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PubMed2026

Unraveling the coastal marine plastisphere archaeome.

Plastic pollution has created an expanding anthropogenic microbial niche, the plastisphere, raising questions about microbial ecology and associated impacts. Archaea, the third domain of life with fundamental ecological and evolutionary significance, remain poorly understood in this habitat. Here, using paired plastic debris and bulk-water samples from coastal marine ecosystems, key archaeal habitats increasingly threatened by plastic pollution, we characterize the plastisphere archaeome through archaeal amplicon sequencing and metagenomics. We show that the archaeome is significantly reshaped in the plastisphere, exhibiting higher taxonomic diversity, greater community heterogeneity, and selective enrichment of Euryarchaeota and Crenarchaeota. Archaeal genes involved in methane, nitrogen, and sulfur cycling are enriched in the plastisphere. Taxonomic and functional divergence between the plastisphere and bulk water increases with anthropogenic chemical stress. These findings suggest that plastic pollution could alter marine archaeal diversity, biogeography, and biogeochemical potential, extending understanding of plastisphere impacts to the archaeal domain.

Archaea↗
PubMed2005

Linkage analysis of the genetic determinants of T-cell IL-4 secretion, and identification of Flj20274 as a putative candidate gene.

The activation-induced differentiation of naive CD4+ T cells generates functionally divergent type 1 helper T cells (Th1) or type 2 helper T cells (Th2) effector cell populations, characterized by secretion of Interferon (IFN)-gamma or Interleukin (IL)-4, respectively. Inappropriate generation of Th subsets may contribute to immune dysfunction. The decision to generate Th1/Th2 lineages is critically regulated by cytokines, such that IL-12 induces Th1 differentiation, while IL-4 induces Th2 differentiation. Genetic factors influence the pathway of Th differentiation, as displayed by the preferential generation of divergent Th populations by different inbred strains of mice. We employ two complementary genetic techniques to identify genes that regulate the default IL-4 secretion profiles of T cells from BALB/c and B6 mice. We performed a genome-wide linkage analysis of the progeny of a backcross between BALB/c and B6 mice to identify three loci, T-cell secretion of interleukin-4 (Tsi)1-3, on chromosomes 7, 19 and 15, respectively, which regulate in vitro T-cell IL-4 production. We have also employed mRNA representational difference analysis to isolate a gene, Flj20274, which is differentially expressed in T cells that secrete high levels of IL-4. Significantly, Flj20274 was mapped to the point of peak linkage within Tsi1 and is a strong candidate for Tsi1.

Animals↗
PubMed1997

Characterisation and chromosome mapping of the human non receptor tyrosine kinase gene, brk.

The brk gene encodes a non-receptor protein tyrosine kinase that consists of single SH3, SH2 and catalytic domains. Although BRK shows strongest sequence similarity to members of the SRC family of PTKs, there are several key structural and regulatory differences that place it on its own amongst non-receptor PTKs. In this study we have isolated genomic DNA clones corresponding to the human brk locus and used these to determine the intron-exon structure of the brk gene. The genomic structure of brk consists of 8 exons, whose boundaries are distinct from other non-receptor PTK family members, again indicating a structural and functional divergence. Alternate splicing of the primary brk transcript generates a distinct mRNA which encodes a truncated protein consisting of an SH3 domain and a novel C-terminal proline rich sequence. Using an antiserum raised to the SH3 domain, we have demonstrated that the product of this alternate brk transcript is expressed in the human breast tumour cell line T-47D. We have previously reported that expression of a tumour derived brk cDNA in mouse embryonic fibroblasts and human mammary epithelial cells supports anchorage independent growth, and in the latter potentiates the mitogenic response to epidermal growth factor. The protein encoded by the genomic sequence derived from normal human tissue is identical to that encoded by the tumour derived cDNA, and therefore the altered growth regulation is not associated with mutations within brk. In addition, we have identified a 5' genomic region that has promoter activity. The brk gene has been assigned to chromosome 20q 13.3 [corrected] using fluorescence in situ hybridisation (FISH).

