Macronuclear genome structure of the ciliate Nyctotherus ovalis: single-gene chromosomes and tiny introns

Abstract

Background: Nyctotherus ovalis is a single-celled eukaryote that has hydrogen-producing mitochondria and lives in the hindgut of cockroaches. Like all members of the ciliate taxon, it has two types of nuclei, a micronucleus and a macronucleus. N. ovalis generates its macronuclear chromosomes by forming polytene chromosomes that subsequently develop into macronuclear chromosomes by DNA elimination and rearrangement.

Results: We examined the structure of these gene-sized macronuclear chromosomes in N. ovalis. We determined the telomeres, subtelomeric regions, UTRs, coding regions and introns by sequencing a large set of macronuclear DNA sequences (4,242) and cDNAs (5,484) and comparing them with each other. The telomeres consist of repeats CCC(AAAACCCC)n, similar to those in spirotrichous ciliates such as Euplotes, Sterkiella (Oxytricha) and Stylonychia. Per sequenced chromosome we found evidence for either a single protein-coding gene, a single tRNA, or the complete ribosomal RNAs cluster. Hence the chromosomes appear to encode single transcripts. In the short subtelomeric regions we identified a few overrepresented motifs that could be involved in gene regulation, but there is no consensus polyadenylation site. The introns are short (21-29 nucleotides), and a significant fraction (1/3) of the tiny introns is conserved in the distantly related ciliate Paramecium tetraurelia. As has been observed in P. tetraurelia, the N. ovalis introns tend to contain in-frame stop codons or have a length that is not dividable by three. This pattern causes premature termination of mRNA translation in the event of intron retention, and potentially degradation of unspliced mRNAs by the nonsense-mediated mRNA decay pathway.

Conclusion: The combination of short leaders, tiny introns and single genes leads to very minimal macronuclear chromosomes. The smallest we identified contained only 150 nucleotides.

Figure 1

a) Structure of N. ovalis macronuclear chromosomes. 1a-0.4% Agarose gel: M1 = λ- DNA (48502 bp). M2 = λ Eco RI/HindIII (sizes of the DNA fragments: 21226, 5148+4973, 4268, 3530, 2027, 1904, 1584 and 1375 bp). Lane 1,2 and 3 contain total DNA of Nyctotherus ovalis. Lane 4 contains total DNA of Euplotes crassus. Arrows indicate the ribosomal clusters (see Materials). b) Length distribution of the sequenced full-length macronuclear chromosomes. c) Southern blotting: unrestricted total DNA of N. ovalis was separated on a 0.7% agarose gel, blotted and hybridized with clones containing the α2 tubulin (α tub) and the β2 tubulin gene of Stylonychia lemnae. The labeling of single, gene-sized bands indicates the presence of tubulin genes on gene-sized DNA molecules.

Figure 2

Nucleotide frequency distributions and Shannon entropy of the frequency distribution of the first 300 nt after telomeres in the 5' of the coding strand.

Figure 3

Schematic view of N. ovalis macronuclear chromosome structure and the purine/pyrimidine ratios. Lengths of the various parts composing the chromosome and cDNA are displayed. The combination of short leaders, short introns and single genes leads to very short chromosomes. In this figure we can observe as well that the purine/pyrimidine skew is higher than 0 in the coding strand, allowing the prediction of coding strands for chromosomes without a homolog among known proteins.

Figure 4

a) Distribution of the intron length (in percentage) within the two sets of introns: The experimental set (48 introns) that was obtained by comparing N. ovalis cDNAs with gDNAs and the predicted set (145 introns) that was obtained by comparison of N. ovalis gDNAs with P. tetraurelia proteins. The predicted set contains three longer introns that are not shown in this histogram (76, 114 and 252 nt long). b) Schematic representation of the exon/intron boundaries with WebLogo in 48 introns. The GTA and TAG motifs are well conserved. c) Conservation of intron location between N. ovalis and P. tetraurelia in two genes that are orthologous to Succinyl-CoA ligase. Introns were considered conserved when they were within 2 amino acids of each other in the protein alignment. Intron I of N. ovalis and II of P. tetraurelia are conserved. Intron II of N. ovalis and I of P. tetraurelia are not conserved. Whether Intron III of P. tetraurelia is conserved or not cannot be determined.

Figure 5

Schematic view of the structure of tRNA macronuclear chromosomes. The motifs found with MEME in more than half of the 12 nr-tRNAs are displayed as a WebLogo picture, together with their positions (from..to (~average)). 0 corresponds to start of the tRNA.

Figure 6

Phylogenetic tree of 18S RNA from the small ribosomal subunit of the most-studied ciliates with respect to their macronuclear structure. The tree was generated with PhyML with 100 bootstraps. The dinoflagellate Symbiodinium is used as an outgroup to root the tree. N. ovalis has a comparable macronuclear structure to Spirotrichea, its sister group in the tree (the lineage containing Euplotes through Sterkiella). MAC stands for macronucleus, MIC for micronucleus.