A thermostable endonuclease III homolog from the archaeon Pyrobaculum aerophilum

Pyrimidine adducts in cellular DNA arise from modification of the pyrimidine 5,6-double bond by oxidation, reduction or hydration. The biological outcome includes increased mutation rate and potential lethality. A major DNA N-glycosylase responsible for the excision of modified pyrimidine bases is the base excision repair (BER) glycosylase endonuclease III, for which functional homologs have been identified and characterized in Escherichia coli, yeast and humans. So far, little is known about how hyperthermophilic Archaea cope with such pyrimidine damage. Here we report characterization of an endonuclease III homolog, PaNth, from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100°C. The predicted product of 223 amino acids shares significant sequence homology with several [4Fe-4S]-containing DNA N-glycosylases including E.coli endonuclease III (EcNth). The histidine-tagged recombinant protein was expressed in E.coli and purified. Under optimal conditions of 80–160 mM NaCl and 70°C, PaNth displays DNA glycosylase/β-lyase activity with the modified pyrimidine base 5,6-dihydrothymine (DHT). This activity is enhanced when DHT is paired with G. Our data, showing the structural and functional similarity between PaNth and EcNth, suggests that BER of modified pyrimidines may be a conserved repair mechanism in Archaea. Conserved amino acid residues are identified for five subfamilies of endonuclease III/UV endonuclease homologs clustered by phylogenetic analysis.

Vipp1 deletion mutant of Synechocystis: A connection between bacterial phage shock and thylakoid biogenesis?

Plant chloroplasts originated from an endosymbiotic event by which an ancestor of contemporary cyanobacteria was engulfed by an early eukaryotic cell and then transformed into an organelle. Oxygenic photosynthesis is the specific feature of cyanobacteria and chloroplasts, and the photosynthetic machinery resides in an internal membrane system, the thylakoids. The origin and genesis of thylakoid membranes, which are essential for oxygenic photosynthesis, are still an enigma. Vipp1 (vesicle-inducing protein in plastids 1) is a protein located in both the inner envelope and the thylakoids of Pisum sativum and Arabidopsis thaliana. In Arabidopsis disruption of the VIPP1 gene severely affects the plant's ability to form properly structured thylakoids and as a consequence to carry out photosynthesis. In contrast, Vipp1 in Synechocystis appears to be located exclusively in the plasma membrane. Yet, as in higher plants, disruption of the VIPP1 gene locus leads to the complete loss of thylakoid formation. So far VIPP1 genes are found only in organisms carrying out oxygenic photosynthesis. They share sequence homology with a subunit encoded by the bacterial phage shock operon (PspA) but differ from PspA by a C-terminal extension of about 30 amino acids. In two cyanobacteria, Synechocystis and Anabaena, both a VIPP1 and a pspA gene are present, and phylogenetic analysis indicates that VIPP1 originated from a gene duplication of the latter and thereafter acquired its new function. It also appears that the C-terminal extension that discriminates VIPP1 proteins from PspA is important for its function in thylakoid formation.

X-ray structure of the human hyperplastic discs protein: An ortholog of the C-terminal domain of poly(A)-binding protein

The poly(A)-binding protein (PABP) recognizes the 3′ mRNA poly(A) tail and plays an essential role in eukaryotic translation initiation and mRNA stabilization/degradation. PABP is a modular protein, with four N-terminal RNA-binding domains and an extensive C terminus. The C-terminal region of PABP is essential for normal growth in yeast and has been implicated in mediating PABP homo-oligomerization and protein–protein interactions. A small, proteolytically stable, highly conserved domain has been identified within this C-terminal segment. Remarkably, this domain is also present in the hyperplastic discs protein (HYD) family of ubiquitin ligases. To better understand the function of this conserved region, an x-ray structure of the PABP-like segment of the human HYD protein has been determined at 1.04-Å resolution. The conserved domain adopts a novel fold resembling a right-handed supercoil of four α-helices. Sequence profile searches and comparative protein structure modeling identified a small ORF from the Arabidopsis thaliana genome that encodes a structurally similar but distantly related PABP/HYD domain. Phylogenetic analysis of the experimentally determined (HYD) and homology modeled (PABP) protein surfaces revealed a conserved feature that may be responsible for binding to a PABP interacting protein, Paip1, and other shared interaction partners.

