Integrating text mining into the MGI biocuration workflow.

A major challenge for functional and comparative genomics resource development is the extraction of data from the biomedical literature. Although text mining for biological data is an active research field, few applications have been integrated into production literature curation systems such as those of the model organism databases (MODs). Not only are most available biological natural language (bioNLP) and information retrieval and extraction solutions difficult to adapt to existing MOD curation workflows, but many also have high error rates or are unable to process documents available in those formats preferred by scientific journals.In September 2008, Mouse Genome Informatics (MGI) at The Jackson Laboratory initiated a search for dictionary-based text mining tools that we could integrate into our biocuration workflow. MGI has rigorous document triage and annotation procedures designed to identify appropriate articles about mouse genetics and genome biology. We currently screen approximately 1000 journal articles a month for Gene Ontology terms, gene mapping, gene expression, phenotype data and other key biological information. Although we do not foresee that curation tasks will ever be fully automated, we are eager to implement named entity recognition (NER) tools for gene tagging that can help streamline our curation workflow and simplify gene indexing tasks within the MGI system. Gene indexing is an MGI-specific curation function that involves identifying which mouse genes are being studied in an article, then associating the appropriate gene symbols with the article reference number in the MGI database.Here, we discuss our search process, performance metrics and success criteria, and how we identified a short list of potential text mining tools for further evaluation. We provide an overview of our pilot projects with NCBO's Open Biomedical Annotator and Fraunhofer SCAI's ProMiner. In doing so, we prove the potential for the further incorporation of semi-automated processes into the curation of the biomedical literature.

The Mouse Genome Database (MGD): from genes to mice--a community resource for mouse biology.

The Mouse Genome Database (MGD) forms the core of the Mouse Genome Informatics (MGI) system (http://www.informatics.jax.org), a model organism database resource for the laboratory mouse. MGD provides essential integration of experimental knowledge for the mouse system with information annotated from both literature and online sources. MGD curates and presents consensus and experimental data representations of genotype (sequence) through phenotype information, including highly detailed reports about genes and gene products. Primary foci of integration are through representations of relationships among genes, sequences and phenotypes. MGD collaborates with other bioinformatics groups to curate a definitive set of information about the laboratory mouse and to build and implement the data and semantic standards that are essential for comparative genome analysis. Recent improvements in MGD discussed here include the enhancement of phenotype resources, the re-development of the International Mouse Strain Resource, IMSR, the update of mammalian orthology datasets and the electronic publication of classic books in mouse genetics.

The Mouse Genome Database (MGD): integrating biology with the genome.

The Mouse Genome Database (MGD) is one component of the Mouse Genome Informatics (MGI) system (http://www.informatics.jax.org), a community database resource for the laboratory mouse. MGD strives to provide a comprehensive knowledgebase about the mouse with experiments and data annotated from both literature and online sources. MGD curates and presents consensus and experimental data representations of genetic, genotype (sequence) and phenotype information including highly detailed reports about genes and gene products. Primary foci of integration are through representations of relationships between genes, sequences and phenotypes. MGD collaborates with other bioinformatics groups to curate a definitive set of information about the laboratory mouse and to build and implement the data and semantic standards that are essential for comparative genome analysis. Recent developments in MGD discussed here include an extensive integration of the mouse sequence data and substantial revisions in the presentation, query and visualization of sequence data.

Initiation of protein synthesis in mammalian cells with codons other than AUG and amino acids other than methionine.

Protein synthesis is initiated universally with the amino acid methionine. In Escherichia coli, studies with anticodon sequence mutants of the initiator methionine tRNA have shown that protein synthesis can be initiated with several other amino acids. In eukaryotic systems, however, a yeast initiator tRNA aminoacylated with isoleucine was found to be inactive in initiation in mammalian cell extracts. This finding raised the question of whether methionine is the only amino acid capable of initiation of protein synthesis in eukaryotes. In this work, we studied the activities, in initiation, of four different anticodon sequence mutants of human initiator tRNA in mammalian COS1 cells, using reporter genes carrying mutations in the initiation codon that are complementary to the tRNA anticodons. The mutant tRNAs used are aminoacylated with glutamine, methionine, and valine. Our results show that in the presence of the corresponding mutant initiator tRNAs, AGG and GUC can initiate protein synthesis in COS1 cells with methionine and valine, respectively. CAG initiates protein synthesis with glutamine but extremely poorly, whereas UAG could not be used to initiate protein synthesis with glutamine. We discuss the potential applications of the mutant initiator tRNA-dependent initiation of protein synthesis with codons other than AUG for studying the many interesting aspects of protein synthesis initiation in mammalian cells.

