UTRdb: a specialized database of 5' and 3' untranslated regions of eukaryotic mRNAs

The 5' and 3' untranslated regions of eukaryotic mRNAs may play a crucial role in the regulation of gene expression controlling mRNA localization, stability and translational efficiency. For this reason we developed UTRdb (http://bigarea.area.ba.cnr.it:8000/BioWWW/#U TRdb), a specialized database of 5' and 3' untranslated sequences of eukaryotic mRNAs cleaned from redundancy. UTRdb entries are enriched with specialized information not present in the primary databases including the presence of nucleotide sequence patterns already demonstrated by experimental analysis to have some functional role. All these patterns have been collected in the UTRsite database so that it is possible to search any input sequence for the presence of annotated functional motifs. Furthermore, UTRdb entries have been annotated for the presence of repetitive elements.     

Amplification of gene expression using both 5'- and 3'-untranslated regions of GLUT1 glucose transporter mRNA

Cis-regulatory elements located at either the 5'- or 3'-untranslated region (UTR) of the GLUT1 glucose transporter mRNA increase the expression of luciferase reporter genes. The aim of the present study was to investigate the possible cooperative effects of 5'- and 3'-UTRs of the GLUT1 mRNA on the expression of a luciferase reporter gene in cultured brain endothelial cells. Luciferase reporter genes containing control elements in nucleotides (nt) 1-171 of GLUT1 5'-UTR, or nt 2100-2300 of GLUT1 3'-UTR produced a 10- and 6-fold increase in the expression of the luciferase reporter gene compared to the control vector containing no GLUT1 regulatory sequences, respectively. The insertion of both GLUT1 mRNA cis-regulatory elements increased 59-fold the activity of luciferase compared to controls. Data presented here demonstrate that cis-regulatory elements located at both the 5'- and 3'-UTR of GLUT1 mRNA increase expression of a reporter gene in an independent manner.     

Mutational analysis of cis-acting sequences in the 3'- and 5'-untranslated regions of RNA2 of red clover necrotic mosaic virus

BACKGROUND/AIMS: Hepatic stellate cells appear to be the main producers of hepatocyte growth factor of the normal liver. Insulin-like growth factors in doses over 20 ng/ml have been reported to stimulate hepatocyte growth factor production in cultured hepatic stellate cells. The aim of the present study was to investigate whether parenchymal cell conditioned medium had insulin-like growth factor-independent effects on hepatic stellate cells. METHODS: Primary rat hepatic stellate cells were cultured for 1-7 days. DNA synthesis was measured by 3H-thymidine incorporation. Hepatocyte growth factor and transforming growth factor beta1 immunoreactivity was quantified by ELISA. Hepatocyte growth factor mRNA levels were determined with gel RNase protection assay. Parenchymal cell conditioned medium was obtained from hepatocytes cultured for 2 days in medium without added serum or hormones. RESULTS: Incubation of 1-7-day-old hepatic stellate cells for 2 days with parenchymal cell conditioned medium enhanced the medium content of hepatocyte growth factor. Parenchymal cell conditioned medium contained less than 5.0 ng/ml immunoreactive insulin-like growth factor-1 as measured by radio immunoassay. Parenchymal cell conditioned medium did not contain any insulin-like growth factor bioactivity measured as phosphorylation of type 1 insulin-like growth factor receptor beta subunit and a protein with a size consistent with that of insulin receptor substrate-1. The stimulatory effect of parenchymal cell conditioned medium on hepatocyte growth factor was time- and dose-dependent. The effects of a high dose of parenchymal cell conditioned medium (dilution 1:2 containing less than 2.5 ng/ml insulin-like growth factor-1) were additive to that of high doses (100 ng/ml) of insulin-like growth factor-1 or des (1-3) insulin-like growth factor-1, an analogue with low affinity to insulin-like growth factor binding proteins. Neither parenchymal cell conditioned medium nor insulin-like growth factor-1 enhanced transforming growth factor beta1 immunoreactivity in the medium. Both parenchymal cell conditioned medium and insulin-like growth factor-1 stimulated DNA synthesis in hepatic stellate cells, confirming previous reports. CONCLUSIONS: The present results indicate that both insulin-like growth factor-1 and insulin-like growth factor-1-independent factors from hepatocytes can stimulate hepatocyte growth factor production by hepatic stellate cells. Therefore, insulin-like growth factor-1 and other hepatocyte-derived factors may indirectly affect hepatocytes via a paracrine loop.     

