Acanthamoebae produce a painful, blinding infection of the cornea. The mannose-binding protein (MBP) of Acanthamoeba is thought to play a key role in the pathogenesis of the infection by mediating the adhesion of parasites to the host cells. We describe here the isolation and molecular cloning of Acanthamoeba MBP. The MBP was isolated by chromatography on the mannose affinity gel. Gel filtration experiments revealed that the Acanthamoeba lectin is a ϳ400-kDa protein that is constituted of multiple 130-kDa subunits. Cloning and sequencing experiments indicated that the Acanthamoeba MBP gene is composed of 6 exons and 5 introns that span 3.6 kb of the amoeba genome and that MBP cDNA codes for a precursor protein of 833 amino acids. That the cloned cDNA encodes authentic MBP was demonstrated by showing that: (i) recombinant MBP possesses mannose binding activity, and (ii) polyclonal antibodies prepared against Acanthamoeba MBP bound to the recombinant protein. Sequence analysis revealed that the MBP contains a large N-terminal extracellular domain, a transmembrane domain, and a short C-terminal cytoplasmic domain. Despite extensive BLAST searches using the MBP sequence, no significant matches were retrieved. The most striking feature of the Acanthamoeba MBP sequence is the presence of a cysteine-rich region containing 14 CXCXC motifs within the extracellular domain. In summary, we have isolated, cloned, and characterized a novel MBP from Acanthamoeba. Because the presence of antibodies to MBP in tears provides protection against infection, the availability of the MBP cDNA sequence and rMBP should help develop: (i) a tear-based test to identify individuals who are at risk of developing the keratitis and (ii) strategies to immunize high-risk individuals.Acanthamoebae are causative agents of two distinct disease entities. In immunocompromised individuals, this protozoan parasite produces chronic granulomatous amoebic encephalitis and disseminating infections including dermatitis and pneumonitis (1, 2). Granulomatous amoebic encephalitis is nearly always fatal because of the lack of an effective treatment. In immunocompetent individuals, Acanthamoebae provoke a debilitating vision-threatening corneal infection known as Acanthamoeba keratitis (AK) 1 (3-5). AK is characterized by intense pain and a slowly worsening clinical course. Contact lens wear is a major risk factor (3, 6, 7). In the developed world, ϳ85% of the cases are diagnosed in contact lens wearers (7). However, in the developing countries, where contact lens use is infrequent, the disease is more commonly found in non-contact lens wearers (8). The mechanism by which Acanthamoebae produce granulomatous amoebic encephalitis and AK has not been fully elucidated. It is generally accepted that the two major predisposing factors in the pathogenesis of AK are minor corneal trauma caused by contact lens wear or other noxious agents and exposure to contaminated solutions including lens care products and tap water (9, 10). The adhesion of parasites to the host cells ...

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The role of the Acanthamoeba casteUlanii TATA-binding protein (TBP) in transcription was examined. Specific antibodies against the nonconserved N-terminal domain of TBP were used to verify the presence of TBP in the fundamental transcription initiation factor for RNA polymerase I, TIF-IB, and to demonstrate that TBP is part of the committed initiation complex on the rRNA promoter. The same antibodies inhibit transcription in all three polymerase systems, but they do so differentially. Oligonucleotide competitors were used to evaluate the accessibility of the TATA-binding site in TIF-IB, TFIID, and TFIIIB. The results suggest that insertion of TBP into the polymerase II and III factors is more similar than insertion into the polymerase I factor.While the transcription systems of eukaryotic RNA polymerases I, II, and III obviously share some characteristics, initiation mechanisms for these transcription systems have been largely studied separately. The polymerases themselves have five subunits in common, seven in the case of the "odd pols," RNA polymerases I and III (45). Nevertheless, the general transcription factors involved with each polymerase have been examined in isolation, perhaps masking important generalizations about their functions. This approach began to change when it was discovered that some of the genes for small nuclear RNAs (snRNAs) are transcribed by polymerase III whereas most are transcribed by polymerase II (27,32,35,47 The full impact of this factor overlap was perhaps not realized because polymerase I still appeared to use dedicated factors. However, a flurry of studies (6,7,44,56) revealed that TBP was required for transcription of all genes. TBP is now known to be a subunit of TFIID (41), TFIIIB (17,24,48,55), and human TIF-IB (SL1) (6). TBP is associated with additional subunits (TAFs) to make up the functional factors (reviewed in reference 41). The TAFs appear to be different for each polymerase system (reviewed in reference 42), although overlap of TAFs has not been rigorously ruled out. All the TBP-containing factors are pivotal for their respective polymerases. Indeed, TFIIIB and TIF-IB are fundamental transcription factors; i.e., they appear to be responsible for the repetitive recruitment of RNA polymerase during successive rounds of initiation (16; reviewed in references 36 and 37).The manner by which the TBP-containing factor is recruited to the promoter differs from gene to gene. For RNA polymerase III genes with type I (SS RNA) or type II (tRNA, VAI, Alu, EBER, 7SL, 4.5S) internal control regions, additional general transcription factors are required to assemble TFIIIB onto the promoter (reviewed in references 10-12). Initiation complex formation on 5S and tRNA genes is an organized process in which the factors bind in an obligatory order, each relying on protein-DNA and protein-protein interactions with a previously bound factor(s) to join the complex. On some genes the DNA interaction site for TFIIIB is sequence specific, whereas on others specific sequence recognition is no...

In this report, we test the hypothesis that TBP binds DNA promiscuously due to its manner of recognition of the DNA minor groove. The experiment performed was to select TBP-binding sequences from a pool of random double stranded oligonucleotides. Sixty two clones from this pool were sequenced. Surprisingly, the results show that TBP has a marked preference for stably binding one sequence (TATATAA) over all others, yet only four classes of TATA box were selected. The features of the selected sequences allow definition of a binding consensus for TBP. The DNA binding properties of TBP to the four TATA variants was examined, the results being in accord with the observed selection frequencies. However, the nature of TBP-DNA binding is strongly affected by ionic strength. We infer that recognition of DNA via the minor groove can be highly selective even where A:T and T:A discrimination is required. Models for how this might be accomplished are discussed.

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