Loss of Octarepeats in two processed prion pseudogenes in the red squirrel, Sciurus vulgaris.

The N-terminal region of the mammalian prion protein (PrP) contains an 'octapeptide' repeat which is involved in copper binding. This eight- or nine-residue peptide is repeated four to seven times, depending on the species, and polymorphisms in repeat number do occur. Alleles with three repeats are very rare in humans and goats, and deduced PrP sequences with two repeats have only been reported in two lemur species and in the red squirrel, Sciurus vulgaris. We here describe that the red squirrel two-repeat PrP sequence actually represents a retroposed pseudogene, and that an additional and older processed pseudogene with three repeats also occurs in this species as well as in ground squirrels. We argue that repeat numbers may tend to contract rather than expand in prion retropseudogenes, and that functional prion genes with two repeats may not be viable.

The platypus is in its place: nuclear genes and indels confirm the sister group relation of monotremes and Therians.

Morphological data supports monotremes as the sister group of Theria (extant marsupials + eutherians), but phylogenetic analyses of 12 mitochondrial protein-coding genes have strongly supported the grouping of monotremes with marsupials: the Marsupionta hypothesis. Various nuclear genes tend to support Theria, but a comprehensive study of long concatenated sequences and broad taxon sampling is lacking. We therefore determined sequences from six nuclear genes and obtained additional sequences from the databases to create two large and independent nuclear data sets. One (data set I) emphasized taxon sampling and comprised five genes, with a concatenated length of 2,793 bp, from 21 species (two monotremes, six marsupials, nine placentals, and four outgroups). The other (data set II) emphasized gene sampling and comprised eight genes and three proteins, with a concatenated length of 10,773 bp or 3,669 amino acids, from five taxa (a monotreme, a marsupial, a rodent, human, and chicken). Both data sets were analyzed by parsimony, minimum evolution, maximum likelihood, and Bayesian methods using various models and data partitions. Data set I gave bootstrap support values for Theria between 55% and 100%, while support for Marsupionta was at most 12.3%. Taking base compositional bias into account generally increased the support for Theria. Data set II exclusively supported Theria, with the highest possible values and significantly rejected Marsupionta. Independent phylogenetic evidence in support of Theria was obtained from two single amino acid deletions and one insertion, while no supporting insertions and deletions were found for Marsupionta. On the basis of our data sets, the time of divergence between Monotremata and Theria was estimated at 231-217 MYA and between Marsupialia and Eutheria at 193-186 MYA. The morphological evidence for a basal position of Monotremata, well separated from Theria, is thus fully supported by the available molecular data from nuclear genes.

Evolutionary diversity of vertebrate small heat shock proteins.

All vertebrates express multiple small heat shock proteins (sHsps), which are important components of the cellular chaperoning machinery and display a spectacular diversity of functions. This ranges from remodeling the cytoskeleton and inhibiting apoptosis to serving as structural proteins in eye lens and sperm tail. Most information is available for the 10 known mammalian sHsps, formally named HspB1-B10. Only three of them (Hsp27/B1, alphaA-crystallin/B4, alphaB-crystallin/B5) have been reported from nonmammalian vertebrates, while an apparent paralog, Hsp30/B11, is found in frogs and teleost fish. To reconstruct the evolutionary diversification of the sHsps in vertebrates, we searched for additional sHsps in genome, protein, and EST databases and sequenced some avian and amphibian sHsps (HspB2, Hsp30/B11). The urochordate Ciona intestinalis was included in the search, as the outgroup of vertebrates. Orthologs of seven mammalian sHsps were now found in other vertebrate classes. Two novel sHsps, named HspB11 and HspB12, were recognized in birds, and four novel sHsps, named HspB12-B15, in teleost fish. Secondary structure predictions of orthologous sHsps from different vertebrate classes indicate conservation of the beta-sandwich structure of the functionally important C-terminal "alpha-crystallin domain," while the N-terminal domains generally have alpha-helical structures, despite their pronounced sequence variation. The constructed chordate sHsp tree is supported by shared introns, indels, and diagnostic sequences. The tree distinguishes putative orthologous and paralogous relationships, which will facilitate the functional and structural comparison of the various vertebrate sHsps. The 15 recognized paralogous vertebrate sHsps reflect the period of extensive gene duplications early in vertebrate evolution. Eleven of these sHsps are grouped in a clade that might be specific for chordates. It is inferred that at least 13 intron insertions have occurred during the evolution of chordate sHsp genes, while a single ancient intron is maintained in some lineages, in line with the general trend of massive intron gain before or during early vertebrate radiation. Interesting is the occurrence of several head-to-head located pairs of chordate sHsp genes.

