Cloning and sequence determination of a cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase.
A partial cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase (ACMPK) was isolated from a lambda ZAP expression library by immunoselection using monospecific polyclonal antibodies to the enzyme. The sequence of both strands of the cDNA (CMKa) was determined. The deduced amino acid (aa) sequence contained all eleven consensus domains found in serine/threonine protein kinases [Hanks et al., Science 241 (1988) 42-52], as well as a putative calmodulin-binding domain. The cDNA contained an intron, lacked an in-frame start codon, and was not polyadenylated. A full-length copy of CMKa was subsequently isolated from a lambda gt10 library of A. nidulans cDNA using a restriction fragment of the first clone as a probe. It contained an in-frame start codon, an open reading frame (ORF) of 1242 bp and was polyadenylated. The ORF encoded a protein of 414 aa residues with an M(r) of 46,895 and an isoelectric point pI = 6.4. These values are in good agreement with that observed for the native enzyme [Bartelt et al., Proc. Natl. Acad. Sci. USA 85 (1988) 3279-3283]. When aligned to optimize homology, 29% of the predicted aa sequence of ACMPK is identical to that of the alpha-subunit of rat brain calmodulin-dependent protein kinase II. ACMPK shares 40 and 44% identity in aa sequence with YCMK1 and YCMK2, respectively, two Ca2+/calmodulin-dependent protein kinases recently cloned from Saccharomyces cerevisiae [Pausch et al., EMBO J. 10 (1991) 1511-1522]. Results of Southern analysis of restriction digests of genomic DNA indicate that ACMPK is encoded by a single-copy gene.
Kornstein
L B
LB
Department of Biological Sciences, St. John's University Jamaica, NY 11439.
Gaiso
M L
ML
Hammell
R L
RL
Bartelt
D C
DC
D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D003001:Cloning, Molecular / Q000379:methods; D004271:DNA, Fungal / Q000235:genetics* / Q000302:isolation & purification; D015723:Gene Library; D007118:Immunoassay; D007438:Introns; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis / Q000235:genetics*; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D004271:DNA, Fungal; D009838:Oligodeoxyribonucleotides; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases