A molecular phylogeny of two extinct sloths.

Xenarthra (Edentata) is an extremely diverse mammalian order whose modern representatives are the armadillos, anteaters, and sloths. The phylogeny of these groups is poorly resolved. This is particularly true for the sloths (phyllophagans), originally a large and diverse group now reduced to two genera in two different families. Both morphological analyses and molecular analyses of rDNA genes of living and extinct sloths have been used with limited success to elucidate their phylogeny. In an attempt to clarify relationships among the sloths, DNA was extracted and mitochondrial cytochrome b gene sequences were determined from representatives of two extinct groups of sloths (Mylodontidae and Megatheriidae), their two living relatives (two-toed sloths [Megalonychidae], three-toed sloths [Bradypodidae]), anteaters and armadillos. A consistent feature of the latter two species was the nuclear copies of cytochrome b gene sequences. Several methods of phylogenetic reconstruction were applied to the sequences determined, and the results were compared with 12S rDNA sequences obtained in previous studies. The cytochrome b gene exhibited a phylogenetic resolving power similar to that of the 12S rDNA sequences. When both data sets were combined, they tended to support the grouping of two-toed sloths with mylodontids and three-toed sloths with megatheriids. The results strengthen the view that the two families of living sloths adapted independently to an arboreal life-style.

Complete sequence of the 24-mer hemocyanin of the tarantula Eurypelma californicum. Structure and intramolecular evolution of the subunits.

Hemocyanins are large oligomeric respiratory proteins found in many arthropods and molluscs. The hemocyanin of the tarantula Eurypelma californicum is a 24-mer protein complex with molecular mass of 1, 726,459 Da that consists of seven different polypeptides (a-g), each occupying a distinct position within the native molecule. Here we report the complete molecular structure of the E. californicum hemocyanin as deduced from the corresponding cDNAs. This represents the first complex arthropod hemocyanin to be completely sequenced. The different subunits display 52-66% amino acid sequence identity. Within the subunits, the central domain, which bears the active center with the copper-binding sites A and B, displays the highest degree of identity. Using a homology modeling approach, the putative three-dimensional structure of individual subunits was deduced and compared. Phylogenetic analyses suggest that differentiation of the individual subunits occurred 400-550 million years ago. The hemocyanin of the stemline Chelicerata was probably a hexamer built up of six distinct subunit types a, b/c, d, e, f, and g, whereas that of the early Arachnida was originally a 24-mer that emerged after the differentiation of subunits b and c.

Complete DNA sequence of the mitochondrial genome of the ascidian Halocynthia roretzi (Chordata, Urochordata).

The complete nucleotide sequence of the 14,771-bp-long mitochondrial (mt) DNA of a urochordate (Chordata)-the ascidian Halocynthia roretzi-was determined. All the Halocynthia mt-genes were found to be located on a single strand, which is rich in T and G rather than in A and C. Like nematode and Mytilus edulis mtDNAs, that of Halocynthia encodes no ATP synthetase subunit 8 gene. However, it does encode an additional tRNA gene for glycine (anticodon TCT) that enables Halocynthia mitochondria to use AGA and AGG codons for glycine. The mtDNA carries an unusual tRNA(Met) gene with a TAT anticodon instead of the usual tRNA(Met)(CAT) gene. As in other metazoan mtDNAs, there is not any long noncoding region. The gene order of Halocynthia mtDNA is completely different from that of vertebrate mtDNAs except for tRNA(His)-tRNA(Ser)(GCU), suggesting that evolutionary change in the mt-gene structure is much accelerated in the urochordate line compared with that in vertebrates. The amino acid sequences of Halocynthia mt-proteins deduced from their gene sequences are quite different from those in other metazoans, indicating that the substitution rate in Halocynthia mt-protein genes is also accelerated.

The mitochondrial genome of the hemichordate Balanoglossus carnosus and the evolution of deuterostome mitochondria.

The complete nucleotide sequence of the mitochondrial genome of the hemichordate Balanoglossus carnosus (acorn worm) was determined. The arrangement of the genes encoding 13 protein, 22 tRNA, and 2 rRNA genes is essentially the same as in vertebrates, indicating that the vertebrate and hemichordate mitochondrial gene arrangement is close to that of their common ancestor, and, thus, that it has been conserved for more than 600 million years, whereas that of echinoderms has been rearranged extensively. The genetic code of hemichordate mitochondria is similar to that of echinoderms in that ATA encodes isoleucine and AGA serine, whereas the codons AAA and AGG, whose amino acid assignments also differ between echinoderms and vertebrates, are absent from the B. carnosus mitochondrial genome. There are three noncoding regions of length 277, 41, and 32 bp: the larger one is likely to be equivalent to the control region of other deuterostomes, while the two others may contain transcriptional promoters for genes encoded on the minor coding strand. Phylogenetic trees estimated from the inferred protein sequences indicate that hemichordates are a sister group of echinoderms.

