Nucleomorph genomes: much ado about practically nothing.

The DNA sequence of one of the smallest eukaryotic genomes has recently been finished - that of the reduced nucleus, or nucleomorph, of an algal endosymbiont that resides within a cryptomonad host cell. Its sequence promises insights into chloroplast acquisition, the constraints on genome size and the basic workings of eukaryotic cells.

Cell division protein FtsZ: running rings around bacteria, chloroplasts and mitochondria.

Of all the proteins involved in prokaryotic cell division FtsZ is one of the earliest acting and most widely distributed, being found in all but a few species. We discuss several recent discoveries of FtsZ in eukaryotic cells and the protein's role in the division of chloroplasts and mitochondria, organelles that are of bacterial origin.

Mitochondrial FtsZ in a chromophyte alga.

A homolog of the bacterial cell division gene ftsZ was isolated from the alga Mallomonas splendens. The nuclear-encoded protein (MsFtsZ-mt) was closely related to FtsZs of the alpha-proteobacteria, possessed a mitochondrial targeting signal, and localized in a pattern consistent with a role in mitochondrial division. Although FtsZs are known to act in the division of chloroplasts, MsFtsZ-mt appears to be a mitochondrial FtsZ and may represent a mitochondrial division protein.

Good things in small packages: the tiny genomes of chlorarachniophyte endosymbionts.

Chlorarachniophytes are amoeboflagellate, marine protists that have acquired photosynthetic capacity by engulfing and retaining a green alga. These green algal endosymbionts are severely reduced, retaining only the chloroplast, nucleus, cytoplasm and plasma membrane. The vestigial nucleus of the endosymbiont, called the nucleomorph, contains only three small linear chromosomes and has a haploid genome size of just 380 kb--the smallest eukaryotic genome known. Initial characterisation of nucleomorph DNA has revealed that all chromosomes are capped with inverted repeats comprising a telomere and a single ribosomal RNA operon. The nucleomorph genome is the quintessence of compactness; average space between genes is a mere 65 bp,- some genes overlap, others are cotranscribed. Intense reductive pressures upon nucleomorph genes have apparently squeezed their spliceosomal-type introns down to only 18, 19 or 20 bases in length. Studies to date indicate the nucleomorph--essentially a stripped-down eukaryotic genome--encodes principally genetic housekeeping functions such as translation, transcription and sing.

Size isn't everything: lessons in genetic miniaturisation from nucleomorphs.

Nucleomorphs are the vestigial nuclear genomes of eukaryotic algal cells now existing as endosymbionts within a host cell. Molecular investigation of the endosymbiont genomes has allowed important insights into the process of eukaryote/eukaryote cell endosymbiosis and has also disclosed a plethora of interesting genetic phenomena. Although nucleomorph genomes retain classic eukaryotic traits such as linear chromosomes, telomeres, and introns, they are highly reduced and modified. Nucleomorph chromosomes are extremely small and encode compacted genes which are disrupted by the tiniest spliceosomal introns found in any eukaryote. Mechanisms of gene expression within nucleomorphs have apparently accommodated increasingly parsimonious DNA usage by permitting genes to become co-transcribed or, in select cases, to overlap.

The miniaturized nuclear genome of eukaryotic endosymbiont contains genes that overlap, genes that are cotranscribed, and the smallest known spliceosomal introns.

Chlorarachniophyte algae contain a complex, multi-membraned chloroplast derived from the endosymbiosis of a eukaryotic alga. The vestigial nucleus of the endosymbiont, called the nucleomorph, contains only three small linear chromosomes with a haploid genome size of 380 kb and is the smallest known eukaryotic genome. Nucleotide sequence data from a subtelomeric fragment of chromosome III were analyzed as a preliminary investigation of the coding capacity of this vestigial genome. Several housekeeping genes including U6 small nuclear RNA (snRNA), ribosomal proteins S4 and S13, a core protein of the spliceosome [small nuclear ribonucleoprotein (snRNP) E], and a cip-like protease (clpP) were identified. Expression of these genes was confirmed by combinations of Northern blot analysis, in situ hybridization, immunocytochemistry, and cDNA analysis. The protein-encoding genes are typically eukaryotic in overall structure and their messenger RNAs are polyadenylylated. A novel feature is the abundance of 18-, 19-, or 20-nucleotide introns; the smallest spliceosomal introns known. Two of the genes, U6 and S13, overlap while another two genes, snRNP E and clpP, are cotranscribed in a single mRNA. The overall gene organization is extraordinarily compact, making the nucleomorph a unique model for eukaryotic genomics.

