No abstract

The nucleotide sequence of a 6156 bp segment of the circular 73 kb DNA from Astasia longa resembling the chloroplast DNA of Euglena was determined. The genes for the plastid elongation factor Tu (tufA) and the ribosomal protein S7 (rps7), six tRNA genes (trnQ, trnS, trnG, trnM, trnT, trnR), and three open reading frames were identified. These genes show a high degree of sequence similarity (73%-99%) to the corresponding genes on the Euglena chloroplast genome. The tufA gene contains two small AT-rich introns within its coding region. Northern analysis revealed the in vivo transcription of the tufA gene and of a reading frame of 456 codons into monocistronic mRNAs of 1.3 and 1.4 kb, respectively. The arrangement and organization of the genes on the 73 kb DNA of the colourless heterotrophic flagellate Astasia and the chloroplast DNA of autotrophic Euglena are compared.

The heterotrophic unicellular alga Prototheca wickerhamii is closely related to the photoautotrophic Chlorella vulgaris but has a 54,100-bp plastid DNA (ptDNA) that is much smaller than the chloroplast DNA of C. vulgaris (150,613 bp). The nucleotide sequence of 28,093 bp of the Prototheca ptDNA has been determined. No genes for photosynthetic functions have been found, except for sequences encoding six subunits of the ATP synthase ( atpA, atpB, atpE, atpF, atpH, and atpI). Transcripts of these atp genes have also been detected. Whether the leucoplasts of Prototheca contain a functional ATP synthase has still to be elucidated. Identified genes further include tufA, minD, cysT, and genes coding for three rRNAs, 22 tRNAs, and 12 ribosomal proteins. The results support the idea that, in the reduced plastid genome of Prototheca, genes coding for components of the plastid translational apparatus have been preferentially retained, and might be needed for the expression of the atp genes and some unassigned ORFs.

Purified chloroplasts from developing leaves of Vicia faba L. were broken and separated into stroma and thylakoid fractions. Both fractions contained polysomes as demonstrated by analytical density gradient centrifugation and in-vitro read-out translation. Messenger RNAs of free and thylakoid-bound polysomes were isolated and analysed by hybridization with heterologous gene probes from spinach and tobacco. Transcripts of the chloroplast genes psaA, psbB, psbC, psbD and petA were found predominantly on thylakoidbound polysomes engaged in the synthesis and the contrasslational integration of membrane proteins. In contrast, transcripts of the genes rbcL, psbE, petD, atpA, atpB, atpE and atpH were found more frequently on free polysomes corresponding to a stroma-located translation of these mRNAs and a posttranslational integration of the encoded intrinsic membrane proteins. We conclude from these findings that chloroplast-encoded membrane proteins are integrated by co-and posttranslational mechanisms.

A gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) was identified on a circular 73 kb DNA from the colourless euglenoid flagellate Astasia longa. The rbcL gene of Astasia extends over 3968 bp. It is a split gene interrupted by seven introns as compared to nine intervening sequences in the rbcL gene of the phylogenetically related Euglena gracilis. Coding sequences as well as the positions of the introns within this gene are highly conserved in comparison with the Euglena rbcL except that two introns are missing in Astasia. The alignment of the amino acid sequences deduced from the nucleotide sequences of rbcL of Astasia and Euglena shows 82% identical amino acids whereas 15% of the amino acids represent conservative changes. A 1.5 kb transcript of the rbcL gene was revealed by northern blot analysis of Astasia RNA. By immunoblot analysis the gene product of rbcL was detected as a 53 kDa polypeptide. Genes for components of the chloroplast transcriptional and translational systems encoded by chloroplast DNA of plants and green algae are conserved on the 73 kb DNA of Astasia [24, 25, 26]. From our finding that Astasia obviously is capable of synthesizing the Rubisco large subunit one must conclude that these genes are expressed and form functional plastid transcriptional and translational systems.