Pyridostatin Gene amplification PCR amplifications were perf
Gene amplification. PCR amplifications were performed using genomic DNA from the different organisms studied as template and two degenerate oligonucleotides (PP1 and PP2) as primersPP1 corresponded to the sense codons of the amino Pyridostatin motif AGGI(A/S)EM, and PP2 to the antisense sequence of the motif GGAWDNA, both located close to the C-terminus of the H+-PPase from higher plants and the photosynthetic bacterium R. rubrum, . As indicated, inosine residues (I) and appropriate nucleotide mixtures were introduced at degenerate positions. Reactions were typically performed as follows: g template DNA was heated up to 95°C for 5min in l Taq polymerase buffer containing DMSO (2% final concentration) and deoxynucleotides (M each); before adding the primers (50pmoles) and 3U of Eco-Taq polymerase (3U) reactions were cooled down to 72°C. Thirty to thirty-five cycles of amplification (95, 42, 72°C) produced DNA fragments which were either subcloned into the cloning site of p-GEM-T plasmid (Promega) or directly sequenced. DNA sequencing. Sequences were obtained by the sequencing service of the Instituto de Parasitologı́a y Biomedicina López-Neyra, CSIC, Granada, Spain. Reactions were performed on both strands with an “ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction” Kit (PE Biosystems) using oligonucleotides PP1 and PP2 as primers when sequencing PCR products and pUC/M13 forward and reverse primers when sequencing bands subcloned in p-GEM-T plasmid. Reactions were run in an “ABI 373 XL Stretch” apparatus and the sequences visualized with the program “Editview” version 1.0.1 for Macintosh computers. Southern blot. Genomic DNA preparations (g) were incubated with different restriction enzymes, electrophoresed in 0.7% agarose gels, transferred to nylon membranes, and hybridized at 65°C as described elsewhere , . Probes were labelled with a “Ready-to-Go” labelling Kit (Pharmacia) following manufacturer\'s instructions. Radioactive (α-32P)dCTP was obtained from Amersham (Bucks, England). Contour-clamped homogeneous electric field (CHEF) electrophoresis. Blocks of T. cruzi and L. major in low melting-point agarose were prepared as described elsewhere . Chromosomes were separated in a 1% agarose gel in 0.5M Tris/borate/EDTA/0.5mg/ml ethidium bromide with a CHEF system (Pharmacia). The following parameters were used: for T. cruzi, frequencies of 350s times for 24h, frequencies of 500s times for 24h, frequencies of 750s times for 24h, and frequencies of 1000s times for 24h at 84V and 13°C; for L. major, frequencies of 75s times for 28h, frequencies of 100s times for 18h, and frequencies of 200s times for 18h at 120V and 13°C. Molecular weights of the chromosomal DNA bands were estimated by comparison with DNA standards from Saccharomyces cerevisiae strain S13. The resulting gels were transferred to a Hybond-N (Amersham) nylon filter and hybridized as described for Southern blots. Sequence comparison and phylogenetic analyses. Multiple sequence alignment of the protein regions deduced from the PCR-amplified fragments of protozoan H+-PPase genes and from selected homologous genes accessible from public databases and genome projects was performed with the CLUSTAL X v.1.8 program . The alignment was used to construct phylogenetic trees using the distance (neighbor-joining, Kimura distance calculations) and maximum parsimony methods with the programs CLUSTAL X v.1.8 and PROTPARS v.3.573c (PHYLIP package v.3.5c (1993) Felsenstein, J., Department of Genetics, University of Washington, Seattle, USA), respectively. Bootstrap analyses (values being presented on a percentage basis) were in both cases computed with 1000 replicates and assigned to each internal branch. The partial H+-PPase gene sequences reported in this work were submitted to databases, the following accession numbers being assigned: AJ245400 (L. major); AJ243978 (T. cruzi); AJ251218 (L. ctenocephali); AJ249334 (Phytomonas sp.); AJ249229 (H. muscarum); AJ249332 (C. fasciculata); AJ249333 (E. schaudinni); AJ251772 (T. pyriformis gene I); AJ251471 (T. pyriformis gene II); AJ251369 (P. tetraurelia gene I); AJ431732 (P. tetraurelia gene II); AJ251219 (H. cavicola); AJ251782 (V. microstoma); AJ249300 (P. falciparum); AJ251783 (A. longa); AJ251725 (O. danica). The corresponding partial H+-PPase sequences from Chlamydomonas reihardtii (green alga) (AJ304836) and Porphyra yezoensis (red alga) (AJ431733) were obtained in our laboratory of IBVF (Sevilla), whereas those of the plant pathogenic oomycete Pytophthora infestans (BE775919) and the pathogenic trypanosomatid Trypanosoma brucei (contig 93A1.TF) were identified in the indicated non-annotated sequence files by searching the corresponding EST or genome projects (see below). Other bacterial or eukaryotic H+-PPase sequences used in the phylogenetic analyses were from GenBank-EMBL and SwissProt databases or were obtained by BLAST sequence searching  on diverse microbial or plant genome projects at the web sites of the National Center of Bioinformatics (NCBI), USA (http://www.ncbi.nih.gov/PMGifs/Genomes/allorg.html), The Joint Genomic Institute (JGI), USA (http://spider.jgi-psf.org/JGI_microbial/html/) or The Institute for Genomic Research (TIGR), USA (http://www.tigr.org/tdb/mdb/mdb.html).