The transcription promoter of the spliced leader gene from Trypanosoma cruzi.

A putative promoter element responsible for transcription of the spliced leader (SL) gene of Trypanosoma cruzi was identified by overlapping deletion and linker scanning analyses of the upstream flanking sequences using the bacterial chloramphenicol acetyltransferase (CAT) gene as a reporter in transient transfections of cultured epimastigotes. Deletion or substitution of a proximal sequence element (PSE) between positions -53 and -40 relative to the transcription start point eliminated CAT gene expression. Comparison of SL genes from several strains of T. cruzi revealed two alternative sequence patterns for the putative SL PSE, both composed of a short run of purines followed by a run of pyrimidines. Moreover, an examination of these sequences supports the subdivision of T. cruzi isolates into two divergent groups. Double-stranded oligonucleotides containing the sequence of the PSE exhibited specific gel mobility shifts after incubation with T. cruzi nuclear extracts, suggesting that a transcription factor binds this site. Finally, experiments designed to increase the level of CAT expression from the SL promoter suggest that it is not a strong promoter in cultured T. cruzi epimastigotes.

Trypanosoma cruzi strains partition into two groups based on the structure and function of the spliced leader RNA and rRNA gene promoters.

We have previously identified a major proximal sequence element (PSE) responsible for transcription of the spliced leader (SL) gene from Trypanosoma cruzi strain CL, and showed that the sequence encompassing this PSE exhibits approximately 30% divergence between two major groups of T. cruzi isolates, but strong conservation within the groups. In this report, we show that the SL RNA gene promoter from the CL strain (group I) is efficiently expressed only in T. cruzi isolates from group I. Similarly, the sequence of the approximately 643 bp promoter region of the T. cruzi rRNA is strongly conserved within, but diverged approximately 20% between, the two groups. Reporter constructs driven by the rRNA promoter sequences from group I strains are strongly expressed after electroporation into other group I strains, but not expressed in group II strains. In contrast, constructs bearing rRNA promoter sequences from group II strains are active in strains from both groups. Phylogenetic analyses performed with both the rRNA and the SL RNA gene promoter sequences yielded similar trees, and these trees strongly reinforce the partitioning of known T. cruzi into two major groups that parallel the observed functional specificity of the promoters. Given the well-documented species specific pattern of both rRNA promoters and PSEs in higher eukaryotes, these results suggest an ancient evolutionary divergence among organisms currently classified as T. cruzi.

The infant vocal-conditioning literature: a theoretical and methodological critique.

Most studies of operant conditioning of infant vocalization do not fully use the experimental-analysis-of-behavior methodology that behavioral researchers have developed to study operant phenomena. This could be a contributing factor in their failure to correctly operationalize the definition of reinforcement, severely limiting the amount of information they can provide about operant learning in infants. Furthermore, single-subject-design studies may be added to supplement or replace group experimental designs in the study of infant vocal conditioning if we are to recognize the different learning processes that affect infant learning. Finally, single-subject experimental designs may be crucial to the development of an effective technology of early language intervention.

Heterogeneous glycosylation of Musca domestica arylphorin.

Two distinct fractions of Musca domestica arylphorin were isolated by affinity chromatography on Concanavalin A-Sepharose column. The results show that in the hexameric arylphorin that do not bind to the lectin there is no Concanavalin A binding subunit and in the majority of the hexamers that bind to the lectin there is only one subunit with Concanavalin A binding site. The results indicate that the carbohydrate moiety of the arylphorin is not involved in its specific uptake by the fat bodies and integument.

kDNA and rDNA sequences reveal a phylogenetic cluster of species originally placed in different genera of trypanosomatids.

Hybridization using kDNA and rDNA sequences as probes was performed to study phylogenetic relatedness of different species of trypanosomatids. Using this approach, we identified five organisms which had been classified as Phytomonas and Herpetomonas that were more closely correlated to each other phylogenetically than to any other species or isolates from either genera. These findings raise doubts about the validity of the current classification of Trypanosomatidae. Finally, we demonstrated the usefulness of kDNA sequences as an alternative to genomic sequences in obtaining phylogenetic information on trypanosomatids.