Sequence and functional conservation of the intergenic region between the head-to-head genes encoding the small heat shock proteins alphaB-crystallin and HspB2 in the mammalian lineage.

An unexpected feature of the large mammalian genome is the frequent occurrence of closely linked head-to-head gene pairs. Close apposition of such gene pairs has been suggested to be due to sharing of regulatory elements. We show here that the head-to-head gene pair encoding two small heat shock proteins, alphaB-crystallin and HspB2, is closely linked in all major mammalian clades, suggesting that this close linkage is of selective advantage. Yet alphaB-crystallin is abundantly expressed in lens and muscle and in response to a heat shock, while HspB2 is abundant only in muscle and not upregulated by a heat shock. The intergenic distance between the genes for these two proteins in mammals ranges from 645 bp (platypus) to 1069 bp (opossum), with an average of about 900 bp; in chicken the distance was the same as in duck (1.6 kb). Phylogenetic footprinting and sequence alignment identified a number of conserved sequence elements close to the HspB2 promoter and two farther upstream. All known regulatory elements of the mouse alphaB-crystallin promoter are conserved, except in platypus and birds. The lens-specific region 1 (LSR1) and the heat shock elements (HSEs) lack in birds; in platypus the LSR1 is reduced to a Pax-6 site, while the Pax-6 site in LSR2 and a HSE are absent. Most likely the primordial mammalian alphaB-crystallin promoter had two LSRs and two HSEs. In transfection experiments the platypus alphaB-crystallin promoter retained heat shock responsiveness and lens expression. It also directed lens expression in Xenopus laevis transgenes, as did the HspB2 promoter of rat or blind mole rat. Deletion of the middle of the intergenic region including the upstream enhancer affected the activity of both the rat alphaB-crystallin and the HspB2 promoters, suggesting sharing of the enhancer region by the two promoters.

Indels in protein-coding sequences of Euarchontoglires constrain the rooting of the eutherian tree.

Despite the availability of large molecular data sets, the position of the root of the eutherian tree remains a controversial issue. Depending on source data, taxon sampling and analytical approach, the root can be placed at either Afrotheria, Xenarthra, Afrotheria+Xenarthra, or murid rodents. We explored the phylogenetic potential of indels in four nuclear protein-coding genes (SCA1, PRNP, TNFalpha, and HspB3) with regard to a possible rooting at the murid branch. According to parsimony principles, five indels were interpreted to contradict such a rooting, and one indel to support it. The results illustrate that indels, despite the occurrence of homoplasy, can be convincing sources of independent molecular evidence to distinguish between alternative phylogenetic hypotheses.

Lactate dehydrogenase A as a highly abundant eye lens protein in platypus (Ornithorhynchus anatinus): upsilon (upsilon)-crystallin.