Organisation and sequence analysis of nuclear-encoded 5s ribosomal RNA genes in cryptomonad algae.

Southern hybridisation, polymerase chain reaction (PCR), and nucleotide sequence, data indicate that the 5s ribosomal RNA (rRNA) gene is linked to the main rRNA gene repeat in the nuclear genome of four cryptomonad algae (Rhinomonas pauca, Storeatula major, Komma caudata, and isolate Cs 134). The 5s gene is apparently transcribed in the same direction as the large and small subunit rRNA genes. The intergenic spacer between the 5s gene and the large subunit rRNA gene exhibits length and sequence polymorphism among the different species. Cryptomonads contain two different eukaryotic genomes: the host nucleus and the nucleus of a eukaryotic endosymbiont. Mapping experiments with isolated chromosomes of the host and endosymbiont genomes showed that the intergenic spacer between the large subunit and the 5s rRNA gene, which was amplified from total DNA by PCR, was derived from the host nuclear genome.

Evidence that an amoeba acquired a chloroplast by retaining part of an engulfed eukaryotic alga.

Chlorarachniophytes are amoeboid algae with unusual chloroplasts. Instead of the usual two membranes that surround the chloroplasts of plants, green algae, and red algae, the chloroplasts of chlorarachniophytes have four bounding membranes. The extra membranes may reflect an unusual origin of chlorarachniophyte chloroplasts. Rather than inheriting the organelle directly from their ancestors, chlorarachniophytes may have adopted the chloroplast of an algal cell ingested as prey. Parts of the algal cell are postulated to remain within the amoeba as a reduced eukaryotic endosymbiont [Hibberd, D. J. & Norris, R. E. (1984) J. Phycol. 20, 310-330]. A small nucleus-like structure, proposed to be a vestige of the endosymbiont's nucleus, is located in a space between the second and third chloroplast membranes. We cloned and sequenced nuclear-type rRNA genes from chlorarachniophytes and found two highly divergent genes. In situ hybridization shows that one gene is expressed by the amoebal (host) nucleus and the other is expressed by the putative endosymbiont nucleus, suggesting that the latter is indeed a foreign genome. Transcripts from the endosymbiont gene accumulate in the small cytoplasmic compartment between the second and third chloroplast membranes, which we believe to be the remnant cytoplasm of the endosymbiont. Using the endosymbiont gene as a probe, we identified three small chromosomes belonging to the endosymbiont nucleus. This knowledge should allow a detailed molecular analysis of the role of the endosymbiont's genome and cytoplasm in the partnership.

The photosynthetic endosymbiont in cryptomonad cells produces both chloroplast and cytoplasmic-type ribosomes.

Cryptomonad algae contain a photosynthetic, eukaryotic endosymbiont. The endosymbiont is much reduced but retains a small nucleus. DNA from this endosymbiont nucleus encodes rRNAs, and it is presumed that these rRNAs are incorporated into ribosomes. Surrounding the endosymbiont nucleus is a small volume of cytoplasm proposed to be the vestigial cytoplasm of the endosymbiont. If this compartment is indeed the endosymbiont's cytoplasm, it would be expected to contain ribosomes with components encoded by the endosymbiont nucleus. In this paper, we used in situ hybridization to localize rRNAs encoded by the endosymbiont nucleus of the cryptomonad alga, Cryptomonas phi. Transcripts of the endosymbiont rRNA gene were observed within the endosymbiont nucleus, and in the compartment thought to represent the endosymbiont's cytoplasm. These results indicate that the endosymbiont produces its own set of cytoplasmic translation machinery. We also localized transcripts of the host nucleus rRNA gene. These transcripts were found in the nucleolus of the host nucleus, and throughout the host cytoplasm, but never in the endosymbiont compartment. Our rRNA localizations indicate that the cryptomonad cell produces two different of sets of cytoplasmic-type ribosomes in two separate subcellular compartments. The results suggest that there is no exchange of rRNAs between these compartments. We also used the probe specific for the endosymbiont rRNA gene to identify chromosomes from the endosymbiont nucleus in pulsed field gel electrophoresis. Like other cryptomonads, the endosymbiont nucleus of Cryptomonas phi contains three small chromosomes.