Molecular cloning and nucleotide sequence of the alpha and beta subunits of allophycocyanin from the cyanelle genome of Cyanophora paradoxa.

Abstract

The genes for the .alpha.- and .beta.-subunit apoproteins of allophycocyanin (AP) were isolated from the cyanelle genome of C. paradoxa and subjected to nucleotide sequence analysis. The AP .beta.-subunit apoprotein gene was localized to a 7.8-kilobase-pair Pst I restriction fragment from cyanelle DNA by hybridization with a tetradecameric oligonucleotide probe. Sequence analysis using that oligonucleotide and its complement as primers for the dideoxy chain-termination sequencing method confirmed the presence of both AP .alpha.- and .beta.-subunit genes on this restriction fragment. Additional oligonucleotide primers were synthesized as sequencing progressed and were used to determine rapidly the nucleotide sequence of a 1336-base-pair region of this cloned fragment. This strategy allowed the sequencing to be completed without a detailed restriction map and without extensive and time-consuming subcloning. The sequenced region contains 2 open reading frames whose deduced amino acid sequences are 81-85% homologous to cyanobacterial and red algal AP subunits whose amino acid sequences were determined. The 2 open reading frames are in the same orientation and are separated by 39 base pairs. AP .alpha. is 5'' to AP .beta. and both coding sequences are preceded by a polypurine, Shine-Dalgarno-type sequence. Sequences upstream from AP .alpha. closely resemble the Escherichia coli consensus promotor sequences and also show considerable homology to promotor sequences for several chloroplast-encoded psbA genes. A 56-base-pair palindromic sequence downstream from the AP .beta. gene could play a role in the termination of transcription or translation. The allophycocyanin apoprotein subunit genes are located on the large single-copy region of the cyanelle genome.