Initiator-elongator discrimination in vertebrate tRNAs for protein synthesis.

Initiator tRNAs are used exclusively for initiation of protein synthesis and not for the elongation step. We show, in vivo and in vitro, that the primary sequence feature that prevents the human initiator tRNA from acting in the elongation step is the nature of base pairs 50:64 and 51:63 in the TpsiC stem of the initiator tRNA. Various considerations suggest that this is due to sequence-dependent perturbation of the sugar phosphate backbone in the TpsiC stem of initiator tRNA, which most likely blocks binding of the elongation factor to the tRNA. Because the sequences of all vertebrate initiator tRNAs are identical, our findings with the human initiator tRNA are likely to be valid for all vertebrate systems. We have developed reporter systems that can be used to monitor, in mammalian cells, the activity in elongation of mutant human initiator tRNAs carrying anticodon sequence mutations from CAU to CCU (the C35 mutant) or to CUA (the U35A36 mutant). Combination of the anticodon sequence mutation with mutations in base pairs 50:64 and 51:63 yielded tRNAs that act as elongators in mammalian cells. Further mutation of the A1:U72 base pair, which is conserved in virtually all eukaryotic initiator tRNAs, to G1:C72 in the C35 mutant background yielded tRNAs that were even more active in elongation. In addition, in a rabbit reticulocyte in vitro protein-synthesizing system, a tRNA carrying the TpsiC stem and the A1:U72-to-G1:C72 mutations was almost as active in elongation as the elongator methionine tRNA. The combination of mutant initiator tRNA with the CCU anticodon and the reporter system developed here provides the first example of missense suppression in mammalian cells.

Amber suppression in mammalian cells dependent upon expression of an Escherichia coli aminoacyl-tRNA synthetase gene.

As an approach to inducible suppression of nonsense mutations in mammalian and in higher eukaryotic cells, we have analyzed the expression of an Escherichia coli glutamine-inserting amber suppressor tRNA gene in COS-1 and CV-1 monkey kidney cells. The tRNA gene used has the suppressor tRNA coding sequence flanked by sequences derived from a human initiator methionine tRNA gene and has two changes in the coding sequence. This tRNA gene is transcribed, and the transcript is processed to yield the mature tRNA in COS-1 and CV-1 cells. We show that the tRNA is not aminoacylated in COS-1 cells by any of the endogenous aminoacyl-tRNA synthetases and is therefore not functional as a suppressor. Concomitant expression of the E. coli glutaminyl-tRNA synthetase gene results in aminoacylation of the suppressor tRNA and its functioning as a suppressor. These results open up the possibility of attempts at regulated suppression of nonsense codons in mammalian cells by regulating expression of the E. coli glutaminyl-tRNA synthetase gene in an inducible, cell-type specific, or developmentally regulated manner.

The role of nucleotides conserved in eukaryotic initiator methionine tRNAs in initiation of protein synthesis.

Mutant human initiator tRNA genes carrying changes in each of the three features unique to eukaryotic initiator tRNAs have been constructed, and introduced into CV-1 monkey kidney cells using SV40 virus vectors. The mutant tRNA genes are expressed, and the mutant tRNAs can all be aminoacylated with both rabbit liver and Escherichia coli methionyl-tRNA synthetases. Based on aminoacylation levels, the tRNAs are expressed to 5-15-fold over the level of endogenous initiator tRNA. The activity of the mutant [35S]methionyl-tRNAs in initiation was studied in rabbit reticulocyte and wheat germ cell-free protein synthesis systems programmed with various mRNAs. Initiation is studied by using a mRNA that codes for a protein whose N-terminal methionine is stable and not removed by methionine aminopeptidase. Changing the A1:U72 base pair to a G1:C72 base pair greatly reduced activity of the tRNA in initiation. Changing the three consecutive G:C base pairs (G29G30G31:C39C40C41) in the anticodon stem to those found in elongator methionine tRNA also reduced initiation activity. Interestingly, changing the A54 and A60 residues in loop IV to T54 and U60 had less of an effect on activity. The tRNA with changes in all three conserved features had virtually no activity in initiation.

Introduction of an intervening sequence into a human serine suppressor tRNA gene: effects on gene expression in vitro and in vivo.

