A new statistical method for estimating divergence dates of species from DNA sequence data by a molecular clock approach is developed. This method takes into account effectively the information contained in a set of DNA sequence data. The molecular clock of mitochondrial DNA (mtDNA) was calibrated by setting the date of divergence between primates and ungulates at the Cretaceous-Tertiary boundary (65 million years ago), when the extinction of dinosaurs occurred. A generalized least-squares method was applied in fitting a model to mtDNA sequence data, and the clock gave dates of 92.3 +/- 11.7, 13.3 +/- 1.5, 10.9 +/- 1.2, 3.7 +/- 0.6, and 2.7 +/- 0.6 million years ago (where the second of each pair of numbers is the standard deviation) for the separation of mouse, gibbon, orangutan, gorilla, and chimpanzee, respectively, from the line leading to humans. Although there is some uncertainty in the clock, this dating may pose a problem for the widely believed hypothesis that the pipedal creature Australopithecus afarensis, which lived some 3.7 million years ago at Laetoli in Tanzania and at Hadar in Ethiopia, was ancestral to man and evolved after the human-ape splitting. Another likelier possibility is that mtDNA was transferred through hybridization between a proto-human and a proto-chimpanzee after the former had developed bipedalism.

The electrochemical behavior of highly conductive, boron‐doped polycrystalline diamond thin films for oxygen reduction was examined in 0.5 M H2SO4 using linear sweep voltammetry. When the potential sweep is confined to the region negative of 0.0 V vs. Ag/AgCl, oxygen reduction is highly inhibited with cathodic current being observed at ∼ −0.6 V vs. Ag/AgCl, as compared with the standard potential for the two‐electron reduction of oxygen false(O2+2H++2e−=H2O2,Eo=0.4V vs. Ag/AgCl at pH 0). The extreme inhibition of oxygen reduction may be due to an absence of catalytic sites. When the potential is swept to potentials positive of + 1.4 V vs. Ag/AgCl, the subsequent sweep into the negative region shows a reduction peak due to oxygen reduction. In this case, catalytic oxygen‐containing functional groups can be formed on sp2 carbon impurities. Relatively mild conditions are required to deactivate the catalytic functional groups, but strong oxidative treatment in base appears to substantially remove the sp2 carbon impurities. The oxygen reduction behavior in acid solution could be useful in characterizing diamond electrodes, i.e., as a diagnostic for the presence of sp2‐normaltype carbon on chemical‐vapor‐deposited diamond thin‐film electrodes. It is proposed that diamond electrode surfaces free of sp2 carbon are highly insensitive to oxygen, which could be a useful feature in electroanalysis. © 1999 The Electrochemical Society. All rights reserved.

Phylogenetic trees among eukaryotic kingdoms were inferred for large- and small-subunit rRNAs by using a maximum-likelihood method developed by Felsenstein. Although Felsenstein's method assumes equal evolutionary rates for transitions and transversions, this is apparently not the case for these data. Therefore, only transversion-type substitutions were taken into account. The molecules used were large-subunit rRNAs from Xenopus laevis (Animalia), rice (Plantae), Saccharomyces cerevisiae (Fungi), Dictyostelium discoideum (Protista), and Physarum polycephalum (Protista); and small-subunit rRNAs from maize (Plantae), S. cerevisiae, X. laevis, rat (Animalia), and D. discoideum. Only conservative regions of the nucleotide sequences were considered for this study. In the maximum-likelihood trees for both large- and small-subunit rRNAs, Animalia and Fungi were the most closely related eukaryotic kingdoms, and Plantae is the next most closely related kingdom, although other branching orders among Plantae, Animalia, and Fungi were not excluded by this work. These three eukaryotic kingdoms apparently shared a common ancestor after the divergence of the two species of Protista, D. discoideum and P. polycephalum. These two species of Protista do not form a clade, and P. polycephalum diverged first and D. discoideum second from the line leading to the common ancestor of Plantae, Animalia, and Fungi. The sequence data indicate that a drastic change occurred in the nucleotide sequences of rRNAs during the evolutionary separation between prokaryote and eukaryote.

Polypeptide tag technology is widely used for protein detection and affinity purification. It consists of two fundamental elements: a peptide sequence and a binder which specifically binds to the peptide tag. In many tag systems, antibodies have been used as binder due to their high affinity and specificity. Recently, we obtained clone Ra48, a high-affinity rabbit monoclonal antibody (mAb) against dopamine receptor D1 (DRD1). Here, we report a novel tag system composed of Ra48 antibody and its epitope sequence. Using a deletion assay, we identified EEAAGIARP in the C-terminal region of DRD1 as the minimal epitope of Ra48 mAb, and we named this sequence the “AGIA” tag, based on its central sequence. The tag sequence does not include the four amino acids, Ser, Thr, Tyr, or Lys, which are susceptible to post-translational modification. We demonstrated performance of this new tag system in biochemical and cell biology applications. SPR analysis demonstrated that the affinity of the Ra48 mAb to the AGIA tag was 4.90 × 10−9 M. AGIA tag showed remarkably high sensitivity and specificity in immunoblotting. A number of AGIA-fused proteins overexpressed in animal and plant cells were detected by anti-AGIA antibody in immunoblotting and immunostaining with low background, and were immunoprecipitated efficiently. Furthermore, a single amino acid substitution of the second Glu to Asp (AGIA/E2D) enabled competitive dissociation of AGIA/E2D-tagged protein by adding wild-type AGIA peptide. It enabled one-step purification of AGIA/E2D-tagged recombinant proteins by peptide competition under physiological conditions. The sensitivity and specificity of the AGIA system makes it suitable for use in multiple methods for protein analysis.

Divergence dates among primates were estimated by molecular clock analysis of DNA sequence data. A molecular clock of eta-globin pseudogene was calibrated by setting the date of divergence between Catarrhini and Platyrrhini at 38 million years (Myr) ago. The clock gave dates of 25.3 +/- 2.4, 11.9 +/- 1.7, 5.9 +/- 1.2, and 4.9 +/- 1.2 Myr ago ( +/- refers to standard error) for the separation of rhesus monkey, orangutan, gorilla, and chimpanzee, respectively, from the line leading to humans. In placing confidence intervals of the estimates in a robust way, a bootstrap method was used. The 95% confidence intervals are 20.5-29.5, 9.0-14.8, 4.1-7.8, and 3.1-7.0 Myr ago for the separation of rhesus monkey, orangutan, gorilla, and chimpanzee, respectively. By a molecular clock dating of the Prosimii-Anthropoidea splitting, it was suggested that the evolutionary rate of the eta-globin gene was high early in primate evolution and subsequently decreased in the line of Anthropoidea. And, by a relative rate test using bootstrap sampling, the possibility of further decrease of the rate (more than 10%) in the line of Hominoidea compared with that of Cercopithecoidea was suggested. Therefore, the above dating of the splittings within Hominoidea may be biased slightly toward younger dates. On the other hand, mitochondrial DNA (mtDNA) seems to have evolved in mammals with a more uniform rate than the eta-globin gene. The ratio of the dates of orangutan splitting to chimpanzee splitting is larger for the mtDNA clock than that for the eta-globin clock, suggesting the possibilities of mtDNA introgression among the early hominids and the early African apes, and/or of mtDNA polymorphism within the common ancestral species of orangutan and the African apes that obscures the date of the true species separation of orangutans.