Amino Acid Sequence↗
PubMed2004

An alternatively spliced survivin variant is positively regulated by p53 and sensitizes leukemia cells to chemotherapy.

Survivin is a unique member of the inhibitor of apoptosis protein family, and its expression is regulated by p53. Recent identification of several functionally divergent survivin variants augments the complexity of survivin action as well as its regulation. Here we report that survivin-2B (retaining a part of intron 2 as a cryptic exon) is positively regulated by p53, and its overexpression plays a role in sensitizing leukemia cells to chemotherapeutic drug doxorubicin. Doxorubicin treatment activated p53, downregulated survivin and survivin-DeltaEx3 but upregulated survivin-2B in EU-3, an acute lymphocytic leukemia (ALL) cell line with wild-type (wt)-p53 phenotype. In contrast, doxorubicin treatment failed to induce these alterations in EU-6 cells, a mutant-p53 ALL cell line. To specify the role of wt-p53 in regulating survivin and its variants, a temperature-sensitive p53 mutant plasmid p53-143 was transfected into EU-4, a p53-null ALL cell line, to establish a subline EU-4/p53-143. When EU-4/p53-143 cell culture was shifted from 37.5 degrees C to the wt-p53-permissive temperature (32.5 degrees C), the expression of survivin and survivin-DeltaEx3 was decreased whereas survivin-2B expression was increased, confirming the distinct regulatory effect of p53 on survivin and its variants. To clarify the role of survivin-2B in the process of apoptosis, survivin-2B cDNA was cloned into pcDNA3HA vector and transfected into EU-4 cells. Enforced expression of survivin-2B in EU-4 cells inhibited cell growth and sensitized these cells to doxorubicin-induced apoptosis. These results suggest that survivin-2B variant is a proapoptotic factor and its expression is upregulated by p53.

Alternative Splicing↗
PubMed2005

Polo-like kinases and oncogenesis.

Polo-like kinases (Plks) play pivotal roles in the regulation of cell cycle progression. Plk1, the best characterized family member among mammalian Plks, strongly promotes the progression of cells through mitosis. Furthermore, Plk1 is found to be overexpressed in a variety of human tumors and its expression correlates with cellular proliferation and prognosis of tumor patients. Although all Plks share two conserved elements, the N-terminal Ser/Thr kinase domain and a highly homologues C-terminal region termed the polo-box motif, their functions diverge considerably. While Plk1 is inhibited by different checkpoint pathways, Plk2 and Plk3 are activated by the spindle checkpoint or the DNA damage checkpoint. Thus, Plk2 and Plk3 seem to inhibit oncogenic transformation. Deregulation of Plk1 activity contributes to genetic instability, which in turn leads to oncogenic transformation. In contrast, Plk2 and Plk3 are involved in checkpoint-mediated cell cycle arrest to ensure genetic stability, thereby inhibiting the accumulation of genetic defects. In this review, we shall discuss the roles of Plks in oncogenesis and Plk1 as a target for therapeutic intervention against cancer.

Animals↗
PubMed2001

Mammalian class Sigma glutathione S-transferases: catalytic properties and tissue-specific expression of human and rat GSH-dependent prostaglandin D2 synthases.