Characterization of Two Divergent Endo-β-1,4-Glucanase cDNA Clones Highly Expressed in the Nonclimacteric Strawberry Fruit

Two cDNAs clones (Cel1 and Cel2) encoding divergent endo-β-1,4-glucanases (EGases) have been isolated from a cDNA library obtained from ripe strawberry (Fragaria x ananassa Duch) fruit. The analysis of the amino acid sequence suggests that Cel1 and Cel2 EGases have different secondary and tertiary structures and that they differ in the presence of potential N-glycosylation sites. By in vitro translation we show that Cel1 and Cel2 bear a functional signal peptide, the cleavage of which yields mature proteins of 52 and 60 kD, respectively. Phylogenetic analysis revealed that the Cel2 EGase diverged early in evolution from other plant EGases. Northern analysis showed that both EGases are highly expressed in fruit and that they have different temporal patterns of accumulation. The Cel2 EGase was expressed in green fruit, accumulating as the fruit turned from green to white and remaining at an elevated, constant level throughout fruit ripening. In contrast, the Cel1 transcript was not detected in green fruit and only a low level of expression was observed in white fruit. The level of Cel1 mRNA increased gradually during ripening, reaching a maximum in fully ripe fruit. The high levels of Cel1 and Cel2 mRNA in ripe fruit and their overlapping patterns of expression suggest that these EGases play an important role in softening during ripening. In addition, the early expression of Cel2 in green fruit, well before significant softening begins, suggests that the product of this gene may also be involved in processes other than fruit softening, e.g. cell wall expansion.

A Millennial Myosin Census

The past decade has seen a remarkable explosion in our knowledge of the size and diversity of the myosin superfamily. Since these actin-based motors are candidates to provide the molecular basis for many cellular movements, it is essential that motility researchers be aware of the complete set of myosins in a given organism. The availability of cDNA and/or draft genomic sequences from humans, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Dictyostelium discoideum has allowed us to tentatively define and compare the sets of myosin genes in these organisms. This analysis has also led to the identification of several putative myosin genes that may be of general interest. In humans, for example, we find a total of 40 known or predicted myosin genes including two new myosins-I, three new class II (conventional) myosins, a second member of the class III/ninaC myosins, a gene similar to the class XV deafness myosin, and a novel myosin sharing at most 33% identity with other members of the superfamily. These myosins are in addition to the recently discovered class XVI myosin with N-terminal ankyrin repeats and two human genes with similarity to the class XVIII PDZ-myosin from mouse. We briefly describe these newly recognized myosins and extend our previous phylogenetic analysis of the myosin superfamily to include a comparison of the complete or nearly complete inventories of myosin genes from several experimentally important organisms.

Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial genome expansions

The evolution of novelty in tightly integrated biological systems, such as hormones and their receptors, seems to challenge the theory of natural selection: it has not been clear how a new function for any one part (such as a ligand) can be selected for unless the other members of the system (e.g., a receptor) are already present. Here I show—based on identification and phylogenetic analysis of steroid receptors in basal vertebrates and reconstruction of the sequences and functional attributes of ancestral proteins—that the first steroid receptor was an estrogen receptor, followed by a progesterone receptor. Genome mapping and phylogenetic analyses indicate that the full complement of mammalian steroid receptors evolved from these ancient receptors by two large-scale genome expansions, one before the advent of jawed vertebrates and one after. Specific regulation of physiological processes by androgens and corticoids are relatively recent innovations that emerged after these duplications. These findings support a model of ligand exploitation in which the terminal ligand in a biosynthetic pathway is the first for which a receptor evolves; selection for this hormone also selects for the synthesis of intermediates despite the absence of receptors, and duplicated receptors then evolve affinity for these substances. In this way, novel hormone-receptor pairs are created, and an integrated system of increasing complexity elaborated. This model suggests that ligands for some “orphan” receptors may be found among intermediates in the synthesis of ligands for phylogenetically related receptors.

