The root of the universal tree and the origin of eukaryotes based on elongation factor phylogeny.

The genes for the protein synthesis elongation factors Tu (EF-Tu) and G (EF-G) are the products of an ancient gene duplication, which appears to predate the divergence of all extant organismal lineages. Thus, it should be possible to root a universal phylogeny based on either protein using the second protein as an outgroup. This approach was originally taken independently with two separate gene duplication pairs, (i) the regulatory and catalytic subunits of the proton ATPases and (ii) the protein synthesis elongation factors EF-Tu and EF-G. Questions about the orthology of the ATPase genes have obscured the former results, and the elongation factor data have been criticized for inadequate taxonomic representation and alignment errors. We have expanded the latter analysis using a broad representation of taxa from all three domains of life. All phylogenetic methods used strongly place the root of the universal tree between two highly distinct groups, the archaeons/eukaryotes and the eubacteria. We also find that a combined data set of EF-Tu and EF-G sequences favors placement of the eukaryotes within the Archaea, as the sister group to the Crenarchaeota. This relationship is supported by bootstrap values of 60-89% with various distance and maximum likelihood methods, while unweighted parsimony gives 58% support for archaeal monophyly.

Dynamic friction and the origin of the complexity of earthquake sources.

We study a simple antiplane fault of finite length embedded in a homogeneous isotropic elastic solid to understand the origin of seismic source heterogeneity in the presence of nonlinear rate- and state-dependent friction. All the mechanical properties of the medium and friction are assumed homogeneous. Friction includes a characteristic length that is longer than the grid size so that our models have a well-defined continuum limit. Starting from a heterogeneous initial stress distribution, we apply a slowly increasing uniform stress load far from the fault and we simulate the seismicity for a few 1000 events. The style of seismicity produced by this model is determined by a control parameter associated with the degree of rate dependence of friction. For classical friction models with rate-independent friction, no complexity appears and seismicity is perfectly periodic. For weakly rate-dependent friction, large ruptures are still periodic, but small seismicity becomes increasingly nonstationary. When friction is highly rate-dependent, seismicity becomes nonperiodic and ruptures of all sizes occur inside the fault. Highly rate-dependent friction destabilizes the healing process producing premature healing of slip and partial stress drop. Partial stress drop produces large variations in the state of stress that in turn produce earthquakes of different sizes. Similar results have been found by other authors using the Burridge and Knopoff model. We conjecture that all models in which static stress drop is only a fraction of the dynamic stress drop produce stress heterogeneity.

Human plectin: organization of the gene, sequence analysis, and chromosome localization (8q24).

Plectin, a 500-kDa intermediate filament binding protein, has been proposed to provide mechanical strength to cells and tissues by acting as a cross-linking element of the cytoskeleton. To set the basis for future studies on gene regulation, tissue-specific expression, and pathological conditions involving this protein, we have cloned the human plectin gene, determined its coding sequence, and established its genomic organization. The coding sequence contains 32 exons that extend over 32 kb of the human genome. Most of the introns reside within a region encoding the globular N-terminal domain of the molecule, whereas the entire central rod domain and the entire C-terminal globular domain were found to be encoded by single exons of remarkable length, >3 kb and >6 kb, respectively. Overall, the organization of the human plectin gene was strikingly similar to that of human bullous pemphigoid antigen 1 (BPAG1), confirming that both proteins belong to the same gene family. Comparison of the deduced protein sequences for human and rat plectin revealed that they were 93% identical. By using fluorescence in situ hybridization, we have mapped the plectin gene to the long arm of chromosome 8 within the telomeric region. This gene locus (8q24) has previously been implicated in the human blistering skin disease epidermolysis bullosa simplex Ogna. Detailed knowledge of the structure of the plectin gene and its chromosome localization will aid in the elucidation of whether this or any other pathological conditions are linked to alterations in the plectin gene.

Cross-modulation by transforming growth factor beta in human tuberculosis: suppression of antigen-driven blastogenesis and interferon gamma production.

