Identification of a Human VPF/VEGF 3′ Untranslated Region Mediating Hypoxia-induced mRNA Stability

Hypoxia is a prominent feature of malignant tumors that are characterized by angiogenesis and vascular hyperpermeability. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) has been shown to be up-regulated in the vicinity of necrotic tumor areas, and hypoxia potently induces VPF/VEGF expression in several tumor cell lines in vitro. Here we report that hypoxia-induced VPF/VEGF expression is mediated by increased transcription and mRNA stability in human M21 melanoma cells. RNA-binding/electrophoretic mobility shift assays identified a single 125-bp AU-rich element in the 3′ untranslated region that formed hypoxia-inducible RNA-protein complexes. Hypoxia-induced expression of chimeric luciferase reporter constructs containing this 125-bp AU-rich hypoxia stability region were significantly higher than constructs containing an adjacent 3′ untranslated region element without RNA-binding activity. Using UV-cross-linking studies, we have identified a series of hypoxia-induced proteins of 90/88 kDa, 72 kDa, 60 kDa, 56 kDa, and 46 kDa that bound to the hypoxia stability region element. The 90/88-kDa and 60-kDa species were specifically competed by excess hypoxia stability region RNA. Thus, increased VPF/VEGF mRNA stability induced by hypoxia is mediated, at least in part, by specific interactions between a defined mRNA stability sequence in the 3′ untranslated region and distinct mRNA-binding proteins in human tumor cells.

Identification of hepatitis B virus indigenous to chimpanzees

Hepatitis B viruses (HBV) and related viruses, classified in the Hepadnaviridae family, are found in a wide variety of mammals and birds. Although the chimpanzee has been the primary experimental model of HBV infection, this species has not been considered a natural host for the virus. Retrospective analysis of 13 predominantly wild-caught chimpanzees with chronic HBV infection identified a unique chimpanzee HBV strain in 11 animals. Nucleotide and derived amino acid analysis of the complete HBV genome and the gene coding for the hepatitis B surface antigen (S gene) identified sequence patterns that could be used to reliably identify chimpanzee HBV. This analysis indicated that chimpanzee HBV is distinct from known human HBV genotypes and is closely related to HBVs previously isolated from a chimpanzee, gibbons, gorillas, and orangutans.

Generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification

We have developed a technique called the generation of longer cDNA fragments from serial analysis of gene expression (SAGE) tags for gene identification (GLGI), to convert SAGE tags of 10 bases into their corresponding 3′ cDNA fragments covering hundred bases. A primer containing the 10-base SAGE tag is used as the sense primer, and a single base anchored oligo(dT) primer is used as an antisense primer in PCR, together with Pfu DNA polymerase. By using this approach, a cDNA fragment extending from the SAGE tag toward the 3′ end of the corresponding sequence can be generated. Application of the GLGI technique can solve two critical issues in applying the SAGE technique: one is that a longer fragment corresponding to a SAGE tag, which has no match in databases, can be generated for further studies; the other is that the specific fragment corresponding to a SAGE tag can be identified from multiple sequences that match the same SAGE tag. The development of the GLGI method provides several potential applications. First, it provides a strategy for even wider application of the SAGE technique for quantitative analysis of global gene expression. Second, a combined application of SAGE/GLGI can be used to complete the catalogue of the expressed genes in human and in other eukaryotic species. Third, it can be used to identify the 3′ cDNA sequence from any exon within a gene. It can also be used to confirm the reality of exons predicted by bioinformatic tools in genomic sequences. Fourth, a combined application of SAGE/GLGI can be applied to define the 3′ boundary of expressed genes in the genomic sequences in human and in other eukaryotic genomes.

Identification of a third distinct estrogen receptor and reclassification of estrogen receptors in teleosts

This paper describes three distinct estrogen receptor (ER) subtypes: ERα, ERβ, and a unique type, ERγ, cloned from a teleost fish, the Atlantic croaker Micropogonias undulatus; the first identification of a third type of classical ER in vertebrate species. Phylogenetic analysis shows that ERγ arose through gene duplication from ERβ early in the teleost lineage and indicates that ERγ is present in other teleosts, although it has not been recognized as such. The Atlantic croaker ERγ shows amino acid differences in regions important for ligand binding and receptor activation that are conserved in all other ERγs. The three ER subtypes are genetically distinct and have different distribution patterns in Atlantic croaker tissues. In addition, ERβ and ERγ fusion proteins can each bind estradiol-17β with high affinity. The presence of three functional ERs in one species expands the role of ER multiplicity in estrogen signaling systems and provides a unique opportunity to investigate the dynamics and mechanisms of ER evolution.