Olfactory Receptor Database: a database of the largest eukaryotic gene family

The Olfactory Receptor Database (ORDB) is a WWW-accessible database that stores data on Olfactory Receptor-like molecules (ORs) and has been open to the public since June 1996. It contains a public and a private area. The public area includes published DNA and protein sequence data for ORs, links to OR models and data on their expression, chromosomal localization and source organism, as well as (i) links to bibliography through PubMed and (ii) interactive WWW-based tools, such as BLAST homology searching. The private area functions as a service to laboratories that are actively cloning receptors. Source laboratories enter the sequences of the receptor clones they have characterized to the private database and can search for identical or near identical OR sequences in both public and private databases. If another laboratory has cloned and deposited an identical or closely matching sequence there are means for communication between the laboratories to help avoid duplication of work. ORDB is available via the WWW at http://crepe.med.yale.edu/ORDB/HTML     

Toxicity of liposomal 3'-5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine in mice

Toxicities of 5-fluoro-2'-deoxyuridine (FUdR) and its liposome incorporated dipalmitoyl derivative (FUdR-dipalmitate) to mouse bone marrow, spleen, liver and ileum were compared after treatment for 6 consecutive days. The applied doses of the two formulations, which were shown earlier to have equal antitumor activity in mouse tumor models, were 600 and 2 mumol/kg respectively. When applied in these doses, toxicity to the hemopoietic system, measured as a decreases in progenitor and precursor cells of the erythroid and granuloid/macrophage lineage in bone marrow and spleen, was more severe for FUdR than for liposomal FUdR-dipalmitate. In the liver, mitotic figures, as indicators of cell division, were absent for both drugs while in control livers the number of cells in mitosis was approximately 2%. Toxicity to the ileum was more severe for liposomal FUdR-dipalmitate than for FUdR and was manifested by granulocyte infiltration, the presence of cell debris, loss of columnar epithelial cells and enlarged nuclei with prominent nucleoli in these cells. Thus, by prolonging the retention time of FUdR in vivo, using liposomes as a vehicle and FUdR-dipalmitate as a lipophilic prodrug, the dose-limiting toxicity appears to shift from bone marrow to the gastrointestinal tract.     

Isolation of a ribozyme with 5'-5' ligase activity

BACKGROUND: Many new ribozymes, including sequence-specific nucleases, ligases and kinases, have been isolated by in vitro selection from large pools of random-sequence RNAs. We are attempting to use in vitro selection to isolate new ribozymes that have, or can be evolved to have, RNA polymerase-like activities. As phosphorimidazolide-activated nucleosides are extensively used to study non-enzymatic RNA replication, we wished to select for a ribozyme that would accelerate the template-directed ligation of 5'-phosphorimidazolide-activated oligonucleotides. RESULTS: Ribozymes selected to perform the desired template-directed ligation reaction instead ligated themselves to the activated substrate oligonucleotide via their 5'-triphosphate, generating a 5'-5' P1,P4-tetraphosphate linkage. Deletion analysis of one of the selected sequences revealed that a 54-nucleotide RNA retained activity; this small ribozyme folds into a pseudoknot secondary structure with an internal binding site for the substrate oligonucleotide. The ribozyme can also synthesize 5'-5' triphosphate and 5'-5' pyrophosphate linkages. CONCLUSIONS: The emergence of ribozymes that accelerate an unexpected 5'-5' ligation reaction from a selection designed to yield template-dependent 3'-5' ligases suggests that it may be much easier for RNA to catalyze the synthesis of 5'-5' linkages than 3'-5' linkages. 5'-5' linkages are found in a variety of contexts in present-day biology. The ribozyme-catalyzed synthesis of such linkages raises the possibility that these 5'-5' linkages originated in the biochemistry of the RNA world.     

Distribution and cloning of eukaryotic mRNAs by means of differential display: refinements and optimization

Differential display has been developed as a tool to detect and characterize altered gene expression in eukaryotic cells. The basic principle is to systematically amplify messenger RNAs and then distribute their 3' termini on a denaturing polyacrylamide gel. Here we provide methodological details and examine in depth the specificity, sensitivity and reproducibility of the method. We show that the number of anchored oligo-dT primers can be reduced from twelve to four that are degenerate at the penultimate base from the 3' end. We also demonstrate that using optimized conditions described here, multiple RNA samples from related cells can be displayed simultaneously. Therefore process-specific rather than cell-specific genes could be more accurately identified. These results enable further streamlining of the technique and make it readily applicable to a broad spectrum of biological systems.     

Removal of 5'-terminal m7G from eukaryotic mRNAs by potato nucleotide pyrophosphatase and its effect on translation

The procedure for isolation of nucleotide pyrophosphatase (E.C. 3.6.1.9.) from potato has been modified to yield an endonuclease-free preparation purified 2300-fold. The enzyme was used for specific cleavage of pyrophosphate linkages in the 5'-terminal cap (m7GpppN) of several eukaryotic messenger RNAs. Enzymatic removal of 5'-terminal pm7G from reovirus, rabbit globin and Artemia salina mRNAs resulted in an almost complete loss (greater than 80%) of their template activities in a cell-free protein synthesizing system from wheat germ. Incubation with nucleotide pyrophosphatase did not decrease the translation of phage f2 RNA in an Escherichia coli cell-free system.     