In tuberculosis, Mycobacterium tuberculosis (MTB)-stimulated T-cell responses are depressed transiently, whereas antibody levels are increased. Lymphoproliferative responses of peripheral blood mononuclear cells (PBMCs) from Pakistani tuberculosis (TB) patients to both mycobacterial and candidal antigens were suppressed by approximately 50% when compared to healthy purified protein derivative (PPD)-positive household contacts. Production of interferon gamma (IFN-gamma) in response to PPD also was depressed by 78%. Stimulation with PPD and the 30-kDa alpha antigen of MTB (30-kDa antigen) induced greater secretion of transforming growth factor beta (TGF-beta), but not interleukin 10 (IL-10) or tumor necrosis factor alpha (TNF-alpha), by PBMCs from TB patients compared to healthy contacts. The degree of suppression correlated with the duration of treatment; patients treated for <1 month had significantly lower T-cell blastogenesis and IFN-gamma production and higher levels of TGF-beta than did patients treated for >1 month. Neutralizing antibody to TGF-beta normalized lymphocyte proliferation in response to PPD, partially restored blastogenesis to candidal antigen, and significantly increased PPD-stimulated production of IFN-gamma in TB patients but not in contacts. Neutralizing antibody to IL-10 augmented, but did not normalize, T-cell responses to both PPD and candida in TB patients and candidal antigen in contacts. TGF-beta, produced in response to MTB antigens, therefore plays a prominent role in down-regulating potentially protective host effector mechanisms and looms as an important mediator of immunosuppression in TB.

Skeletal matrices, muci, and the origin of invertebrate calcification.

The sudden appearance of calcified skeletons among many different invertebrate taxa at the Precambrian-Cambrian transition may have required minor reorganization of preexisting secretory functions. In particular, features of the skeletal organic matrix responsible for regulating crystal growth by inhibition may be derived from mucous epithelial excretions. The latter would have prevented spontaneous calcium carbonate overcrusting of soft tissues exposed to the highly supersaturated Late Proterozoic ocean [Knoll, A. H., Fairchild, I. J. & Swett, K. (1993) Palaios 8, 512-525], a putative function for which we propose the term "anticalcification." We tested this hypothesis by comparing the serological properties of skeletal water-soluble matrices and mucous excretions of three invertebrates--the scleractinian coral Galaxea fascicularis and the bivalve molluscs Mytilus edulis and Mercenaria mercenaria. Crossreactivities recorded between muci and skeletal water-soluble matrices suggest that these different secretory products have a high degree of homology. Furthermore, freshly extracted muci of Mytilus were found to inhibit calcium carbonate precipitation in solution.

On the origin of the order Artiodactyla.

The first known members of the order Artiodactyla appeared suddenly throughout the Holarctic region at the beginning of the Eocene. They are characterized by distinctive cursorial skeletal specializations. Owing to their abrupt appearance and the lack of transitional forms, the origin of the order is problematic. Descent from a "condylarth," specifically the arctocyonid Chriacus, has been suggested based on dental resemblances, but until now postcranial anatomy seemed to preclude close relationship between Arctocyonidae and Artiodactyla. A middle Paleocene specimen of a small arctocyonid (?Chriacus) reported here is much more similar to the oldest artiodactyl, Diacodexis, in the derived condition of the hindlimb, reviving the possibility that Artiodactyla evolved from an arctocyonid.

Archaeal-eubacterial mergers in the origin of Eukarya: phylogenetic classification of life.

A symbiosis-based phylogeny leads to a consistent, useful classification system for all life. "Kingdoms" and "Domains" are replaced by biological names for the most inclusive taxa: Prokarya (bacteria) and Eukarya (symbiosis-derived nucleated organisms). The earliest Eukarya, anaerobic mastigotes, hypothetically originated from permanent whole-cell fusion between members of Archaea (e.g., Thermoplasma-like organisms) and of Eubacteria (e.g., Spirochaeta-like organisms). Molecular biology, life-history, and fossil record evidence support the reunification of bacteria as Prokarya while subdividing Eukarya into uniquely defined subtaxa: Protoctista, Animalia, Fungi, and Plantae.

Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends.

