Comparative genomic studies suggest that the cyanobacterial endosymbionts of the amoeba Paulinella chromatophora possess an import apparatus for nuclear-encoded proteins.

Plastids evolved from free-living cyanobacteria through a process of primary endosymbiosis. The most widely accepted hypothesis derives three ancient lineages of primary plastids, i.e. those of glaucophytes, red algae and green plants, from a single cyanobacterial endosymbiosis. This hypothesis was originally predicated on the assumption that transformations of endosymbionts into organelles must be exceptionally rare because of the difficulty in establishing efficient protein trafficking between a host cell and incipient organelle. It turns out, however, that highly integrated endosymbiotic associations are more common than once thought. Among them is the amoeba Paulinella chromatophora, which harbours independently acquired cyanobacterial endosymbionts functioning as plastids. Sequencing of the Paulinella endosymbiont genome revealed an absence of essential genes for protein trafficking, suggesting their residence in the host nucleus and import of protein products back into the endosymbiont. To investigate this hypothesis, we searched the Paulinella endosymbiont genome for homologues of higher plant translocon proteins that form the import apparatus in two-membrane envelopes of primary plastids. We found homologues of Toc12, Tic21 and Tic32, but genes for other key translocon proteins (e.g. Omp85/Toc75 and Tic20) were missing. We propose that these missing genes were transferred to the Paulinella nucleus and their products are imported and integrated into the endosymbiont envelope membranes, thereby creating an effective protein import apparatus. We further suggest that other bacterial/cyanobacterial endosymbionts found in protists, plants and animals could have evolved efficient protein import systems independently and, therefore, reached the status of true cellular organelles.

Are red algae plants? A critical evaluation of three key molecular data sets.

Whether red algae are related to green plants has been debated for over a century. Features present due to their shared photosynthetic habit have been interpreted as support for an evolutionary sisterhood of the two groups but, until very recently, characters endogenous to the host cell have provided no reliable indication of such a relationship. In this investigation, we examine three molecular data sets that have provided key evidence of a possible relationship between green plants and red algae. Analyses of an expanded alignment of DNA-dependent RNA polymerase II largest subunit sequences indicate that their support for independent origins of rhodophytes and chlorophytes is not the result of long-branch attraction, as has been proposed elsewhere. Differences in the pol II C-terminal domain, an essential component of plant mRNA transcription, also suggest different host cell ancestors for the two groups. In contrast, concatenated sequences of two groups of mitochondrial genes, those encoding subunits of NADH-dehydrogenase as well as cytochrome c oxidase subunits plus apocytochrome B, appear to cluster red algal and green plant sequences together because both groups have evolved relatively slowly and share a super-abundance of ancestral positions. Finally, analyses of elongation factor 2 sequences demonstrate a strong phylogenetic signal favoring a rhodophyte/chlorophyte sister relationship, but that signal is restricted to a contiguous segment comprising approximately half of the EF2 gene. These results argue for great caution in the interpretation of phylogenetic analyses of ancient evolutionary events but, in combination, indicate that there is no emerging consensus from molecular data supporting a sister relationship between red algae and green plants.

Evolutionary complementation for polymerase II CTD function.

The C-terminal domain (CTD) of the largest subunit (RPB1) of eukaryotic RNA polymerase II is essential for pol II function and has been shown to play a number of important roles in the mRNA transcription cycle. The CTD is composed of a tandemly repeated heptapeptide that is conserved in yeast, animals, plants and several protistan organisms. Some eukaryotes, however, have what appear to be degenerate or deviant CTD regions, and others have no CTD at all. The functional and evolutionary implications of this variation among RPB1 C-termini is largely unexplored. We have transformed yeast cells with a construct consisting of the yeast RPB1 gene with 25 heptads from the primitive protist Mastigamoeba invertens in place of the wild-type CTD. The Mastigamoeba heptads differ from the canonical CTD by the invariable presence of alanines in place of threonines at position 4, and in place of serines at position 7 of each heptad. Despite this double substitution, mutants are viable even under conditions of temperature and nutrient stress. These results provide new insights into the relative functional importance of several of the conserved CTD residues, and indicate that in vivo expression of evolutionary variants in yeast can provide important clues for understanding the origin, evolution and function of the pol II CTD.

Long-branch attraction and the rDNA model of early eukaryotic evolution.

