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.
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
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Shapira
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D006360:Heat-Shock Proteins / Q000235:genetics*; D007438:Introns; D007700:Kinetics; D007894:Leishmania mexicana / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D003062:Codon; D006360:Heat-Shock Proteins; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D015500:Chloramphenicol O-Acetyltransferase