Chemical composition of Fe-Mn ores from the North Pacific seamounts

Ore crusts from the Mid-Pacific Seamounts were studied by scanning electron microscopy and by atomic-absorption and chemical analysis. Characteristic ultramicroscopic structures of ore material of these crusts are globular, fibrous, conchoidal and cellular. Non-ore components are represented by fragments of bedrocks, zeolites, biogenic carbonates, and apatite. Contents of ore elements are: Fe 5.53-15.82%; Mn 14.92-23.45%; Co 0.32-0.82%; Ni 0.22-0.70%; Cu 0.02-0.12%, Mn/Fe ratio varies from 1.02 to 3.39. In general elevated contents of Co (>0.55%) in Fe-Mn crusts correspond to elevated (>1) Mn/Fe ratios.

Probing the Saccharomyces cerevisiae centromeric DNA (CEN DNA)–binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy

Yeast centromeric DNA (CEN DNA) binding factor 3 (CBF3) is a multisubunit protein complex that binds to the essential CDEIII element in CEN DNA. The four CBF3 proteins are required for accurate chromosome segregation and are considered to be core components of the yeast kinetochore. We have examined the structure of the CBF3–CEN DNA complex by atomic force microscopy. Assembly of CBF3–CEN DNA complexes was performed by combining purified CBF3 proteins with a DNA fragment that includes the CEN region from yeast chromosome III. Atomic force microscopy images showed DNA molecules with attached globular bodies. The contour length of the DNA containing the complex is ≈9% shorter than the DNA alone, suggesting some winding of DNA within the complex. The measured location of the single binding site indicates that the complex is located asymmetrically to the right of CDEIII extending away from CDEI and CDEII, which is consistent with previous data. The CEN DNA is bent ≈55° at the site of complex formation. A significant fraction of the complexes are linked in pairs, showing three to four DNA arms, with molecular volumes approximately three times the mean volumes of two-armed complexes. These multi-armed complexes indicate that CBF3 can bind two DNA molecules together in vitro and, thus, may be involved in holding together chromatid pairs during mitosis.

A single-headed dimer of Escherichia coli ribosomal protein L7/L12 supports protein synthesis

During protein synthesis, the two elongation factors Tu and G alternately bind to the 50S ribosomal subunit at a site of which the protein L7/L12 is an essential component. L7/L12 is present in each 50S subunit in four copies organized as two dimers. Each dimer consists of distinct domains: a single N-terminal (“tail”) domain that is responsible for both dimerization and binding to the ribosome via interaction with the protein L10 and two independent globular C-terminal domains (“heads”) that are required for binding of elongation factors to ribosomes. The two heads are connected by flexible hinge sequences to the N-terminal domain. Important questions concerning the mechanism by which L7/L12 interacts with elongation factors are posed by us in response to the presence of two dimers, two heads per dimer, and their dynamic, mobile properties. In an attempt to answer these questions, we constructed a single-headed dimer of L7/L12 by using recombinant DNA techniques and chemical cross-linking. This chimeric molecule was added to inactive core particles lacking wild-type L7/L12 and shown to restore activity to a level approaching that of wild-type two-headed L7/L12.

Molecular evolution of integrins: Genes encoding integrin β subunits from a coral and a sponge

The integrin family of cell surface receptors is strongly conserved in higher animals, but the evolutionary history of integrins is obscure. We have identified and sequenced cDNAs encoding integrin β subunits from a coral (phylum Cnidaria) and a sponge (Porifera), indicating that these proteins existed in the earliest stages of metazoan evolution. The coral βCn1 and, especially, the sponge βPo1 sequences are the most divergent of the “β1-class” integrins and share a number of features not found in any other vertebrate or invertebrate integrins. Perhaps the greatest difference from other β subunits is found in the third and fourth repeats of the cysteine-rich stalk, where the generally conserved spacings between cysteines are highly variable, but not similar, in βCn1 and βPo1. Alternatively spliced cDNAs, containing a stop codon about midway through the full-length translated sequence, were isolated from the sponge library. These cDNAs appear to define a boundary between functional domains, as they would encode a protein that includes the globular ligand-binding head but would be missing the stalk, transmembrane, and cytoplasmic domains. These and other sequence comparisons with vertebrate integrins are discussed with respect to models of integrin structure and function.