The TIGR Gene Indices: analysis of gene transcript sequences in highly sampled eukaryotic species

While genome sequencing projects are advancing rapidly, EST sequencing and analysis remains a primary research tool for the identification and categorization of gene sequences in a wide variety of species and an important resource for annotation of genomic sequence. The TIGR Gene Indices (http://www.tigr.org/tdb/tgi.shtml) are a collection of species-specific databases that use a highly refined protocol to analyze EST sequences in an attempt to identify the genes represented by that data and to provide additional information regarding those genes. Gene Indices are constructed by first clustering, then assembling EST and annotated gene sequences from GenBank for the targeted species. This process produces a set of unique, high-fidelity virtual transcripts, or Tentative Consensus (TC) sequences. The TC sequences can be used to provide putative genes with functional annotation, to link the transcripts to mapping and genomic sequence data, to provide links between orthologous and paralogous genes and as a resource for comparative sequence analysis.

Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor

The corticotropin-releasing factor (CRF) family of neuropeptides includes the mammalian peptides CRF, urocortin, and urocortin II, as well as piscine urotensin I and frog sauvagine. The mammalian peptides signal through two G protein-coupled receptor types to modulate endocrine, autonomic, and behavioral responses to stress, as well as a range of peripheral (cardiovascular, gastrointestinal, and immune) activities. The three previously known ligands are differentially distributed anatomically and have distinct specificities for the two major receptor types. Here we describe the characterization of an additional CRF-related peptide, urocortin III, in the human and mouse. In searching the public human genome databases we found a partial expressed sequence tagged (EST) clone with significant sequence identity to mammalian and fish urocortin-related peptides. By using primers based on the human EST sequence, a full-length human clone was isolated from genomic DNA that encodes a protein that includes a predicted putative 38-aa peptide structurally related to other known family members. With a human probe, we then cloned the mouse ortholog from a genomic library. Human and mouse urocortin III share 90% identity in the 38-aa putative mature peptide. In the peptide coding region, both human and mouse urocortin III are 76% identical to pufferfish urocortin-related peptide and more distantly related to urocortin II, CRF, and urocortin from other mammalian species. Mouse urocortin III mRNA expression is found in areas of the brain including the hypothalamus, amygdala, and brainstem, but is not evident in the cerebellum, pituitary, or cerebral cortex; it is also expressed peripherally in small intestine and skin. Urocortin III is selective for type 2 CRF receptors and thus represents another potential endogenous ligand for these receptors.

Identification of a Saccharomyces cerevisiae Gene that Is Required for G1 Arrest in Response to the Lipid Oxidation Product Linoleic Acid Hydroperoxide*

Reactive oxygen species cause damage to all of the major cellular constituents, including peroxidation of lipids. Previous studies have revealed that oxidative stress, including exposure to oxidation products, affects the progression of cells through the cell division cycle. This study examined the effect of linoleic acid hydroperoxide, a lipid peroxidation product, on the yeast cell cycle. Treatment with this peroxide led to accumulation of unbudded cells in asynchronous populations, together with a budding and replication delay in synchronous ones. This observed modulation of G1 progression could be distinguished from the lethal effects of the treatment and may have been due to a checkpoint mechanism, analogous to that known to be involved in effecting cell cycle arrest in response to DNA damage. By examining several mutants sensitive to linoleic acid hydroperoxide, the YNL099c open reading frame was found to be required for the arrest. This gene (designated OCA1) encodes a putative protein tyrosine phosphatase of previously unknown function. Cells lacking OCA1 did not accumulate in G1 on treatment with linoleic acid hydroperoxide, nor did they show a budding, replication, or Start delay in synchronous cultures. Although not essential for adaptation or immediate cellular survival, OCA1 was required for growth in the presence of linoleic acid hydroperoxide, thus indicating that it may function in linking growth, stress responses, and the cell cycle. Identification of OCA1 establishes cell cycle arrest as an actively regulated response to oxidative stress and will enable further elucidation of oxidative stress-responsive signaling pathways in yeast.