An assay that allows measurement of absolute induction frequencies for DNA double-strand breaks (dsbs) in defined regions of the genome and that quantitates rejoining of correct DNA ends has been used to study repair of dsbs in normal human fibroblasts after x-irradiation. The approach involves hybridization of single-copy DNA probes to Not I restriction fragments separated according to size by pulsed-field gel electrophoresis. Induction of dsbs is quantitated from the decrease in the intensity of the hybridizing restriction fragment and an accumulation of a smear below the band. Rejoining of dsbs results in reconstitution of the intact restriction fragment only if correct DNA ends are joined. By comparing results from this technique with results from a conventional electrophoresis assay that detects all rejoining events, it is possible to quantitate the misrejoining frequency. Three Not I fragments on the long arm of chromosome 21 were investigated with regard to dsb induction, yielding an identical induction rate of 5.8 X 10(-3) break per megabase pair per Gy. Correct dsb rejoining was measured for two of these Not I fragments after initial doses of 80 and 160 Gy. The misrejoining frequency was about 25% for both fragments and was independent of dose. This result appears to be representative for the whole genome as shown by analysis of the entire Not I fragment distribution. The correct rejoining events primarily occurred within the first 2 h, while the misrejoining kinetics included a much slower component, with about half of the events occurring between 2 and 24 h. These misrejoining kinetics are similar to those previously reported for production of exchange aberrations in interphase chromosomes.

A single-stranded DNA binding protein binds the origin of replication of the duplex kinetoplast DNA.

Replication of the kinetoplast DNA (kDNA) minicircle of trypanosomatids initiates at a conserved 12-nt sequence, 5'-GGGGTTGGTGTA-3', termed the universal minicircle sequence (UMS). A sequence-specific single-stranded DNA-binding protein from Crithidia fasciculata binds the heavy strand of the 12-mer UMS. Whereas this UMS-binding protein (UMSBP) does not bind a duplex UMS dodecamer, it binds the double-stranded kDNA minicircle as well as a duplex minicircle fragment containing the origin-associated UMS. Binding of the minicircle origin region by the single-stranded DNA binding protein suggested the local unwinding of the DNA double helix at this site. Modification of thymine residues at this site by KMnO4 revealed that the UMS resides within an unwound or otherwise sharply distorted DNA at the minicircle origin region. Computer analysis predicts the sequence-directed curving of the minicircle origin region. Electrophoresis of a minicircle fragment containing the origin region in polyacrylamide gels revealed a significantly lower electrophoretic mobility than expected from its length. The fragment anomalous electrophoretic mobility is displayed only in its native conformation and is dependent on temperature and gel porosity, indicating the local curving of the DNA double helix. We suggest that binding of UMSBP at the minicircle origin of replication is possible through local unwinding of the DNA double helix at the UMS site. It is hypothesized here that this local melting is initiated through the untwisting of unstacked dinucleotide sequences at the bent origin site.

Subunit-specific functions of N-linked oligosaccharides in human thyrotropin: role of terminal residues of alpha- and beta-subunit oligosaccharides in metabolic clearance and bioactivity.

The recombinant human thyroid stimulating hormone (rhTSH) containing oligosaccharides terminated with NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc beta 1 showed higher in vivo activity and lower metabolic clearance rate (MCR) than pituitary human TSH (phTSH), which contains oligosaccharides terminating predominantly in SO(4)4GalNAc(beta 1-4)GlcNAc beta 1. To elucidate the relative contribution of the sulfated and sialylated carbohydrate chains of each subunit in the MCR and bioactivity of the hormone, the alpha and beta subunits of phTSH, rhTSH, and enzymatically desialylated rhTSH (asialo-rhTSH; asrhTSH) were isolated, their oligosaccharides were analyzed, and the respective subunits were dimerized in various combinations. The hybrids containing alpha subunit from phTSH or asrhTSH showed higher in vitro activity than those with alpha subunit from rhTSH, indicating that sialylation of alpha but not beta subunit attenuates the intrinsic activity of TSH. In contrast, hybrids with beta subunit from rhTSH displayed lower MCR compared to those with beta subunit from phTSH. The phTSH alpha-rhTSH beta hybrid had the highest in vivo bioactivity followed by rhTSH alpha-rhTSH beta, rhTSH alpha-phTSH beta, phTSH alpha-phTSH beta, and asrhTSH dimers. These differences indicated that hybrids with beta subunit from rhTSH displayed the highest in vivo activity and relatively low MCR, probably due to higher sialylation, more multiantennary structure, and/or the unique location of the beta-subunit oligosaccharide chain in the molecule. Thus, the N-linked oligosaccharides of the beta subunit of glycoprotein hormones have a more pronounced role than those from the alpha subunit in the metabolic clearance and thereby in the in vivo bioactivity. In contrast, the terminal residues of alpha-subunit oligosaccharides have a major impact on TSH intrinsic potency.

