Nuclear factor I is a cellular site-specific DNA-binding protein required for the efficient in vitro replication of adenovirus DNA. We have characterized human DNA sequences to which nuclear factor I binds. Three nuclear factor I binding sites (FIB sites), isolated from HeLa cell DNA, each contain the sequence TGG(N)67GCCAA. Comparison with other known and putative FIB sites suggests that this sequence is importadt for the binding of nuclear factor I. Nuclear factor I protects a 25-to 30-base-pair region surrounding this sequence from digestion by DNase I. Methylation protection studies suggest that nuclear factor I interacts with guanine residues within the TGG(N)67GCCAA consensus sequence. One binding site (FIB-2) contained a restriction endonuclease HaeIII cleavage site (GGCC) at the 5' end of the GCCAA motif. Digestion of FIB-2 with HaeIII abolished the binding of nuclear factor I. Southern blot analyses indicate that the cellular FIB sites described here are present within single-copy DNA in the HeLa cell genome.Considerable progress has been made in the purification and characterization of procaryotic DNA replication proteins. (12). However, the lack of both genetic analysis and in vitro replication systems has hampered similar studies on DNA synthesis in eucaryotes. Adenovirus (Ad) DNA replication is the only system in eucaryotes that is amenable to genetic manipulation (5, 22, 30) and for which the initiation and elongation of DNA chains have been reconstituted with purified proteins (20; for reviews see references 3, 9, and 28). We have fractionated and purified the viral and cellular proteins required for the in vitro replication of Ad DNA with the goal of isolating proteins involved in host DNA synthesis (7,9,16). A protein of particular interest is nuclear factor I, a cellular site-specific DNA-binding protein that is required for the efficient in vitro initiation of Ad DNA replication (19,21,23).Nuclear factor I was purified from nuclear extracts of uninfected HeLa cells by its ability to support the replication of Ad DNA in vitro (19). The synthesis of full-length Ad DNA in vitro requires five purified proteins (4,20). Three of these proteins are viral coded, the 80,000-dalton precursor to the 55,000-dalton terminal protein found at the 5' end of the Ad genome (pTP), the Ad DNA polymerase, and the Ad DNA-binding protein. The two remaining proteins, nuclear factors I and II, are host coded and have been purified from nuclear extracts of uninfected HeLa cells. The initiation of Ad DNA synthesis occurs by the covalent attachment of dCMP, the 5'-terminal deoxynucleotide of Ad DNA, to the pTP (2, 15). This initiation reaction is catalyzed by the Ad DNA polymerase and, in the presence of the Ad DNA-binding protein, is completely dependent on nuclear factor 1 (19). Nuclear factor I has been shown to specifically bind, and protect from DNase I digestion, a 32-base-pair (bp) DNA sequence located in the replication origin present at the termini of the 36,000-bp Ad genome (21. 23). The ability of nuclear fact...

Following sequence analysis of a Leishmania donovani kinetoplast DNA (kDNA) minicircle, we have developed synthetic oligonucleotides for use in the polymerase chain reaction (PCR). With these primers, we have amplified L. donovani kDNA from splenic aspirates and blood samples taken from kala-azar patients. Treatment of the samples for PCR requires only limited DNA purification by lysis in SDS, digestion with proteinase K, phenol extraction and ethanol precipitation of the resulting nucleic acid. We have obtained amplified product routinely with DNA prepared from the equivalent of 2.5-25 microliters of splenic aspirate or of 50-500 microliters of blood from infected patients. In dilution experiments a visible product has been obtained on amplification of DNA from the equivalent of 2.5 x 10(-7) microliters of splenic material. We therefore propose the amplification of L. donovani kDNA by PCR as a rapid and highly sensitive method for the diagnosis of kala-azar.