Probes bound to myosin Cys-707 rotate during length transients in contraction

It is widely conjectured that muscle shortens because portions of myosin molecules (the “cross-bridges”) impel the actin filament to which they transiently attach and that the impulses result from rotation of the cross-bridges. Crystallography indicates that a cross-bridge is articulated–consisting of a globular catalytic/actin-binding domain and a long lever arm that may rotate. Conveniently, a rhodamine probe with detectable attitude can be attached between the globular domain and the lever arm, enabling the observer to tell whether the anchoring region rotates. Well-established signature effects observed in shortening are tension changes resulting from the sudden release or quick stretch of active muscle fibers. In this investigation we found that closely correlated with such tension changes are changes in the attitude of the rhodamine probes. This correlation strongly supports the conjecture about how shortening is achieved.

The C-type lectin domains of lecticans, a family of aggregating chondroitin sulfate proteoglycans, bind tenascin-R by protein– protein interactions independent of carbohydrate moiety

The lecticans are a family of chondroitin sulfate proteoglycans including aggrecan, versican, neurocan, and brevican. The C-terminal globular domains of lecticans are structurally related to selectins, consisting of a C-type lectin domain flanked by epidermal growth factor and complement regulatory protein domains. The C-type lectin domain of versican has been shown to bind tenascin-R, an extracellular matrix protein specifically expressed in the nervous system, and the interaction was presumed to be mediated by a carbohydrate–protein interaction. In this paper, we show that the C-type lectin domain of brevican, another lectican that is specifically expressed in the nervous system, also binds tenascin-R. Surprisingly, this interaction is mediated by a protein–protein interaction through the fibronectin type III domains 3–5 of tenascin-R, independent of any carbohydrates or sulfated amino acids. The lectin domains of versican and other lecticans also bind the same domain of tenascin-R by protein–protein interactions. Surface plasmon resonance analysis revealed that brevican lectin has at least a 10-fold higher affinity than the other lectican lectins. Tenascin-R is coprecipitated with brevican from adult rat brain extracts, suggesting that tenascin-R and brevican form complexes in vivo. These results demonstrate that the C-type lectin domain can interact with fibronectin type III domains through protein–protein interactions, and suggest that brevican is a physiological tenascin-R ligand in the adult brain.

Mutations of Arabidopsis thaliana that transform leaves into cotyledons

We describe mutations of three genes in Arabidopsis thaliana—extra cotyledon1 (xtc1), extra cotyledon2 (xtc2), and altered meristem programming1 (amp1)—that transform leaves into cotyledons. In all three of these mutations, this transformation is associated with a change in the timing of events in embryogenesis. xtc1 and xtc2 delay the morphogenesis of the embryo proper at the globular-to-heart transition but permit the shoot apex to develop to an unusually advanced stage late in embryogenesis. Both mutations have little or no effect on seed maturation and do not affect the viability of the shoot or the rate of leaf initiation after germination. amp1 perturbs the pattern of cell division at an early globular stage, dramatically increases the size of the shoot apex and, like xtc1 and xtc2, produces enlarged leaf primordia during seed development. These unusual phenotypes suggest that these genes play important regulatory roles in embryogenesis and demonstrate that the development of the shoot apical meristem and the development of the embryo proper are regulated by independent processes that must be temporally coordinated to ensure normal organ identity.

Proteins unfold in steps

I present results from an experiment on the dynamics of folding of a globular protein (bovine serum albumin). Employing a micro-mechanical technique, I perform the measurements on very few molecules (1–100). I observed a sequence of steps in time for both unfolding and refolding. The overall characteristic time of the process is thus built up of waiting times between successive steps. The pattern of steps is reproducible, demonstrating the existence of deterministic pathways for folding and unfolding. Certain symmetries in the patterns of steps may reflect the architecture of the protein’s structure.

The terminal tail region of a yeast myosin-V mediates its attachment to vacuole membranes and sites of polarized growth

The Saccharomyces cerevisiae myosin-V, Myo2p, has been implicated in the polarized movement of several organelles and is essential for yeast viability. We have shown previously that Myo2p is required for the movement of a portion of the lysosome (vacuole) into the bud and consequently for proper inheritance of this organelle during cell division. Class V myosins have a globular carboxyl terminal tail domain that is proposed to mediate localization of the myosin, possibly through interaction with organelle-specific receptors. Here we describe a myo2 allele whose phenotypes support this hypothesis. vac15–1/myo2–2 has a single mutation in this globular tail domain, causing defects in vacuole movement and inheritance. Although a portion of wild-type Myo2p fractionates with the vacuole, the myo2–2 gene product does not. In addition, the mutant protein does not concentrate at sites of active growth, the predominant location of wild-type Myo2p. Although deletion of the tail domain is lethal, the myo2–2 gene product retains the essential functions of Myo2p. Moreover, myo2–2 does not cause the growth defects and lethal genetic interactions seen in myo2–66, a mutant defective in the actin-binding domain. These observations suggest that the myo2–2 mutation specifically disrupts interactions with selected myosin receptors, namely those on the vacuole membrane and those at sites of polarized growth.

