STRUCTURAL CHARACTERIZATION OF THE TERMINAL DOMAINS OF LINEAR PLASMID‐LIKE DNA FROM THE GREEN ALGA ERNODESMIS (CHLOROPHYTA)¹

Abstract

Terminal regions of linear plasmid-like DNA molecules from chloroplasts of the green alga Ernodesmis verticillata (Kützing) Børgesen were cloned and structurally characterized. Phosphorylation experiments with polynucleotide kinase indicated the presence of a 5 0 -phosphate, but the data did not reveal any protective protein(s) at the 5 0 end. To characterize the 3 0 end of these molecules, homopolymer-(poly(G)-) tailed molecules were annealed to and cloned into a linearized vector that was poly(C) tailed with terminal deoxynucleotidyl transferase. Sequencing analyses verified the heterogeneous nature of the molecules. Two distinct clones displayed extensive terminal inverted repeats (TIRs) at the 3 0 end (94 and 433 nt). Shorter TIRs (3-6 nt) were identified at the 3 0 end of most clones, which may serve to protect the ends. In fact, exonuclease III and k exonuclease digested the plasmid-like DNAs only after heat denaturation, signifying that conformational changes due to such treatment potentially make the 3 0 and 5 0 ends (respectively) susceptible to degradation. Multiple tandem and direct repeats were evident near the 3 0 ends. A consensus sequence of 18 þ nt was discovered in nearly every clone opposite the poly(G) tail, suggesting that this sequence has structural and/or functional significance. Pair-wise sequence comparisons and the presence of repeats indicated that these novel molecules may be highly recombinant.

The plastid genome in Cladophorales green algae is encoded by hairpin plasmids

“…observations of abundant LMW DNA in chloroplasts of several species of Cladophorales 34 . The hairpin configuration of the plasmids derived from our sequence data corresponds with earlier data based on electron microscopy, endo-and exonuclease digestion experiments, acridine orange staining, and denaturing gel electrophoresis 34,37 . Fluorescence in situ hybridization, and Southern blot hybridisation indicated that these plasmids were present within the chloroplast only 35 , supporting the congruence between chloroplast 454 contigs and sequences from the LMW fraction ( Fig.…”

“…1d). Pioneering work revealed that these plasmids are singlestranded DNA (ssDNA) molecules of 1.5-3.0 kb that fold in a hairpin configuration and lack sequence similarity to the nuclear DNA 36,37 . Some of the hairpin plasmids contain putatively transcribed sequences with similarity to chloroplast genes encoding subunits of Photosystems I and II (psaB, psbB, psbC and psbF) 36 .…”

Multi-marker metabarcoding of coral skeletons reveals a rich microbiome and diverse evolutionary origins of endolithic algae

“…S3 ). Like dinoflagellates, this green algal order possesses an unusual plastid configuration that prevents amplification with standard plastid markers 33 . We found 5 OTUs in the Cladophorales and this is the first record of their occurrence in coral skeletons.…”

Phylogenetic position of the coral symbiont Ostreobium (Ulvophyceae) inferred from chloroplast genome data

“…The chloroplast genomes of two other closely related orders, Cladophorales and Trentepohliales, have also not been sequenced or characterized in detail (but see La Claire et al. , , La Claire and Wang ). The Ulvales–Ulotrichales lineage, which is more distantly related in phylogenies of nuclear genes (Cocquyt et al.…”