In kinetoplastid protozoa, import of cytosolic tRNAs into mitochondria occurs through tRNAs interacting with membrane-bound proteins, the identities of which are unknown. The inner membrane RNA import complex of Leishmania tropica contains multiple proteins and is active for import in vitro . RIC1, the largest subunit of this complex, is structurally homologous to the conserved α subunit of F1 ATP synthase. The RIC1 gene complemented an atpA mutation in Escherichia coli . Antisense-mediated knockdown of RIC1/F1α in Leishmania resulted in depletion of several mitochondrial tRNAs belonging to distinct subsets (types I and II) that interact cooperatively or antagonistically within the import complex. The knockdown-induced defect in import of type I tRNAs was rectified in a reconstituted system by purified RIC1/F1α alone, but recovery of type II tRNA import additionally required a type I tRNA. RIC1/F1α formed stable complexes with type I, but not type II, tRNAs through the cooperation of its nucleotide binding and C-terminal domains. Thus, RIC1/F1α is a type I tRNA import receptor. As expected of a bifunctional protein, RIC1/F1α is shared by both the import complex and by respiratory complex V. Alternative use of ancient respiratory proteins may have been an important step in the evolution of tRNA import.

Intracellular parasitic protozoans of the genus Leishmania depend for their survival on the elaboration of enzymic and other mechanisms for evading toxic free-radical damage inflicted by their phagocytic macrophage host. One such mechanism may involve superoxide dismutase (SOD), which detoxifies reactive superoxide radicals produced by activated macrophages, but the role of this enzyme in parasite survival has not yet been demonstrated. We have cloned a SOD gene from L. tropica and generated SOD-deficient parasites by expressing the corresponding antisense RNA from an episomal vector. Such parasites have enhanced sensitivity to menadione and hydrogen peroxide in axenic culture, and a markedly reduced survival in mouse macrophages. These results indicate that SOD is a major determinant of intracellular survival of Leishmania.

The mitochondrial genomes of a wide variety of species contain an insufficient number of functional tRNA genes, and translation of mitochondrial mRNAs is sustained by import of nucleus-encoded tRNAs. In Leishmania, transfer of tRNAs across the inner membrane can be regulated by positive and negative interactions between them. To define the factors involved in such interactions, a large multisubunit complex (molecular mass, ϳ640 kDa) from the inner mitochondrial membrane of the kinetoplastid protozoon Leishmania, consisting of ϳ130-Å particles, was isolated. The complex, when incorporated into phospholipid vesicles, induced specific, ATP-and proton motive force-dependent transfer of Leishmania There is remarkable diversity in the scope and mechanism of mitochondrial tRNA import (reviewed in reference 18). Human mitochondria do not import tRNA, but a number of neuromuscular degenerative and metabolic diseases are caused by mutations in mitochondrial tRNA genes (21). In yeast, a single tRNA is imported, apparently through protein import channels and requiring at least two soluble factors, including the mitochondrial form of the cognate aminoacyl-tRNA synthetase (8). By contrast, in kinetoplastid protozoa (Leishmania and trypanosomes), import of a whole spectrum of tRNAs is necessitated by the complete lack of mitochondrial tRNA genes (5,19). In this system, membrane-bound tRNA binding proteins recognize specific structural motifs (import signals) on tRNA, soluble factors are not required, and the translocation pathway appears to be distinct from that for protein import (11,14,17). Moreover, the sequence and bioenergetic requirements for outer and inner membrane transfer are nonidentical (2), indicating the presence of a distinct transport machinery (the RNA import complex [RIC]) at the inner membrane, a situation similar to the TOM and TIM complexes for protein import (15). A 15-kDa polypeptide has been shown to be required for import into Leishmania mitochondria (1); otherwise, the import machinery remains undefined.Using an in vitro evolution protocol, it was recently shown that Leishmania mitochondria recognize a number of short sequence motifs homologous to multiple domains in tRNAs, suggesting the presence of several import signals (3). Moreover, novel positive and negative allosteric interactions between these aptamers, as well as between intact tRNAs, at the inner membrane were described (3). The RNAs could be classified into two types: type I RNAs are efficiently transferred through the inner membrane but are inhibited by type II. In contrast, type II RNAs have poor inner membrane transfer efficiencies and are stimulated by type I. For example, tRNA Tyr (GUA) is a type I RNA containing the conserved motif UA GAGC in the D domain, while tRNA Ile (UAU) is type II with the sequence UCGCGGGUU in the variable loop-T domain (V-T) region (3). The mechanism of these allosteric interactions is unknown, but there are several possibilities. A single conformationally flexible dimeric or multimeric receptor could bind t...