Phylogenetic analyses of ribosomal RNA genes have become widely accepted as a framework for understanding broad-scale eukaryotic evolution. Nevertheless, conflicts exist between the phylogenetic placement of certain taxa in rDNA trees and their expected position based on fossils, cytology, or protein-encoding gene sequences. For example, pelobiont amoebae appear to be an ancient group based on cytologic features, but they are not among the early eukaryotic brances in rDNA analyses. In this report, the derived position of pelobionts in rDNA trees is shown to be unreliable and likely due to long-branch attraction among more deeply branching sequences. All sequences that branch near the base of the tree suffer from relatively high apparent substitution rates and exhibit greater variation in ssu rDNA sequence length. Moreover, the order of the branches leading from the root of the eukaryotic tree to the base of the so-called "crown taxa" is consistent with a sequential attachment, due to "long-branch" effects, of sequences with increasing rates of evolution. These results suggest that the basal eurkaryotic topology drawn from rDNA analyses may be, in reality, an artifact of variation in the rate of molecular evolution among eukaryotic taxa.

Amitochondriate amoebae and the evolution of DNA-dependent RNA polymerase II.

Unlike parasitic protist groups that are defined by the absence of mitochondria, the Pelobiontida is composed mostly of free-living species. Because of the presence of ultrastructural and cellular features that set them apart from all other eukaryotic organisms, it has been suggested that pelobionts are primitively amitochondriate and may represent the earliest-evolved lineage of extant protists. Analyses of rRNA genes, however, have suggested that the group arose well after the diversification of the earliest-evolved protists. Here we report the sequence of the gene encoding the largest subunit of DNA-dependent RNA polymerase II (RPB1) from the pelobiont Mastigamoeba invertens. Sequences within RPB1 encompass several of the conserved catalytic domains that are common to eubacterial, archaeal, and eukaryotic nuclear-encoded RNA polymerases. In RNA polymerase II, these domains catalyze the transcription of all nuclear pre-mRNAs, as well as the majority of small nuclear RNAs. In contrast with rDNA-based trees, phylogenetic analyses of RPB1 sequences indicate that Mastigamoeba represents an early branch of eukaryotic evolution. Unlike sequences from parasitic amitochondriate protists that were included in our study, there is no indication that Mastigamoeba RPB1 is attracted to the base of the eukaryotic tree artifactually. In addition, the presence of introns and a heptapeptide C-terminal repeat in the Mastigamoeba RPB1 sequence, features that are typically associated with more recently derived eukaryotic groups, raise provocative questions regarding models of protist evolution that depend almost exclusively on rDNA sequence analyses.

The origin of red algae: implications for plastid evolution.

The origin of the red algae has remained an enigma. Historically the Rhodophyta were classified first as plants and later as the most ancient eukaryotic organisms. Recent molecular studies have indicated similarities between red and green plastids, which suggest that there was a single endosymbiotic origin for these organelles in a common ancestor of the rhodophytes and green plants. Previous efforts to confirm or reject this effort by analyses of nuclear DNA have been inconclusive; thus, additional molecular markers are needed to establish the relationship between the host cell lineages, independent of the evolutionary history of their plastids. To furnish such a data set we have sequenced the largest subunit of RNA polymerase II from two red algae, a green alga and a relatively derived amoeboid protist. Phylogenetic analyses provide strong statistical support for an early evolutionary emergence of the Rhodophyta that preceded the origin of the line that led to plants, animals, and fungi. These data, which are congruent with results from extensive analyses of nuclear rDNA, argue for a reexamination of current models of plastid evolution.

Boxing and chronic brain damage.

A chronic, and at times, progressive neurologic syndrome associated with boxing has been recognized for some time by boxing fans and people involved with the sport. Since Martland's first description of the syndrome in 1929, there has been only one randomly selected study of ex-boxers, that of Roberts, which showed a 17 per cent prevalence of this syndrome among boxers who fought between 1929 and 1955. This syndrome can be progressive but often is not. Furthermore, the extent of occupational exposure is probably a significant risk factor. Because of this, it would be expected that the prevalance of the syndrome in the modern boxer, as well as the amateur, would be significantly less than during the first half of the century, and, indeed, several studies appear to support this. Recent studies provide evidence that brain damage does exist in modern boxers and suggests that "subclinical" brain damage is likely to be more prevalent than obvious clinical dysfunction. There is clearly a discrepancy between subclinical evidence of neurologic involvement (for example, an abnormal CT scan) and signs of clinical neurologic dysfunction (for example, clinical exam and neuropsychological testing). The latter tend to show less frequent and consistent evidence of brain damage in boxers than does the CT scan. Although it is tempting to assume that an abnormal CT scan presages the development of neurologic dysfunction, it is not clear that this is the case. The prevalence of the syndrome, risk for progression to functional deficit, warning signs, and the natural history cannot be defined at this time. The only way to better define these parameters would be a controlled prospective study, which has yet to be undertaken.