GSH-dependent prostaglandin D(2) synthase (PGDS) enzymes represent the only vertebrate members of class Sigma glutathione S-transferases (GSTs) identified to date. Complementary DNA clones encoding the orthologous human and rat GSH-dependent PGDS (hPGDS and rPGDS, respectively) have been expressed in Escherichia coli, and the recombinant proteins isolated by affinity chromatography. The purified enzymes were both shown to catalyse specifically the isomerization of prostaglandin (PG) H(2) to PGD(2). Each transferase also exhibited GSH-conjugating and GSH-peroxidase activities. The ability of hPGDS to catalyse the conjugation of aryl halides and isothiocyanates with GSH was found to be less than that of the rat enzyme. Whilst there is no difference between the enzymes with respect to their K(m) values for 1-chloro-2,4-dinitrobenzene, marked differences were found to exist with respect to their K(m) for GSH (8 mM versus 0.3 mM for hPGDS and rPGDS, respectively). Using molecular modelling techniques, amino acid substitutions have been identified in the N-terminal domain of these enzymes that lie outside the proposed GSH-binding site, which may explain these catalytic differences. The tissue-specific expression of PGDS also varies significantly between human and rat; amongst the tissues examined, variation in expression between the two species was most apparent in spleen and bone marrow. Differences in catalytic properties and tissue-specific expression of hPGDS and rPGDS appears to reflect distinct physiological roles for class Sigma GST between species. The evolution of divergent functions for the hPGDS and rPGDS is discussed in the context of the orthologous enzyme from chicken.

Amino Acid Sequence↗
PubMed2006

Comparative characterization and expression analysis of the four Old Yellow Enzyme homologues from Shewanella oneidensis indicate differences in physiological function.

Shewanella oneidensis contains four genes that encode proteins that have high sequence identity with yeast OYE (Old Yellow Enzyme, an NADPH oxidoreductase), the well-studied archetype of the OYE protein family. The present paper describes the first comparative study of OYEs that are present in a single bacterial species, performed to gain insight into their biochemical properties and physiological importance. The four proteins [named SYE1-SYE4 (Shewanella Yellow Enzyme 1-4)] were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The yield of SYE2, however, was too low for further characterization, even after expression attempts in S. oneidensis. The SYE1, SYE3 and SYE4 proteins were found to have characteristics similar to those of other OYE family members. They were identified as flavoproteins that catalyse the reduction of different alpha,beta-unsaturated carbonyl compounds and form charge transfer complexes with a range of phenolic compounds. Whereas the properties of SYE1 and SYE3 were very similar, those of SYE4 were clearly different in terms of ligand binding, catalytic efficiency and substrate specificity. Also, the activity of SYE4 was found to be NADPH-dependent, whereas SYE1 and SYE3 had a preference for NADH. It has been suggested that yeast OYE protects the actin cytoskeleton from oxidative stress. There are indications that bacterial OYEs are also involved in the oxidative stress response, but their exact role is unclear. Induction studies in S. oneidensis revealed that yeast and bacterial OYEs may share a common physiological role, i.e. the protection of cellular components against oxidative damage. As only SYE4 was induced under oxidative stress conditions, however, a functional divergence between bacterial OYEs is likely to exist.

Amino Acid Sequence↗
PubMed2000

Two zebrafish (Danio rerio) antizymes with different expression and activities.

Cellular polyamines are regulated by a unique feedback mechanism involving ornithine decarboxylase (ODC) antizyme. The synthesis of mammalian antizyme requires a programmed translational frameshift event induced by polyamines. Antizyme represses ODC, a key enzyme for polyamine synthesis, through accelerating enzyme degradation by the 26 S proteasome. Antizyme also inhibits the cellular uptake of polyamines. In the present study we isolated two distinct zebrafish (Danio rerio) antizyme cDNA clones (AZS and AZL) from an embryonic library. Their sequences revealed that both clones required translational frameshifting for expression. Taking account of +1 frameshifting, AZS and AZL products were 214 and 218 residues long respectively and shared 51.8% amino acid identity. In rabbit reticulocyte lysates, both mRNA species were translated through spermidine-induced frameshifting. The presence of the two antizyme mRNA species in embryos, adult fish and a cultured cell line was confirmed by Northern blot analysis. The ratio of AZS mRNA to AZL mRNA in the adult fish was 1.8-fold higher than in the embryos. Whole-mount hybridization in situ demonstrated that both mRNA species are expressed in every tissue in embryo, but predominantly in the central nervous system and the eyes. Bacterial expression products of both cDNA species inhibited ODC activity, but only the AZS product accelerated ODC degradation in vitro. These results show that both zebrafish antizymes are induced by polyamines but their mRNA species are expressed differently during development. The difference in activities on ODC degradation suggests their functional divergence.

