Intergenic sequences from the heat-shock protein 83-encoding gene cluster in Leishmania mexicana amazonensis promote and regulate reporter gene expression in transfected parasites.

Regulation of expression from hsp83 gene cluster encoding heat-shock protein (HSP) 83 of the protozoan parasite Leishmania mexicana amazonensis (L.m.a) was examined. The first gene from this cluster, along with 8 kb of flanking sequences, was cloned, and intergenic region (IR) sequences were found upstream from the cluster. L.m.a. parasites were electroporated with a plasmid (pICI) in which the chloramphenicol acetyltransferase (CAT)-encoding gene (cat) was cloned between two IRs derived from an internal repeat unit of the hsp83 cluster, resulting in CAT activity at 26 degrees C. Exposure of cells transfected with this plasmid to a 35 degrees C heat shock led to an increase in CAT activity, within a range similar to that observed for the accumulation of hsp83 steady-state mRNA at 35 degrees C. S1 analysis of the hsp83 mRNA showed that the major part of the IR was transcribed and mostly present as 3' non-translated extensions. Deletion analysis of the flanking regions indicated that the presence of IR sequences, both upstream and downstream from cat, was critical to its expression. Partial deletions that removed the original AG splice acceptor site (leaving 289 bp upstream) and downstream IR sequences (leaving 200 bp) did not eliminate CAT activity. However, this combined deletion altered the effect of temperature on cat expression in transfected cells, as compared with the activity measured in cells transfected with the original plasmid.

Leishmania mexicana amazonensis: effect of heat shock on the spliced leader RNA and its ribonucleoprotein particle SL RNP.

Trypanosomatid parasites of the genus Leishmania experience a temperature shift from 22-38 degrees C to 33-37 degrees C while being transmitted from the invertebrate vector to the mammalian host. Expression of many protein-coding genes in protozoan parasites that cycle between two hosts have been shown to be thermosensitive, and temperature changes most probably serve as a major regulatory factor during stage differentiation. We present here our studies on effects of physiological temperature shift on the steady-state and nascent synthesis of the spliced leader SL RNA, as well as on its small RNP particle, SL RNP. Northern blot analysis showed no significant changes in the steady-state level of SL RNA at elevated temperatures. Neither were any alterations detected at the two different temperatures in nascent transcription of the SL RNA gene, examined in cells made permeable by treatment with lysolecithin. Fractionation of cell extracts on Cs2SO4 gradients indicated that temperature elevation led to changes in SL RNP particles. Alterations in these particles upon heat shock were also observed by separation on polyacrylamide gels, but only in the presence of urea, indicating that the differences caused by elevation of temperature were revealed exclusively under stringent fractionation conditions.

Temperature-induced expression of proteins in Leishmania mexicana amazonensis. A 22-kDa protein is possibly localized in the mitochondrion.

Temperature increase is an integral part of Leishmania life cycle, and plays a major role in stage transformation. Analysis of the temperature-dependent pattern of protein synthesis on two-dimensional gel electrophoresis shows that, in addition to the conserved heat-shock type of response in which expression of the major 70-kDa and 83-kDa heat-shock proteins is observed, a group of low-molecular-mass (17-40 kDa) proteins is induced in promastigotes of Leishmania mexicana amazonensis at elevated temperatures. Immuno-gold labelling with antibodies raised against the heat-induced 22-kDa proteins was localized mainly in the mitochondrion of Leishmania parasites, though labelling was observed also in the nucleus. The correlation of this finding with various reports on induction of mitochondrial enzymes in response to temperature stress in other organisms is discussed.

Sequence analysis and transcriptional activation of heat shock protein 83 of Leishmania mexicana amazonensis.

Changes in environmental temperature regulate the differential expression of genes during Leishmania stage differentiation. Therefore, molecular analysis of the heat shock proteins (HSPs) in these parasites is of interest as a model for thermoregulation of gene expression. Sequences of the HSP83 repetitive unit in the genome of Leishmania mexicana amazonensis, including both the coding and intergenic regions, are described. The 5' boundary of the message was mapped by S1 analysis, to potential AG splice sites located 293, 295 and 321 nucleotides upstream of the first ATG. A high degree of conservation (84%) is present between the coding sequence of HSP83 from L. mexicana amazonensis and similar sequences from Trypanosoma cruzi. The intergenic leishmanial sequences, however, were not homologous to similar sequences from HSP83 of trypanosomes, or from HSP70 of Leishmania major. A search for sequences that resemble eukaryote thermoregulated promoters was made and several regions with dyad symmetry were detected. However, only one of these regions was partially homologous with the consensus heat shock element present upstream of all eukaryotic HSPs studied to date.

Heat-shock protein 83 of Leishmania mexicana amazonensis is an abundant cytoplasmic protein with a tandemly repeated genomic arrangement.

The 83-kDa heat-shock protein HSP83 is a highly abundant protein in Leishmania amastigotes and promastigotes exposed to elevated temperature. Antibodies against this protein were obtained by immunization with a synthetic peptide derived from a conserved region. These antibodies recognized both the denatured and the native form of the molecule and were used for immunofluorescence analysis. These experiments, together with analysis by cell fractionation, show that HSP83 is distributed in the cytoplasm of Leishmania parasites. The gene for HSP83 in Leishmania mexicana amazonensis has been cloned from a genomic library, and molecular characterization shows it is present in several copies of 4-kb repeats arranged in tandem.