Is there a Population II analogy to the F star lithium dip?

Observers have found a small number of lithium-depleted halo stars in the temperature range of the Spite plateau. The current status of the mass-loss hypothesis for producing the observed lithium dip in Population (Pop) I stars is briefly discussed and extended to Pop II stars as a possible explanation for these halo objects. Based on detections of F-type main-sequence variables, mass loss is assumed to occur in a narrow temperature region corresponding to this “instability strip.” As Pop II main-sequence stars evolve to the blue, they enter this narrow temperature region, then move back through the lower temperature area of the Spite plateau. If 0.05 M⊙ (solar mass) or more have been lost, they will show lithium depletion. This hypothesis affects the lithium-to- beryllium abundance, the ratio of high- to low-lithium stars, and the luminosity function. Constraints on the mass-loss hypothesis due to these effects are discussed. Finally, mass loss in this temperature range would operate in stars near the turnoff of metal-poor globular clusters, resulting in apparent ages 2 to 3 Gyr (gigayears) older than they actually are.

A Cell Signal Pathway Involving Laminin-5, α3β1 Integrin, and Mitogen-activated Protein Kinase Can Regulate Epithelial Cell Proliferation

Laminin-5 (LN5) is a matrix component of epithelial tissue basement membranes and plays an important role in the initiation and maintenance of epithelial cell anchorage to the underlying connective tissue. Here we show that two distinct LN5 function-inhibitory antibodies, both of which bind the globular domain of the α3 subunit, inhibit proliferation of epithelial cells. These same antibodies also induce a decrease in mitogen-activated protein kinase activity. Inhibition of proliferation by the function-perturbing LN5 antibodies is reversed upon removal of the antibodies and can be overcome by providing the antibody-treated cells with exogenous LN5 and rat tail collagen. Because epithelial cells use the integrin receptor α3β1 to interact with both LN5 and rat tail collagen, we next investigated the possibility that integrin α3β1 is involved in mediating the proliferative impact of LN5. Proliferation of human epithelial cells is significantly inhibited by a function-perturbing α3 integrin antibody. In addition, antibody activation of β1 integrin restores the proliferation of epithelial cells treated with LN5 function-perturbing antibodies. These data indicate that a complex comprising LN5 and α3β1 integrin is multifunctional and contributes not only to epithelial cell adhesion but also to the regulation of cell growth via a signaling pathway involving mitogen-activated protein kinase. We discuss our study in light of recent evidence that LN5 expression is up-regulated at the leading tips of tumors, where it may play a role in tumor cell proliferation.

Folding of Active β-Lactamase in the Yeast Cytoplasm before Translocation into the Endoplasmic Reticulum

Polypeptides targeted to the yeast endoplasmic reticulum (ER) posttranslationally are thought to be kept in the cytoplasm in an unfolded state by Hsp70 chaperones before translocation. We show here that Escherichia coli β-lactamase associated with Hsp70, but adopted a native-like conformation before translocation in living Saccharomyces cerevisiae cells. β-Lactamase is a globular trypsin-resistant molecule in authentic form. For these studies, it was linked to the C terminus of a yeast polypeptide Hsp150Δ, which conferred posttranslational translocation and provided sites for O-glycosylation. We devised conditions to retard translocation of Hsp150Δ-β-lactamase. This enabled us to show by protease protection assays that an unglycosylated precursor was associated with the cytoplasmic surface of isolated microsomes, whereas a glycosylated form resided inside the vesicles. Both proteins were trypsin resistant and had similar β-lactamase activity and Km values for nitrocefin. The enzymatically active cytoplasmic intermediate could be chased into the ER, followed by secretion of the activity to the medium. Productive folding in the cytoplasm occurred in the absence of disulfide formation, whereas in the ER lumen, proper folding required oxidation of the sulfhydryls. This suggests that the polypeptide was refolded in the ER and consequently, at least partially unfolded for translocation.