Demonstration of the extrinsic coagulation pathway in teleostei: Identification of zebrafish coagulation factor VII

It is not known whether the mammalian mechanism of coagulation initiation is conserved in fish. Identification of factor VII is critical in providing evidence for such a mechanism. A cDNA was cloned from a zebrafish (teleost) library that predicted a protein with sequence similarity to human factor VII. Factor VII was shown to be present in zebrafish blood and liver by Western blot analysis and immunohistochemistry. Immunodepletion of factor VII from zebrafish plasma selectively inhibited thromboplastin-triggered thrombin generation. Heterologous expression of zebrafish factor VII demonstrated a secreted protein (50 kDa) that reconstituted thromboplastin-triggered thrombin generation in immunodepleted zebrafish plasma. These results suggest conservation of the extrinsic coagulation pathway between zebrafish and humans and add credence to the zebrafish as a model for mammalian hemostasis. The structure of zebrafish factor VIIa predicted by homology modeling was consistent with the overall three-dimensional structure of human factor VIIa. However, amino acid disparities were found in the epidermal growth factor-2/serine protease regions that are present in the human tissue factor–factor VIIa contact surface, suggesting a structural basis for the species specificity of this interaction. In addition, zebrafish factor VII demonstrates that the Gla-EGF-EGF-SP domain structure, which is common to coagulation factors VII, IX, X, and protein C, was present before the radiation of the teleosts from the tetrapods. Identification of zebrafish factor VII significantly narrows the evolutionary window for development of the vertebrate coagulation cascade and provides insight into the structural basis for species specificity in the tissue factor–factor VIIa interaction.

Identification of a new calcitonin gene in the salmon Oncorhynchus gorbuscha.

Three isoforms of calcitonin (CT) exist in salmonids. Isohormones I and II are expressed in the pink salmon Oncorhynchus gorbuscha. We report here the existence in this species of a CT gene and of its transcripts, which encode for a fourth isohormone, the salmon CT (sCT) IV. This new CT gene was identified by PCR from genomic DNA and by sequencing the amplified DNA. The expression of this CT gene was established in ultimobranchial body and brain, by reverse transcription-PCR, hybridization and sequencing. The sCT IV gene, like the sCT I gene, is a complex transcription unit, containing exons encoding for a CT as a calcitonin gene-related peptide (CGRP) molecule. The predicted peptide, sCT IV, has a greater homology with the eel CT and the sCT II than with the sCT I. Alignment of the sCT IV with other fish and chicken CT showed amino acid modifications in similar positions as those found during evolution. The predicted salmon CGRP IV peptide is highly homologous to the known CGRP molecules in other species, confirming the high conservation of the molecule during evolution. This identification of a new salmon CT gene is interesting both for the therapeutic potential represented by the new molecules encoded by this gene and for phylogenetic studies.

Identification of interdomain sequences promoting the intronless evolution of a bacterial protein family.

In the evolution of eukaryotic genes, introns are believed to have played a major role in increasing the probability of favorable duplication events, chance recombinations, and exon shuffling resulting in functional hybrid proteins. As a rule, prokaryotic genes lack introns, and the examples of prokaryotic introns described do not seem to have contributed to gene evolution by exon shuffling. Still, certain protein families in modern bacteria evolve rapidly by recombination of genes, duplication of functional domains, and as shown for protein PAB of the anaerobic bacterial species Peptostreptococcus magnus, by the shuffling of an albumin-binding protein module from group C and G streptococci. Characterization of a protein PAB-related gene in a P. magnus strain with less albumin-binding activity revealed that the shuffled module was missing. Based on this fact and observations made when comparing gene sequences of this family of bacterial surface proteins interacting with albumin and/or immunoglobulin, a model is presented that can explain how this rapid intronless evolution takes place. A new kind of genetic element is introduced: the recer sequence promoting interdomain, in frame recombination and acting as a structure-less flexibility-promoting spacer in the corresponding protein. The data presented also suggest that antibiotics could represent the selective pressure behind the shuffling of protein modules in P. magnus, a member of the indigenous bacterial flora.

Identification of functional domains and evolution of Tc1-like transposable elements.

Tc1-like transposable elements from teleost fish have been phylogenetically examined to determine the mechanisms involved in their evolution and conserved domains of function. We identified two new functional domains in these elements. The first is a bipartite nuclear localization signal, indicating that transposons can take advantage of the transport machinery of host cells for nuclear uptake of their transposases. The second is a novel combination of a paired domain-related protein motif juxtaposed to a leucine zipper-like domain located in the putative DNA-binding regions of the transposases. This domain coexists with a special inverted repeat structure in certain transposons in such phylogenetically distant hosts as fish and insects. Our data indicate that reassortment of functional domains and horizontal transmission between species are involved in the formation and spread of new types of transposable elements.