Codon reassignment and amino acid composition in hemichordate mitochondria.

In the mitochondrial genome of the hemichordate Balanoglossus carnosus, the codon AAA, which is assigned to lysine in most metazoans but to asparagine in echinoderms, is absent. Furthermore, the lysine tRNA gene carries an anticodon substitution that renders its gene product unable to decode AAA codons, whereas the asparagine tRNA gene has not changed to encode a tRNA with the ability to recognize AAA codons. Thus, the hemichordate mitochondrial genome can be regarded as an intermediate in the process of reassignment of mitochondrial AAA codons, where most metazoans represent the ancestral situation and the echinoderms the derived situation. This lends support to the codon capture hypothesis. We also show that the reassignment of the AAA codon is associated with a reduction in the relative abundance of lysine residues in mitochondrial proteins.

The mitochondrial genome of a monotreme--the platypus (Ornithorhynchus anatinus).

The complete nucleotide sequence of the mitochondrial genome of a platypus (Ornithorhynchus anatinus) was determined. Its overall genomic organization is similar to that of placental mammals, Xenopus laevis, and fishes. However, it contains an apparently noncoding sequence of 88 base pairs located between the genes for tRNA(Leu)(UUR) and ND1. The base composition of this sequence and its conservation among monotremes, as well as the existence of a transcript from one of the strands, indicate that it may have a hitherto-unknown function. When the protein-coding sequences are used to reconstruct a phylogeny of mammals, the data suggest that monotremes and marsupials are sister groups and thus that placental mammals represent the most ancient divergence among mammals.

The marsupial mitochondrial genome and the evolution of placental mammals.

The entire nucleotide sequence of the mitochondrial genome of the American opossum, Didelphis virginiana, was determined. Two major features distinguish this genome from those of other mammals. First, five tRNA genes around the origin of light strand replication are rearranged. Second, the anticodon of tRNA(Asp) is posttranscriptionally changed by an RNA editing process such that its coding capacity is altered. When the complete protein-coding region of the mitochondrial genome is used as an outgroup for placental mammals it can be shown that rodents represent an earlier branch among placental mammals than primates and artiodactyls and that artiodactyls share a common ancestor with carnivores. The overall rates of evolution of most of the mitochondrial genome of placentals are clock-like. Furthermore, the data indicate that the lineages leading to the mouse and rat may have diverged from each other as much as 35 million years ago.

Arthropod hemocyanins. Molecular cloning and sequencing of cDNAs encoding the tarantula hemocyanin subunits a and e.

cDNA clones comprising the entire coding region of two out of the seven heterogeneous subunits of hemocyanin from the tarantula, Eurypelma californicum, were isolated from four cDNA libraries constructed from total RNA from the heart tissue of single spiders. Hybridization was first carried out using a tarantula hemocyanin subunit e partial cDNA, and several positive clones were isolated, including one containing a 2.2-kilobase full-length cDNA (lambda M1). The cDNA comprises an open reading frame for 623 amino acids, 34 nucleotides of the 5'noncoding region, and 286 nucleotides of the 3'-noncoding region. To select for other hemocyanin subunits, two 17-mer oligonucleotide mixtures, corresponding to the conserved regions in the copper A and copper B oxygen-binding site of chelicerate hemocyanins, were used as probes. Among the positive clones obtained, full-length cDNAs coding for subunit a were identified. The cDNA sequence determined from clone lambda K1 provides an open reading frame coding for 630 amino acids and includes the 5'- and 3'-noncoding regions. Northern blot analysis revealed single transcripts for subunits a and e, each 2.3 kilobases long. The cDNAs for subunits a and e were both found to lack any leader peptide sequence. This supports the idea that the mature protein accumulates in the cytoplasm and is released by cell rupture.

cDNA cloning and sequencing of tarantula hemocyanin subunits.

Tarantula heart cDNA libraries were screened with synthetic oligonucleotide probes deduced from the highly conserved amino acid sequences of the two copper-binding sites, copper A and copper B, found in chelicerate hemocyanins. Positive cDNA clones could be obtained and four different cDNA types were characterized.