Perspectives on archaeal diversity, thermophily and monophyly from environmental rRNA sequences.

Phylogenetic analysis of ribosomal RNA sequences obtained from uncultivated organisms of a hot spring in Yellowstone National Park reveals several novel groups of Archaea, many of which diverged from the crenarchaeal line of descent prior to previously characterized members of that kingdom. Universal phylogenetic trees constructed with the addition of these sequences indicate monophyly of Archaea, with modest bootstrap support. The data also show a specific relationship between low-temperature marine Archaea and some hot spring Archaea. Two of the environmental sequences are enigmatic: depending upon the data set and analytical method used, these sequences branch deeply within the Crenarchaeota, below the bifurcation between Crenarchaeota and Euryarchaeota, or even as the sister group to Eukaryotes. If additional data confirm either of the latter two placements, then the organisms represented by these ribosomal RNA sequences would merit recognition as a new kingdom, provisionally named "Korarchaeota."

16S rRNA genes reveal stratified open ocean bacterioplankton populations related to the Green Non-Sulfur bacteria.

Microorganisms play an important role in the biogeochemistry of the ocean surface layer, but spatial and temporal structures in the distributions of specific bacterioplankton species are largely unexplored, with the exceptions of those organisms that can be detected by either autofluorescence or culture methods. The use of rRNA genes as genetic markers provides a tool by which patterns in the growth, distribution, and activity of abundant bacterioplankton species can be studied regardless of the ease with which they can be cultured. Here we report an unusual cluster of related 16S rRNA genes (SAR202, SAR263, SAR279, SAR287, SAR293, SAR307) cloned from seawater collected at 250 m in the Sargasso Sea in August 1991, when the water column was highly stratified and the deep chlorophyll maximum was located at a depth of 120 m. Phylogenetic analysis and an unusual 15-bp deletion confirmed that the genes were related to the Green Non-Sulfur phylum of the domain Bacteria. This is the first evidence that representatives of this phylum occur in the open ocean. Oligonucleotide probes were used to examine the distribution of the SAR202 gene cluster in vertical profiles (0-250 m) from the Atlantic and Pacific Oceans, and in discrete (monthly) time series (O and 200 m) (over 30 consecutive months in the Western Sargasso Sea. The data provide robust statistical support for the conclusion that the SAR202 gene cluster is proportionately most abundant at the lower boundary of the deep chlorophyll maximum (P = 2.33 x 10(-5)). These results suggest that previously unsuspected stratification of microbial populations may be a significant factor in the ecology of the ocean surface layer.

cDNA structure, tissue distribution, and chromosomal localization of rat PC7, a novel mammalian proprotein convertase closest to yeast kexin-like proteinases.

By using reverse transcription-coupled PCR on rat anterior pituitary RNA, we isolated a 285-bp cDNA coding for a novel subtilisin/kexin-like protein convertase (PC), called rat (r) PC7. By screening rat spleen and PC12 cell lambda gt11 cDNA libraries, we obtained a composite 3.5-kb full-length cDNA sequence of rPC7. The open reading frame codes for a prepro-PC with a 36-amino acid signal peptide, a 104-amino acid prosegment ending with a cleavable RAKR sequence, and a 747-amino acid type I membrane-bound glycoprotein, representing the mature form of this serine proteinase. Phylogenetic analysis suggests that PC7 represents the most divergent enzyme of the mammalian convertase family and that it is the closest member to the yeast convertases krp and kexin. Northern blot analyses demonstrated a widespread expression with the richest source of rPC7 mRNA being the colon and lymphoid-associated tissues. In situ hybridization revealed a distinctive tissue distribution that sometimes overlaps with that of furin, suggesting that PC7 has widespread proteolytic functions. The gene for PC7 (Pcsk7) was mapped to mouse chromosome 9 by linkage analysis of an interspecific backcross DNA panel.