The 13 nucleotide Xenopus laevis tyrosine tRNA gene intervening sequence was into a human serine suppressor tRNA gene which lacked an intron, by site-directed mutagenesis. Analysis of the products of in vitro transcription in a HeLa cell extract indicates that the intervening sequence is accurately removed to generate a mature sized RNA identical to that obtained from an intron-less gene. Analysis of the transcripts obtained in vitro and in vivo shows that the U in the CUA anticodon sequence is partially modified to psi. Total TRNA isolated from cells infected with recombinant SV40 viruses carrying the mutant tRNA genes is active in suppression of UAG codons in a reticulocyte cell-free system. Cotransfection of COS cells with the mutant tRNA genes and a mutant chloramphenicol acetyltransferase gene containing the termination codon UAG demonstrated that the tRNA functions as a UAG suppressor in vivo. Analysis of 32P-labeled RNA obtained from infected cells showed, however, that cells infected with the intron-containing gene accumulate less mature tRNA than cells infected with the intron-less tRNA genes.

Attempted expression of a human initiator tRNA gene in Saccharomyces cerevisiae.

In attempts to overproduce the wild type and, eventually, mutant human initiator methionine tRNAs for use in structure-function relationship studies, we have investigated the expression of the wild type human initiator tRNA gene in the yeast Saccharomyces cerevisiae, both in vitro and in vivo. We find that the yeast extract, while capable of accurately transcribing several yeast tRNA genes, does not transcribe the human initiator tRNA gene. In addition, when the human initiator tRNA gene is introduced into yeast as part of a 2 mu vector, no expression of the human tRNA gene was detected. A yeast alanine tRNA gene similarly introduced into yeast is expressed efficiently. The block in expression of the human tRNA gene is at the level of transcription and not processing. The yeast cell-free extract can accurately process precursors of the same human initiator tRNA made in a HeLa cell-free extract. Surprisingly, although the human tRNA gene has essentially the same intragenic control elements as the yeast initiator tRNA gene, the human tRNA gene competes extremely poorly for transcription factors in yeast extracts. In the course of screening a yeast DNA bank for initiator tRNA clones we have isolated and sequenced three yeast tRNA genes corresponding to glycine, alanine, and aspartic acid tRNAs. The sequence of glycine tRNA gene differs from the published tRNA sequence in having an additional nucleotide in the variable loop. The alanine tRNA gene codes for a new tRNA. All three genes are transcriptionally active in yeast extracts.

Site-specific mutagenesis on a human initiator methionine tRNA gene within a sequence conserved in all eukaryotic initiator tRNAs and studies of its effects on in vitro transcription.

We have used oligonucleotide-directed site-specific mutagenesis to generate a mutant human initiator tRNA gene in which the sequence GATCG corresponding to the universal GAUCG found in loop IV of eukaryotic cytoplasmic initiator tRNAs is changed to GTTCG. The mutant tRNA gene has been characterized by restriction mapping and by DNA sequencing. We show that this mutation has no effect on in vitro transcription of the tRNA gene in HeLa cell extracts. Transcripts derived from both the wild type (A54) and the mutant (T54) initiator tRNA genes are processed in vitro to produce mature tRNAs with the correct 5'-and 3'-termini. Fingerprint analysis of in vitro transcripts shows that the mutant RNA has the expected nucleotide change. Modified nucleotide composition analyses on the RNAs show that when A54 is changed to U54, the neighboring nucleotide U55 is modified quantitatively to psi 55 in the in vitro extracts; U54 itself is partially modified to ribo-T. Other modified bases identified in the in vitro transcripts include m1G, m2G, m7G, D, and m5C.

Expression in vivo of a mutant human initiator tRNA gene in mammalian cells using a simian virus 40 vector.

We have cloned both the wild type (A54) and mutant (T54) human initiator genes described in the preceding paper (Drabkin, H. J., and RajBhandary, U. L. (1985) J. Biol. Chem. 260, 5580-5587) as 141-base pair fragments into the SV40-pBR322 vector pSV1GT3. These vectors were subsequently used to transfect monkey kidney CV-1 cells to obtain recombinant virus stocks carrying each of the initiator tRNA genes. Following infection of CV-1 cells by the recombinant virus stocks, both the wild type and mutant tRNAs are produced in large quantities during a 48-h period. Fingerprint analysis of 32P-labeled tRNAs was used to characterize the tRNAs made in vivo and to show that the sequence AUCG in loop IV of the wild type tRNA is replaced by T psi CG in the mutant tRNA. Modified nucleotide composition analysis of the [32P]tRNAs overproduced in vivo shows that they contain all the modified nucleotides found in human placenta initiator tRNA. Both wild type and mutant initiator tRNAs can be aminoacylated by either mammalian or Escherichia coli methionyl-tRNA synthetases. Furthermore, the mutant tRNA can be easily separated from the endogenous monkey initiator tRNA by RPC-5 column chromatography.