The effect of (2'-5') and (3'-5') phosphodiester linkages on conformational and stacking properties of cytidylyl-cytidine in aqueous solution

Conformational properties of (2'-5') and (3'-5') CpC have been determined by proton magnetic resonance spectroscopy at 220 MHz. The ribose ring structures are predominantly 3E with the exception of the ring from the 2'-phosphate fragment of C(2'-5')pC which exhibits an 2E pucker. Bases are oriented anti with respect to the ribose and the conformations about C4'-C5', C5'-O5', C3'-O3' (C2'-O2') are gg, g'g', and g+ in equilibrium g-, respectively. The dimers exist as mixtures of stacked (g+g+ and g-g- about the P-O(C) bonds) and unstacked species at 20 degrees C. Stacking is estimated to be 35% in both dimers.     

Oligoribonuclease is encoded by a highly conserved gene in the 3'-5' exonuclease superfamily

Oligoribonuclease, a 3'-to-5' exoribonuclease specific for small oligoribonucleotides, was purified to homogeneity from extracts of Escherichia coli. The purified protein is an alpha2 dimer of 40 kDa. NH2-terminal sequence analysis of the protein identified the gene encoding oligoribonuclease as yjeR (o204a), a previously reported open reading frame located at 94 min on the E. coli chromosome. However, as a consequence of the sequence information, the translation start site of this open reading frame has been revised. Cloning of yjeR led to overexpression of oligoribonuclease activity, and interruption of the cloned gene with a kanamycin resistance cassette eliminated the overexpression. On the basis of these data, we propose that yjeR be renamed orn. Orthologs of oligoribonuclease are present in a wide range of organisms, extending up to humans.     

p59OASL, a 2'-5' oligoadenylate synthetase like protein: a novel human gene related to the 2'-5' oligoadenylate synthetase family

The 2'-5' oligoadenylate synthetases form a well conserved family of interferon induced proteins, presumably present throughout the mammalian class. Using the Expressed Sequence Tag databases, we have identified a novel member of this family. This protein, which we named p59 2'-5' oligoadenylate synthetase-like protein (p59OASL), shares a highly conserved N-terminal domain with the known forms of 2'-5' oligoadenylate synthetases, but differs completely in its C-terminal part. The C-terminus of p59OASL is formed of two domains of ubiquitin-like sequences. Here we present the characterisation of a full-length cDNA clone, the genomic sequence and the expression pattern of this gene. We have addressed the evolution of the 2'-5' oligoadenylate synthetase gene family, in the light of both this new member and new 2'-5' oligoadenylate synthetase sequence data from other species, which have recently appeared in the databases.     

The role of 3'-5' exonucleolytic proofreading and mismatch repair in yeast mitochondrial DNA error avoidance

In the D171G/D230A mutant generated at conserved aspartate residues in the Exo1 and Exo2 sites of the 3'-5' exonuclease domain of the yeast mitochondrial DNA (mtDNA) polymerase (pol-gamma), the mitochondrial genome is unstable and the frequency of mtDNA point mutations is 1500 times higher than in the wild-type strain and 10 times higher than in single substitution mutants. The 10(4)-fold decrease in the 3'-5' exonuclease activity of the purified mtDNA polymerase is associated with mismatch extension and high rates of base misincorporation. Processivity of the purified polymerase on primed single-stranded DNA is decreased and the Km for dNTP is increased. The sequencing of mtDNA point mutations in the wild-type strain and in proofreading and mismatch-repair deficient mutants shows that mismatch repair contributes to elimination of the transitions while exonucleolytic proofreading preferentially repairs transversions, and more specifically A to T (or T to A) transversions. However, even in the wild-type strain, A to T (or T to A) transversions are the most frequent substitutions, suggesting that they are imperfectly repaired. The combination of both mismatch repair and proofreading deficiencies elicits a mitochondrial error catastrophe. These data show that the faithful replication of yeast mtDNA requires both exonucleolytic proofreading and mismatch repair.     

A 5'-3' exonuclease activity involved in forming the 3' products of histone pre-mRNA processing in vitro

Histone RNA 3' processing in vitro produces one or more 5' cleavage products corresponding to the mature histone mRNA 3' end, and a group of 3' cleavage products whose 5' ends are mostly located several nucleotides downstream of the mRNA 3' end. The formation of these 3' products is coupled to the formation of 5' products and dependent on the U7 snRNP and a heat-labile processing factor. These short 3' products therefore are a true and general feature of the processing reaction. Identical 3' products are also formed from a model RNA containing all spacer nucleotides downstream of the mature mRNA 3' end, but no sequences from the mature mRNA. Again, this reaction is dependent on both the U7 snRNP and a heat-labile factor. Unlike the processing with a full-length histone pre-mRNA, this reaction produces only 3' but no 5' fragments. In addition, product formation is inhibited by addition of cap structures at the model RNA 5' end, indicating that product formation occurs by 5'-3' exonucleolytic degradation. This degradation of a model 3' product by a 5'-3' exonuclease suggests a mechanism for the release of the U7 snRNP after processing by shortening the cut-off histone spacer sequences base paired to U7 RNA.     