The virion glycoproteins of Ebola viruses are encoded in two reading frames and are expressed through transcriptional editing.

In late 1994 and early 1995, Ebola (EBO) virus dramatically reemerged in Africa, causing human disease in the Ivory Coast and Zaire. Analysis of the entire glycoprotein genes of these viruses and those of other EBO virus subtypes has shown that the virion glycoprotein (130 kDa) is encoded in two reading frames, which are linked by transcriptional editing. This editing results in the addition of an extra nontemplated adenosine within a run of seven adenosines near the middle of the coding region. The primary gene product is a smaller (50-70 kDa), nonstructural, secreted glycoprotein, which is produced in large amounts and has an unknown function. Phylogenetic analysis indicates that EBO virus subtypes are genetically diverse and that the recent Ivory Coast isolate represents a new (fourth) subtype of EBO virus. In contrast, the EBO virus isolate from the 1995 outbreak in Kikwit, Zaire, is virtually identical to the virus that caused a similar epidemic in Yambuku, Zaire, almost 20 years earlier. This genetic stability may indicate that EBO viruses have coevolved with their natural reservoirs and do not change appreciably in the wild.

Evidence that two present-day components needed for the genetic code appeared after nucleated cells separated from eubacteria.

The trinucleotide/amino acid relationships of the present-day genetic code are established by the amino-acylation reactions of tRNA synthetases, whereby each of 20 specific amino acids is attached to its cognate tRNAs, which bear anticodon trinucleotides. Because of its universality, the appearance of the modern genetic code is thought to predate the separation of prokaryotic and eukaryotic organisms in the universal phylogenetic tree. In the light of new sequence information, we present here a phylogenetic analysis that shows an unusual picture for tyrosyl- and tryptophanyl-tRNA synthetases. Ij particular, the eukaryotic tyrosyl- and tryptophanyl-tRNA synthetases are more related to each other than to their respective prokaryotic counterparts. In contrast, each of the other 18 eukaryotic synthetases is more related to its prokaryotic counterpart than to any eukaryotic synthetase specific for a different amino acid. Our results raise the possibility that present day tyrosyl- and tryptophanyl-tRNA synthetases appeared after the separation of nucleated cells from eubacteria. The results have implications for the development of the genetic code.

Intracellular coexistence of methano- and thioautotrophic bacteria in a hydrothermal vent mussel.

The coexistence of two phylogenetically distinct symbiont species within a single cell, a condition not previously known in any metazoan, is demonstrated in the gills of a Mid-Atlantic Ridge hydrothermal vent mussel (family Mytilidae). Large and small symbiont morphotypes within the gill bacteriocytes are shown to be separate bacterial species by molecular phylogenetic analysis and fluorescent in situ hybridization. The two symbiont species are affiliated with thioautotrophic and methanotrophic symbionts previously found in monospecific associations with closely related mytilids from deep-sea hydrothermal vents and hydrocarbon seeps.

Identification of planarian homeobox sequences indicates the antiquity of most Hox/homeotic gene subclasses.