Trypanosomatidae: a spliced-leader-derived probe specific for the genus Phytomonas.

We probed DNA from all trypanosomatid genera by slot blot hybridization with an oligonucleotide (SL3') complementary to a sequence of the Phytomonas spliced-leader or mini-exon RNA. The 19-nucleotide probe target site was previously shown to be highly conserved among a limited number of Phytomonas isolates, but diverges in other kinetoplastid genera. Our examination of 84 isolates of various genera of trypanosomatids showed hybridization of this probe exclusively with isolates from plants or insects which could, by morphological, biochemical, and molecular criteria, be considered to belong to the genus Phytomonas. In contrast, no hybridization was observed with flagellates of the genera Blastocrithidia, Crithidia, Endotrypanum, Herpetomonas, Leptomonas, Leishmania, and Trypanosoma. The method detected DNA quantities as low as 50 ng using either radioactive or nonradioactive probes, and was effective with as few as 10(4) intact flagellates. Together, these results suggest that this probe will serve as a convenient marker for taxonomic and epidemiological studies requiring reliable identification of Phytomonas spp. in plants or in putative insect vectors.

Trypanosomatidae: Phytomonas detection in plants and phytophagous insects by PCR amplification of a genus-specific sequence of the spliced leader gene.

In this paper we describe a method for the detection of Phytomonas spp. from plants and phytophagous insects using the PCR technique by targeting a genus-specific sequence of the spliced leader (SL) gene. PCR amplification of DNA from 48 plant and insect isolates previously classified as Phytomonas by morphological, biochemical, and molecular criteria resulted in all cases in a 100-bp fragment that hybridized with the Phytomonas-specific spliced leader-derived probe SL3'. Moreover, this Phytomonas-specific PCR could also detect Phytomonas spp. in crude preparations of naturally infected plants and insects. This method shows no reaction with any other trypanosomatid genera or with plant and insect host DNA, revealing it to be able to detect Phytomonas spp. from fruit, latex, or phloem of various host plants as well as from salivary glands and digestive tubes of several species of insect hosts. Results demonstrated that SLPCR is a simple, fast, specific, and sensitive method that can be applied to the diagnosis of Phytomonas among cultured trypanosomatids and directly in plants and putative vector insects. Therefore, the method was shown to be a very specific and sensitive tool for diagnosis of Phytomonas without the need for isolation, culture, and DNA extraction of flagellates, a feature that is very convenient for practical and epidemiological purposes.

Comparative analyses of the complete genome sequences of Pierce's disease and citrus variegated chlorosis strains of Xylella fastidiosa.

Xylella fastidiosa is a xylem-dwelling, insect-transmitted, gamma-proteobacterium that causes diseases in many plants, including grapevine, citrus, periwinkle, almond, oleander, and coffee. X. fastidiosa has an unusually broad host range, has an extensive geographical distribution throughout the American continent, and induces diverse disease phenotypes. Previous molecular analyses indicated three distinct groups of X. fastidiosa isolates that were expected to be genetically divergent. Here we report the genome sequence of X. fastidiosa (Temecula strain), isolated from a naturally infected grapevine with Pierce's disease (PD) in a wine-grape-growing region of California. Comparative analyses with a previously sequenced X. fastidiosa strain responsible for citrus variegated chlorosis (CVC) revealed that 98% of the PD X. fastidiosa Temecula genes are shared with the CVC X. fastidiosa strain 9a5c genes. Furthermore, the average amino acid identity of the open reading frames in the strains is 95.7%. Genomic differences are limited to phage-associated chromosomal rearrangements and deletions that also account for the strain-specific genes present in each genome. Genomic islands, one in each genome, were identified, and their presence in other X. fastidiosa strains was analyzed. We conclude that these two organisms have identical metabolic functions and are likely to use a common set of genes in plant colonization and pathogenesis, permitting convergence of functional genomic strategies.

Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis.

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide O side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.