Considerations on the folding topology and evolutionary origin of cadherin domains.

Cell-cell adhesion in zonula adherens and desmosomal junctions is mediated by cadherins, and recent crystal structures of the first domain from murine N-cadherin provide a plausible molecular basis for this adhesive action. A structure-based sequence analysis of this adhesive domain indicates that its fold is common to all extracellular cadherin domains. The cadherin folding topology is also shown to be similar to immunoglobulin-like domains and to other Greek-key beta-sandwich structures, as diverse as domains from plant cytochromes, bacterial cellulases, and eukaryotic transcription factors. Sequence similarities between cadherins and these other molecules are very low, however, and intron patterns are also different. On balance, independent origins for a favorable folding topology seem more likely than evolutionary divergence from an ancestor common to cadherins and immunoglobulins.

Evolutionary origin of Plasmodium and other Apicomplexa based on rRNA genes.

We have explored the evolutionary history of the Apicomplexa and two related protistan phyla, Dinozoa and Ciliophora, by comparing the nucleotide sequences of small subunit ribosomal RNA genes. We conclude that the Plasmodium lineage, to which the malarial parasites belong, diverged from other apicomplexan lineages (piroplasmids and coccidians) several hundred million years ago, perhaps even before the Cambrian. The Plasmodium radiation, which gave rise to several species parasitic to humans, occurred approximately 129 million years ago; Plasmodium parasitism of humans has independently arisen several times. The origin of apicomplexans (Plasmodium), dinoflagellates, and ciliates may be > 1 billion years old, perhaps older than the three multicellular kingdoms of animals, plants, and fungi. Digenetic parasitism independently evolved several times in the Apicomplexa.

Genetics and the origin of European languages.

A new set of European genetic data has been analyzed to dissect independent patterns of geographic variation. The most important cause of European genetic variation has been confirmed to correspond to the migration of Neolithic farmers from the area of origin of agriculture in the Middle East. The next most important component of genetic variation is apparently associated with a north-south gradient possibly due to adaptation to cold climates but also to the differentiation of the Uralic and the Indo-European language-speaking people; however, the relevant correlations are not significantly different from zero after elimination of the spatial autocorrelation. The third component is highly correlated with the infiltration of the Yamna ("Kurgan") people, nomadic pastoralists who domesticated the horse and who have been claimed to have spread Indo-European languages to Europe; this association, which is statistically significant even when taking spatial autocorrelations into account, does not completely exclude the hypothesis of Indo-European as the language of Neolithic farmers. It is possible that both expansions were responsible for the spread of different subfamilies of Indo-European languages, but our genetic data cannot resolve their relative importance.

In vitro cleavage and joining at the viral origin of replication by the replication initiator protein of tomato yellow leaf curl virus.

Replication of the single-stranded DNA genome of geminiviruses occurs via a double-stranded intermediate that is subsequently used as a template for rolling-circle replication of the viral strand. Only one of the proteins encoded by the virus, here referred to as replication initiator protein (Rep protein), is indispensable for replication. We show that the Rep protein of tomato yellow leaf curl virus initiates viral-strand DNA synthesis by introducing a nick in the plus strand within the nonanucleotide 1TAATATT decreases 8AC, identical among all geminiviruses. After cleavage, the Rep protein remains bound to the 5' end of the cleaved strand. In addition, we show that the Rep protein has a joining activity, suggesting that it acts as a terminase, thus resolving the nascent viral single strand into genome-sized units.