MMDB: Entrez's 3D structure database

The three dimensional structures for representatives of nearly half of all protein families are now available in public databases. Thus, no matter which protein one investigates, it is increasingly likely that the 3D structure of a homolog will be known and may reveal unsuspected structure-function relationships. The goal of Entrez's 3D-structure database is to make this information accessible and usable by molecular biologists (http://www.ncbi.nlm.nih.gov/Entrez). To this end Entrez provides two major analysis tools, a search engine based on sequence and structure 'neighboring' and an integrated visualization system for sequence and structure alignments. From a protein's sequence 'neighbors' one may rapidly identify other members of a protein family, including those where 3D structure is known. By comparing aligned sequences and/or structures in detail, using the visualization system, one may identify conserved features and perhaps infer functional properties. Here we describe how these analysis tools may be used to investigate the structure and function of newly discovered proteins, using the PTEN gene product as an example.     

Efficient translesion replication in the absence of Escherichia coli Umu proteins and 3'-5' exonuclease proofreading function

Translesion replication (TR) past a cyclobutane pyrimidine dimer in Escherichia coli normally requires the UmuD'2C complex, RecA protein, and DNA polymerase III holoenzyme (pol III). However, we find that efficient TR can occur in the absence of the Umu proteins if the 3'-5' exonuclease proofreading activity of the pol III epsilon-subunit also is disabled. TR was measured in isogenic uvrA6 DeltaumuDC strains carrying the dominant negative dnaQ allele, mutD5, or DeltadnaQ spq-2 mutations by transfecting them with single-stranded M13-based vectors containing a specifically located cis-syn T-T dimer. As expected, little TR was observed in the DeltaumuDC dnaQ+ strain. Surprisingly, 26% TR occurred in UV-irradiated DeltaumuDC mutD5 cells, one-half the frequency found in a uvrA6 umuDC+mutD5 strain. lexA3 (Ind-) derivatives of the strains showed that this TR was contingent on two inducible functions, one LexA-dependent, responsible for approximately 70% of the TR, and another LexA-independent, responsible for the remaining approximately 30%. Curiously, the DeltaumuDC DeltadnaQ spq-2 strain exhibited only the LexA-independent level of TR. The cause of this result appears to be the spq-2 allele, a dnaE mutation required for viability in DeltadnaQ strains, since introduction of spq-2 into the DeltaumuDC mutD5 strain also reduces the frequency of TR to the LexA-independent level. The molecular mechanism responsible for the LexA-independent TR is unknown but may be related to the UVM phenomenon [Palejwala, V. A., Wang, G. E., Murphy, H. S. & Humayun, M. Z. (1995) J. Bacteriol. 177, 6041-6048]. LexA-dependent TR does not result from the induction of pol II, since TR in the DeltaumuDC mutD5 strain is unchanged by introduction of a DeltapolB mutation.     

Human RAD2 homolog 1 5'- to 3'-exo/endonuclease can efficiently excise a displaced DNA fragment containing a 5'-terminal abasic lesion by endonuclease activity

Repair of abasic lesions, one of the most common types of damage found in DNA, is crucial to an organism's well-being. Studies in vitro indicate that after apurinic-apyrimidinic endonuclease cleaves immediately upstream of a baseless site, removal of the 5'-terminal sugar-phosphate residue is achieved by deoxyribophosphodiesterase activity, an enzyme-mediated beta-elimination reaction, or by endonucleolytic cleavage downstream of the baseless sugar. Synthesis and ligation complete repair. Eukaryotic RAD2 homolog 1 (RTH1) nuclease, by genetic and biochemical evidence, is involved in repair of modified DNA. Efficient endonucleolytic cleavage by RTH1 nuclease has been demonstrated for annealed primers that have unannealed 5'-tails. In vivo, such substrate structures could result from repair-related strand displacement synthesis. Using 5'-tailed substrates, we examined the ability of human RTH1 nuclease to efficiently remove 5'-terminal abasic residues. A series of upstream primers were used to increasingly displace an otherwise annealed downstream primer containing a 5'-terminal deoxyribose-5-phosphate. Until displacement of the first annealed nucleotide, substrates resisted cleavage. With further displacement, efficient cleavage occurred at the 3'-end of the tail. Therefore, in combination with strand displacement activity, RTH1 nucleases may serve as an important alternative to other pathways in repair of abasic sites in DNA.