ABSTRACT
Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37 percent of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89 percent similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.
Subject(s)
Animals , Mice , Calmodulin/metabolism , HSP90 Heat-Shock Proteins/metabolism , Saccharomyces cerevisiae/chemistry , Amino Acid Sequence , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Calmodulin/genetics , Electrophoresis, Polyacrylamide Gel , HSP90 Heat-Shock Proteins/genetics , /genetics , /metabolism , Mass Spectrometry , Phylogeny , Sequence Alignment , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/geneticsABSTRACT
The sequencing of Trypanosoma cruzi genome has been completed and a great deal of information is now available. However, the organization of protozoa genomes is somewhat elusive and much effort must be applied to reveal all the information coded in the nucleotide sequences. Among the DNA segments that needs further investigation are the untranslated regions of genes. Many of the T. cruzi genes that were revealed by the genome sequencing lack information about the untranslated regions. In this paper, some features of these untranslated segments as well as their applications in T. cruzi populations are discussed.
Subject(s)
Animals , Calmodulin/genetics , Genes, Protozoan/genetics , Mutation , Trypanosoma cruzi/genetics , Untranslated Regions/genetics , Calmodulin/chemistry , Untranslated Regions/chemistryABSTRACT
En el presente trabajo se reporta el primer DNA satélite (PFCOL692) aislado para Plasmodium falciparum. La unidad básica es una secuencia de aproximadamente 700 pares de bases (pb). Las dos variantes secuenciadas presentan una homología del 85 por ciento. Los patrones de bandas producidos al hibridar digestiones de DNA del parásito con el DNA satélite marcado radioactivamente, permitieron detectar la existencia de muchas más variantes. Además, las variantes se encuentran a nivel subtelomérico organizadas en bloques de seres unidos cabeza a cola. Por la localización esta secuencia debe jugar un papel importante en los rearreglos que producen polimorfismo cromosómico y antigénico en Plasmodium falciparum. De los patrones de bandas obtenidos por la hibridación de esta secuencia con DNA de diferentes aislados digeridos con enzima de restricción, se pudo ver la utilidad de esta secuencia para caraterizar cepas en el laboratorio. Se construyó una genoteca de Plasmodium falciparum en el fago Lambda EMBL-4 y fueron aislados 28 clonos que contienen la secuencia PFCOL692. El resultado del manejo de restricción de los clonos (en otro trabajo) confirman la organzación planteada. Como otra parte del trabajo se construyó una genoteca de Plasmodium falciparum en el fago Lamda gt10. Se diseño una sonda de oligonucleótidos con base en la secuencia del gen de calmodulina de Plasmodium falciparum. Con la sonda, se aisló un clon que contiene la mayoría del gen de calmodulina del parásito