Characterization of a second secreted IgE isoform and identification of an asymmetric pathway of IgE assembly.

A number of alternatively spliced epsilon transcripts have been detected in IgE-producing B cells, in addition to the mRNAs encoding the classical membrane and secreted IgE heavy (H) chains. In a recent study, we examined the protein products of three of these alternatively spliced isoforms and found that they are intracellularly retained and degraded because of their inability to assemble into complete IgE molecules. We have now similarly examined a more recently described epsilon mRNA species that is generated by splicing between a donor splice site immediately upstream of the stop codon in the H-chain constant region exon 4 (CH4) and an acceptor site located in the 3' part of the second membrane exon. We show that this isoform is efficiently secreted by both plasma cells and B lymphocytes and therefore represents a second secreted IgE isoform (epsilon S2). The epsilon S2 H chain is only six amino acids longer than the classical secreted Ig H chain (epsilon S1) and contains a C-terminal cysteine, which is a characteristic sequence feature of mu and alpha H chains. However, unlike IgM and IgA, the epsilon S2 C-terminal cysteine (Cys-554) does not induce polymerization of H2L2 molecules (where L is light chain), but rather creates a disulfide bond between the two H chains that increases the rate of association into covalently bound H2L2 monomers. This C-terminal cysteine also does not function as an intracellular retention element because the epsilon S2 isoform was secreted in amounts equal to that of the epsilon S1, both in B lymphocytes and in plasma cells. The epsilon S2 H chains secreted by B lymphocytes differed from the epsilon S1 H chains in the extent of glycosylation. Interestingly, a difference in glycosylation between B-lymphocytes and plasma cells was also noted for both isoforms. The presence of the Cys-554 also allowed the identification of a distinctive asymmetric pathway of IgE assembly, common to both types of epsilon H chains.

Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium.

Mapping the insertion points of 16 signature-tagged transposon mutants on the Salmonella typhimurium chromosome led to the identification of a 40-kb virulence gene cluster at minute 30.7. This locus is conserved among all other Salmonella species examined but is not present in a variety of other pathogenic bacteria or in Escherichia coli K-12. Nucleotide sequencing of a portion of this locus revealed 11 open reading frames whose predicted proteins encode components of a type III secretion system. To distinguish between this and the type III secretion system encoded by the inv/spa invasion locus known to reside on a pathogenicity island, we refer to the inv/spa locus as Salmonella pathogenicity island (SPI) 1 and the new locus as SPI2. SPI2 has a lower G+C content than that of the remainder of the Salmonella genome and is flanked by genes whose products share greater than 90% identity with those of the E. coli ydhE and pykF genes. Thus SPI2 was probably acquired horizontally by insertion into a region corresponding to that between the ydhE and pykF genes of E. coli. Virulence studies of SPI2 mutants have shown them to be attenuated by at least five orders of magnitude compared with the wild-type strain after oral or intraperitoneal inoculation of mice.

Identification and characterization of putative transposable DNA elements in solanaceous plants and Caenorhabditis elegans.

Several families of putative transposable elements (TrEs) in both solanaceous plants and Caenorhabditis elegans have been identified by screening the DNA data base for inverted repeated domains present in multiple copies in the genome. The elements are localized within intron and flanking regions of many genes. These elements consist of two inverted repeats flanking sequences ranging from 5 bp to > 500 bp. Identification of multiple elements in which sequence conservation includes both the flanking and internal regions implies that these TrEs are capable of duplicative transposition. Two of the elements were identified in promoter regions of the tomato (Lycoperiscon esculentum) polygalacturonase and potato (Solanum tuberosum) Win1 genes. The element in the polygalacturonase promoter spans a known regulatory region. In both cases, ancestral DNA sequences, which represent potential recombination target sequences prior to insertion of the elements, have been cloned from related species. The sequences of the inverted repeated domains in plants and C. elegans show a high degree of phylogenetic conservation. While frequency of the different elements is variable, some are present in very high copy number. A member of a single C. elegans TrE family is observed approximately once every 20 kb in the genome. The abundance of the described TrEs suggests utility in the genomic analysis of these and related organisms.

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.