The homeotic gene complex (HOM-C) is a cluster of genes involved in the anteroposterior axial patterning of animal embryos. It is composed of homeobox genes belonging to the Hox/HOM superclass. Originally discovered in Drosophila, Hox/HOM genes have been identified in organisms as distantly related as arthropods, vertebrates, nematodes, and cnidarians. Data obtained in parallel from the organization of the complex, the domains of gene expression during embryogenesis, and phylogenetic relationships allow the subdivision of the Hox/HOM superclass into five classes (lab, pb/Hox3, Dfd, Antp, and Abd-B) that appeared early during metazoan evolution. We describe a search for homologues of these genes in platyhelminths, triploblast metazoans emerging as an outgroup to the great coelomate ensemble. A degenerate PCR screening for Hox/HOM homeoboxes in three species of triclad planarians has revealed 10 types of Antennapedia-like genes. The homeobox-containing sequences of these PCR fragments allowed the amplification of the homeobox-coding exons for five of these genes in the species Polycelis nigra. A phylogenetic analysis shows that two genes are clear orthologues of Drosophila labial, four others are members of a Dfd/Antp superclass, and a seventh gene, although more difficult to classify with certainty, may be related to the pb/Hox3 class. Together with previously identified Hox/HOM genes in other flatworms, our analyses demonstrate the existence of an elaborate family of Hox/HOM genes in the ancestor of all triploblast animals.

Direct evidence for secondary loss of mitochondria in Entamoeba histolytica.

Archezoan protists are though to represent lineages that diverged from other eukaryotes before acquisition of the mitochondrion and other organelles. The parasite Entamoeba histolytica was originally included in this group. Ribosomal RNA based phylogenies, however, place E. histolytica on a comparatively recent branch of the eukaryotic tree, implying that its ancestors had these structures. In this study, direct evidence for secondary loss of mitochondrial function was obtained by isolating two E. histolytica genes encoding proteins that in other eukaryotes are localized in the mitochondrion: the enzyme pyridine nucleotide transhydrogenase and the chaperonin cpn60. Phylogenetic analysis of the E. histolytica homolog of cpn60 confirmed that it is specifically related to the mitochondrial lineage. The data suggest that a mitochondrial relic may persist in this organism. Similar studies are needed in archezoan protists to ascertain which, if any, eukaryotic lineages primitively lack mitochondria.

Cloning of a receptor for amphibian [Phe13]bombesin distinct from the receptor for gastrin-releasing peptide: identification of a fourth bombesin receptor subtype (BB4).

Bombesin is a tetradecapeptide originally isolated from frog skin and demonstrated to have a wide range of actions in mammals. Based on structural homology and similar biological activities, gastrin-releasing peptide (GRP) has been considered the mammalian equivalent of bombesin. We previously reported that frogs have both GRP and bombesin, which therefore are distinct peptides. We now report the cloning of a bombesin receptor subtype (BB4) that has higher affinity for bombesin than GRP. PCR was used to amplify cDNAs related to the known bombesin receptors from frog brain. Sequence analysis of the amplified cDNAs revealed 3 classes of receptor subtypes. Based on amino acid homology, two classes were clearly the amphibian homologs of the GRP and neuromedin B receptors. The third class was unusual and a full-length clone was isolated from a Bombina orientalis brain cDNA library. Expression of the receptor in Xenopus oocytes demonstrated that the receptor responded to picomolar concentrations of [Phe13]-bombesin, the form of bombesin most prevalent in frog brain. The relative rank potency of bombesin-like peptides for this receptor was [Phe13]bombesin > [Leu13]bombesin > GRP > neuromedin B. In contrast, the rank potency for the GRP receptor is GRP > [Leu13]bombesin > [Phe13]bombesin > neuromedin B. Transient expression in CHOP cells gave a Ki for [Phe13]bombesin of 0.2 nM versus a Ki of 2.1 nM for GRP. Distribution analysis showed that this receptor was expressed only in brain, consistent with the distribution of [Phe13]-bombesin. Thus, based on distribution and affinity, this bombesin receptor is the receptor for [Phe13]bombesin. Phylogenetic analysis suggests that this receptor separated prior to separation of the GRP and neuromedin B receptors; thus, BB4 receptors and their cognate ligands may also exist in mammals.