Amino Acid Sequence↗
PubMed2003

Biochemical Society Special Lecture. Nitrate- and nitrite-responsive sensors NarX and NarQ of proteobacteria.

Nitrate and nitrite are efficient respiratory oxidants for anaerobic growth. In Escherichia coli, the homologous nitrate reductase (Nar) two-component regulatory systems NarX-NarL and NarQ-NarP collaborate to control anaerobic respiratory gene expression in response to nitrate and nitrite. Several other species classified in the gamma and beta subdivisions of the proteobacteria contain only a single Nar two-component regulatory pair. This raises questions concerning the physiology of anaerobic respiration as well as the evolution, function and cross-regulation of two-component regulatory systems. Here, I focus on the sensor histidine kinases NarX and NarQ, and present a comparison of the deduced NarX and NarQ primary sequences from a broad sampling of proteobacteria. This comparison defines shared features, including a large central region of unknown function that appears to be unique to this family of sensor kinases. I then consider the phylogenetic distribution of narX and narQ genes in relation to anaerobic respiratory enzyme repertoire and physiological function. One noteworthy observation is that narXL genes are specifically associated with the structural genes for membrane-bound nitrate reductase, narGHJI, whereas organization and linkage of the narQ and narP genes is quite variable. I conclude with some speculative thoughts on the evolutionary and functional divergence of the NarX-NarL and NarQ-NarP regulatory systems. Overall, this analysis aims to provide a basis for future hypothesis and experimentation in this area.

Amino Acid Sequence↗
PubMed2003

Evolutionary relationships, but functional differences, between the Drosophila and human Toll-like receptor families.

The Toll receptor was first found to function in the dorsoventral patterning pathway of Drosophila embryos. It is activated by a specific protein ligand, Spätzle, generated at ventral positions in the early embryo. Drosophila Toll (dToll) also functions in innate immune responses to Gram-positive bacteria and fungi, and Spätzle is required for this response. We have shown that Spätzle is necessary and sufficient for activation of the dToll pathway, and that it probably acts by cross-linking two molecules of Toll to form homodimers. In the present paper, we contrast this mode of regulation with that proposed for the vertebrate Toll-like receptor family, which mediate analogous responses to pathogen pattern antigens. In contrast with dToll, these receptors appear to be activated by direct exposure to pathogen patterns, such as peptidoglycan and lipopolysaccharide. We discuss the evolutionary basis of this functional divergence of the vertebrate and invertebrate Toll-like receptors.

Amino Acid Sequence↗
PubMed2003

Sprouty: a controversial role in receptor tyrosine kinase signalling pathways.

Sprouty was first identified in Drosophila as a novel antagonist of the fibroblast growth factor signalling pathway. Sprouty proteins comprise a big family, members of which are characterized by a cysteine-rich domain which confers inhibitory activity, whereas differences in the N-terminal region may be responsible for functional divergence. The role of Sprouty in RTK (receptor tyrosine kinase) signalling pathways is still controversial. Sprouty may negatively or positively regulate RTK signalling via differential interaction with different signalling molecules, and hence exert different mechanism of action.

Animals↗
PubMed2006

P450ome of the white rot fungus Phanerochaete chrysosporium: structure, evolution and regulation of expression of genomic P450 clusters.