Vertebrate eye lenses mostly contain two abundant types of proteins, the alpha-crystallins and the beta/gamma-crystallins. In addition, certain housekeeping enzymes are highly expressed as crystallins in various taxa. We now observed an unusual approximately 41-kd protein that makes up 16% to 18% of the total protein in the platypus eye lens. Its cDNA sequence was determined, which identified the protein as muscle-type lactate dehydrogenase A (LDH-A). It is the first observation of LDH-A as a crystallin, and we designate it upsilon (upsilon)-crystallin. Interestingly, the related heart-type LDH-B occurs as an abundant lens protein, known as epsilon-crystallin, in many birds and crocodiles. Thus, two members of the ldh gene family have independently been recruited as crystallins in different higher vertebrate lineages, suggesting that they are particularly suited for this purpose in terms of gene regulatory or protein structural properties. To establish whether platypus LDH-A/upsilon-crystallin has been under different selective constraints as compared with other vertebrate LDH-A sequences, we reconstructed the vertebrate ldh-a gene phylogeny. No conspicuous rate deviations or amino acid replacements were observed.

Molecular evolution of the mammalian prion protein.

Prion protein (PrP) sequences are until now available for only six of the 18 orders of placental mammals. A broader comparison of mammalian prions might help to understand the enigmatic functional and pathogenic properties of this protein. We therefore determined PrP coding sequences in 26 mammalian species to include all placental orders and major subordinal groups. Glycosylation sites, cysteines forming a disulfide bridge, and a hydrophobic transmembrane region are perfectly conserved. Also, the sequences responsible for secondary structure elements, for N- and C-terminal processing of the precursor protein, and for attachment of the glycosyl-phosphatidylinositol membrane anchor are well conserved. The N-terminal region of PrP generally contains five or six repeats of the sequence P(Q/H)GGG(G/-)WGQ, but alleles with two, four, and seven repeats were observed in some species. This suggests, together with the pattern of amino acid replacements in these repeats, the regular occurrence of repeat expansion and contraction. Histidines implicated in copper ion binding and a proline involved in 4-hydroxylation are lacking in some species, which questions their importance for normal functioning of cellular PrP. The finding in certain species of two or seven repeats, and of amino acid substitutions that have been related to human prion diseases, challenges the relevance of such mutations for prion pathology. The gene tree deduced from the PrP sequences largely agrees with the species tree, indicating that no major deviations occurred in the evolution of the prion gene in different placental lineages. In one species, the anteater, a prion pseudogene was present in addition to the active gene.

Spawning times, reproductive compatibilities and genetic structuring in the Acropora aspera group: evidence for natural hybridization and semi-permeable species boundaries in corals.

Species boundaries among five sympatric coral species of the Indo-Pacific Acropora aspera group were examined by a combination of in vitro breeding trials, comparisons of spawning times and DNA sequence analysis of ribosomal DNA internal transcribed spacer (rDNA ITS) and 5.8S regions. The breeding trials showed that reproductive compatibility exists between at least some colonies of all the species pairs tested, suggesting a large potential for natural hybridization and introgression. The Acropora ITS regions exhibited extremely high levels of variability (up to approximately 62% for ITS1, approximately 11% for 5.8S and approximately 43% for ITS2), but most of the variation was shared among four of the five species, A. millepora, A. papillare, A. pulchra and A. spathulata, consistent with extensive introgression. Phylogenetic analyses did not resolve these four species as distinct clusters across a wide biogeographic region stretching from the southern Great Barrier Reef to Papua New Guinea. However, most colonies of the fifth species, A. aspera, constituted a distinct clade in phylogenetic analyses. This is consistent with our observations of a semi-permeable temporal barrier involving differences in spawning times between this and the other four species. Although the majority of colonies of all five species generally spawned within 90 min of each other, in two out of four years, gametes were absent prior to mass spawning episodes from at least some A. aspera colonies. Hence, our data suggest that transient reproductive barriers may be the result of year-to-year variation in the date of spawning and that this difference in spawning time contributes to the genetic structure detected among Acropora species in this group. Occasional leakage through the reproductive barrier was confirmed by the observation of A. aspera xA. pulchra F1 hybrids, identified based on additivity of ITS sequences.