Organization of Highly Acetylated Chromatin around Sites of Heterogeneous Nuclear RNA Accumulation

Histones found within transcriptionally competent and active regions of the genome are highly acetylated. Moreover, these highly acetylated histones have very short half-lives. Thus, both histone acetyltransferases and histone deacetylases must enrich within or near these euchromatic regions of the interphase chromatids. Using an antibody specific for highly acetylated histone H3, we have investigated the organization of transcriptionally active and competent chromatin as well as nuclear histone acetyltransferase and deacetylase activities. We observe an exclusion of highly acetylated chromatin around the periphery of the nucleus and an enrichment near interchromatin granule clusters (IGCs). The highly acetylated chromatin is found in foci that may reflect the organization of highly acetylated chromatin into “chromonema” fibers. Transmission electron microscopy of Indian muntjac fibroblast cell nuclei indicates that the chromatin associated with the periphery of IGCs remains relatively condensed, most commonly found in domains containing chromatin folded beyond 30 nm. Using electron spectroscopic imaging, we demonstrate that IGCs are clusters of ribonucleoprotein particles. The individual granules comprise RNA-rich fibrils or globular regions that fold into individual granules. Quantitative analysis of individual granules indicates that they contain variable amounts of RNA estimated between 1.5 and >10 kb. We propose that interchromatin granules are heterogeneous nuclear RNA-containing particles, some of which may be pre-mRNA generated by nearby transcribed chromatin. An intermediary zone between the IGC and surrounding chromatin is described that contains factors with the potential to provide specificity to the localization of sequences near IGCs.

The EF-hand Ca2+-binding Protein p22 Associates with Microtubules in an N-Myristoylation–dependent Manner

Proteins containing the EF-hand Ca2+-binding motif, such as calmodulin and calcineurin B, function as regulators of various cellular processes. Here we focus on p22, an N-myristoylated, widely expressed EF-hand Ca2+-binding protein conserved throughout evolution, which was shown previously to be required for membrane traffic. Immunofluorescence studies show that p22 distributes along microtubules during interphase and mitosis in various cell lines. Moreover, we report that p22 associates with the microtubule cytoskeleton indirectly via a cytosolic microtubule-binding factor. Gel filtration studies indicate that the p22–microtubule-binding activity behaves as a 70- to 30-kDa globular protein. Our results indicate that p22 associates with microtubules via a novel N-myristoylation–dependent mechanism that does not involve classic microtubule-associated proteins and motor proteins. The association of p22 with microtubules requires the N-myristoylation of p22 but does not involve p22’s Ca2+-binding activity, suggesting that the p22–microtubule association and the role of p22 in membrane traffic are functionally related, because N-myristoylation is required for both events. Therefore, p22 is an excellent candidate for a protein that can mediate interactions between the microtubule cytoskeleton and membrane traffic.

Defensive extrusive ectosymbionts of Euplotidium (Ciliophora) that contain microtubule-like structures are bacteria related to Verrucomicrobia

Epixenosomes, ectosymbionts on hypotrich ciliates (genus Euplotidium) defend their host against the ciliate predator Litonotus lamella. Although here only Euplotidium itoi and Euplotidium arenarium from tide pools along a rocky shore near Leghorn (Ligurian sea) were studied in detail, these epibionts are certainly present on specimens of E. itoi and on other Euplotidium species in similar north coastal habitats. The complex life history of epixenosomes has two main stages. In stage I, cells with typical prokaryotic structure divide by binary fission. Stage II cells show complex organization with different cytoplasmic compartments where an extrusive apparatus within a proteinaceous matrix, although not membrane-bounded, differs from the remaining cytoplasm. The ejection process is involved in defense; extrusive apparatus is surrounded by a basket consisting of bundles of tubules. These tubules, 22 ± 3 nm in diameter, delimited by a wall made up of globular structures, are sensitive to inhibitor of tubulin polymerization (nocodazole/4°C temperature) and react positively with different antitubulin antibodies, two of which are monoclonal. The prokaryotic vs. eukaryotic nature of epixenosomes was resolved by comparative sequence analysis of amplified small subunit rRNA genes and in situ hybridization with fluorescently labeled rRNA-targeted polynucleotide probes. These unique ectosymbionts are phylogenetically related to Verrucomicrobia. Epixenosomes represent marine symbionts in this recently discovered division of the Bacteria.