Cloning from Leishmania major of a developmentally regulated gene, c-lpk2, for the catalytic subunit of the cAMP-dependent protein kinase.
Protein kinases are important in the regulation of cellular processes including growth and differentiation. Using the polymerase chain reaction with oligonucleotide primers derived from conserved regions of cAMP-dependent protein kinases (PKAs), three different DNA fragments were amplified from leishmanial genomic DNA. One fragment was used to isolate a stage specific gene, c-lpk2, from a Leishmania major genomic library. This gene shows high homology to other eukaryotic PKAs, and the open reading frame encodes a 332 amino acid protein with a predicted molecular mass of 38.2 kDa. When aligned with other PKAs the leishmanial enzyme has a unique eight amino acid extension at the carboxy terminus. The c-lpk2 gene is present as a single copy in L. major, L. donovani and L. amazonensis. The 5'-flanking region contains a polypyrimidine rich tract upstream from the predicted ATG start codon. The gene is highly expressed in promastigotes and barely detectable in amastigotes of L. major. Temperature increase was shown to rapidly down-regulate c-lpk2 expression. Transfer of L. amazonensis promastigotes to 35 degrees C resulted in the rapid disappearance of c-lpk2 mRNA (> 70% in 1 h), while at 26 degrees C the mRNA was more stable. The strict temperature dependence of mRNA degradation rate suggests that PKA expression is regulated post-transcriptionally.
Siman-Tov
M M
MM
Department of Parasitology, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Aly
R
R
Shapira
M
M
Jaffe
C L
CL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics*; D016054:DNA, Protozoan / Q000032:analysis; D018628:Gene Dosage; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D017125:Genes, Protozoan / Q000235:genetics*; D006358:Hot Temperature; D007891:Leishmania / Q000235:genetics; D018320:Leishmania major / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D016053:RNA, Protozoan / Q000032:analysis; D015183:Restriction Mapping; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D016054:DNA, Protozoan; D012333:RNA, Messenger; D016053:RNA, Protozoan; D017868:Cyclic AMP-Dependent Protein Kinases