating the iron-inducible activity of an ap65-1 reporter gene. Intriguingly, either of these elements was shown to be sufficient to repress basal activity, but together they were also shown to activate growth-related activity of the reporter gene in iron-depleted cells. A myb1 gene which encodes a 24-kDa protein containing a Myb-like R2R3 DNA binding domain was identified from Southwestern screening of MRE-2f-binding proteins. The Myb1 protein was detected as a major 35-kDa protein which exhibited variations in nuclear concentration with changes in the iron supply. A recombinant Myb1 protein was shown to differentially interact with MRE-1/MRE-2r and MRE-2f in vitro. Overexpression of hemagglutinin-tagged Myb1 in T. vaginalis resulted in repression or activation of ap65-1 transcription in iron-depleted cells at an early and a late stage of cell growth, respectively, while iron-inducible ap65-1 transcription was constitutively repressed. The hemagglutinin-tagged Myb1 protein was found to constantly occupy the chromosomal ap65-1 promoter at a proximal site, but it also selected two more distal sites only at the late growth stage. Together, these observations suggest that Myb1 critically regulates multifarious ap65-1 transcription, possibly via differential selection of multiple promoter sites upon environmental changes.As the most common sexually transmitted disease of nonviral origin in humans, trichomoniasis caused by infection with the protozoan parasite Trichomonas vaginalis is an important risk factor for the transmission of human immunodeficiency virus (33). Along with increasing numbers of drug-resistant clinical T. vaginalis isolates (7, 9), trichomoniasis is emerging as a major threat to public health.Iron availability, which periodically varies in the human vagina, where the parasite colonizes, regulates the cytoadherence of T. vaginalis, possibly through controlling the expression of several adhesion proteins in transcription initiation and protein trafficking steps (2,11,18,35). Although only a few T. vaginalis genes have been characterized in detail, the most common example seems to suggest that T. vaginalis uses a conserved initiator (Inr) sequence as the sole core promoter element to regulate the basal transcription of protein-coding genes via interaction of the Inr with a unique Inr-binding protein, IBP39, to recruit ␣-ammanitin-resistant RNA polymerase II (17,20,22,30). This basal transcription machinery significantly differs from that of higher eukaryotic systems, in which the ␣-ammanitin-sensitive RNA polymerase II machinery exhibits considerable diversity both in the core promoter context and in the components of the promoter-recognition TFIID complex (15,24,31). Thus, the transcription efficiency of a particular type II...