The model white rot fungus Phanerochaete chrysosporium has the extraordinary ability to degrade (to CO(2)) lignin and detoxify a variety of chemical pollutants. Whole genome sequencing of this fungus has revealed the presence of the largest P450ome in fungi comprising approx. 150 P450 genes, most of which have unknown function. On the basis of our genome-wide structural and evolutionary analysis, these P450 genes could be classified into 12 families and 23 subfamilies and under 11 fungal P450 clans. The analysis further revealed an extensive gene clustering with a total of 16 P450 clusters constituted of up to 11 members per cluster. In particular, evidence and role of gene duplications and horizontal gene transfer in the evolution of these P450 clusters have been discussed using two of the P450 families [CYP63 and CYP505 (where CYP is cytochrome P450)] as examples. In addition, the observed differential transcriptional induction of the clustered members of the CYP63 gene family, in response to different xenobiotic chemicals and carbon sources, indicated functional divergence within the P450 clusters, of this basidiomycete fungus.

Cytochrome P-450 Enzyme System↗
PubMed2005

Enzyme redesign and interactions of substrate analogues with sterol methyltransferase to understand phytosterol diversity, reaction mechanism and the nature of the active site.

Several STM (sterol methyltransferase) genes have been cloned, sequenced and expressed in bacteria recently, making it possible to address questions of the relationship between sterol structure and function. The active site and mechanism of action of a set of phylogenetically diverse SMTs have been probed by site-directed mutagenesis as well as by using substrate and related analogues of the SMT-catalysed reaction. An active-site model has been developed that is in accord with the results presented, which is consistent with the hypothesis that SMTs are bifunctional enzymes kinetically responsible to bind Delta24-acceptor sterols of specific steric and electronic character and rigid orientation imposed by multiple hydrophobic active site contacts exacted from a common waxy core. Functional divergence influenced by the architectural role of sterols in membranes is considered to govern the evolution of product distribution and specificity of individual SMTs as discussed.

Amino Acid Sequence↗
PubMed2001

Overlapping and distinct roles of the duplicated yeast transcription factors Ace2p and Swi5p.

The yeast transcription factors Ace2p and Swi5p regulate the expression of several target genes involved in mating type switching, exit from mitosis and cell wall function. We describe the analysis of 12 novel targets, some regulated by Ace2p or Swi5p alone and some by both. We show that Ace2p is the major regulator of four genes (CTS1, YHR143W, SCW11 and YER124C). Expression of all four is inhibited by Swi5p. Like Cts1p and Scw11p, the two new Ace2p targets are associated with cell wall metabolism. Yhr143p is localized to the cell wall, and deletion affects cell separation and enhances pseudohyphal growth. Deleting YER124C also affects cell separation and sensitivity to drugs targeted against the cell wall. Expression of PIR1, YPL158C and YNL046W is dependent on Swi5p alone. In contrast, expression of YBR158W, YNL078W and YOR264W is minimized when both ace2 and swi5 are disrupted. We propose that, although Ace2p and Swi5p co-operate to induce the expression of a subset of genes, some functional divergence has occurred. This results in a delay in the expression of those genes predominantly regulated by Ace2p, compared with those predominantly regulated by Swi5p.

Cell Cycle Proteins↗
PubMed2003

Novel Proteorhodopsin variants from the Mediterranean and Red Seas.

Proteorhodopsins, ubiquitous retinylidene photoactive proton pumps, were recently found in the widespread uncultured SAR86 bacterial group in oceanic surface waters. To survey proteorhodopsin diversity, new degenerate sets of proteorhodopsin primers were designed based on a genomic proteorhodopsin gene sequence originating from an Antarctic fosmid library. New proteorhodopsin variants were identified in Red Sea samples that were most similar to the original green-light absorbing proteorhodopsins found in Monterey Bay California. Unlike green-absorbing proteorhodopsins however, these new variants contained a glutamine residue at position 105, the same site recently shown to control spectral tuning in naturally occurring proteorhodopsins. Different proteorhodopsin variants were also found in the Mediterranean Sea. These proteorhodopsins formed new and distinctive proteorhodopsin groups. Phylogenetic analyses show that some of the new variants were very different from previously characterized proteorhodopsins, and formed the deepest branching groups found so far among marine proteorhodopsins. The existence of these varied proteorhodopsin sequences suggests that this class of proteins has undergone substantial evolution. These variants could represent functionally divergent paralogous genes, derived from the same or similar species, or orthologous proteorhodopsins that are distributed amongst divergent planktonic microbial taxa.

Amino Acid Sequence↗
PubMed2006

On the existence and scaling of structure functions in turbulence according to the data.

We sample a velocity field that has an inertial spectrum and a skewness that matches experimental data. In particular, we compute a self-consistent correction to the Kolmogorov exponent and find that for our model it is zero. We find that the higher-order structure functions diverge for orders larger than a certain threshold, as theorized in some recent work. The significance of the results for the statistical theory of homogeneous turbulence is reviewed.

Journal Article↗
PubMed2001

Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster.

Streptomyces lavendulae produces complestatin, a cyclic peptide natural product that antagonizes pharmacologically relevant protein-protein interactions including formation of the C4b,2b complex in the complement cascade and gp120-CD4 binding in the HIV life cycle. Complestatin, a member of the vancomycin group of natural products, consists of an alpha-ketoacyl hexapeptide backbone modified by oxidative phenolic couplings and halogenations. The entire complestatin biosynthetic and regulatory gene cluster spanning ca. 50 kb was cloned and sequenced. It consisted of 16 ORFs, encoding proteins homologous to nonribosomal peptide synthetases, cytochrome P450-related oxidases, ferredoxins, nonheme halogenases, four enzymes involved in 4-hydroxyphenylglycine (Hpg) biosynthesis, transcriptional regulators, and ABC transporters. The nonribosomal peptide synthetase consisted of a priming module, six extending modules, and a terminal thioesterase; their arrangement and domain content was entirely consistent with functions required for the biosynthesis of a heptapeptide or alpha-ketoacyl hexapeptide backbone. Two oxidase genes were proposed to be responsible for the construction of the unique aryl-ether-aryl-aryl linkage on the linear heptapeptide intermediate. Hpg, 3,5-dichloro-Hpg, and 3,5-dichloro-hydroxybenzoylformate are unusual building blocks that repesent five of the seven requisite monomers in the complestatin peptide. Heterologous expression and biochemical analysis of 4-hydroxyphenylglycine transaminon confirmed its role as an aminotransferase responsible for formation of all three precursors. The close similarity but functional divergence between complestatin and chloroeremomycin biosynthetic genes also presents a unique opportunity for the construction of hybrid vancomycin-type antibiotics.

Base Sequence↗
PubMed1982

Ascorbic acid sulfate sulfohydrolase (C2 sulfatase): the modulator of cellular levels of L-ascorbic acid in rainbow trout.

The enzyme L-ascorbic acid 2-sulfate sulfohydrolase (C2 sulfatase) was purified from rainbow trout liver. The enzyme catalyzes the hydrolysis of L-ascorbic acid 2-sulfate and has a pH optimum at 6.0. It has a molecular weight of about 117,500 at pH 5.0 and is inhibited by a number of sulfhydryl blocking agents including L-ascorbic acid. C2 sulfatase activity was observed in most metabolic organs of rainbow trout. These findings suggest that the physiologic role of the enzyme is to maintain adequate cellular concentrations of L-ascorbic acid in the fish. The activity of the enzyme is controlled by L-ascorbic acid through feedback inhibition. Comparison of kinetic constants and inhibition patterns suggests that C2 sulfatase is structurally identical to human arylsulfatase A. However, unlike C2 sulfatase, human arylsulfatase A may not be involved in ascorbate metabolism. Its physiologic substrate is reported to be cerebroside-3-sulfate, not L-ascorbic acid 2-sulfate. A scheme is proposed to account for the functional divergence of these two structurally identical enzymes.

Animals↗