Synergy between medical informatics and bioinformatics: facilitating genomic medicine for future health care.

In this paper, we review the results of BIOINFOMED, a study funded by the European Commission (EC) with the purpose to analyse the different issues and challenges in the area where Medical Informatics and Bioinformatics meet. Traditionally, Medical Informatics has been focused on the intersection between computer science and clinical medicine, whereas Bioinformatics have been predominantly centered on the intersection between computer science and biological research. Although researchers from both areas have occasionally collaborated, their training, objectives and interests have been quite different. The results of the Human Genome and related projects have attracted the interest of many professionals, and introduced new challenges that will transform biomedical research and health care. A characteristic of the 'post genomic' era will be to correlate essential genotypic information with expressed phenotypic information. In this context, Biomedical Informatics (BMI) has emerged to describe the technology that brings both disciplines (BI and MI) together to support genomic medicine. In recognition of the dynamic nature of BMI, institutions such as the EC have launched several initiatives in support of a research agenda, including the BIOINFOMED study.

The Gcn5 bromodomain co-ordinates nucleosome remodelling.

The access of transcription factors to eukaryotic promoters often requires modification of their chromatin structure, which is accomplished by the action of two general classes of multiprotein complexes. One class contains histone acetyltransferases (HATs), such as Gcn5 in the SAGA complex, which acetylate nucleosomal histones. The second class contains ATPases, such as Swi2 in the Swi/Snf complex, which provide the energy for nucleosome remodelling. In several promoters these two complexes cooperate but their functional linkage is unknown. A protein module that is present in all nuclear HATs, the bromodomain, could provide such a link. The recently reported in vitro binding of a HAT bromodomain with acetylated lysines within H3 and H4 amino-terminal peptides indicates that this interaction may constitute a targeting step for events that follow histone acetylation. Here we use a suitable promoter to show that bromodomain residues essential for acetyl-lysine binding are not required in vivo for Gcn5-mediated histone acetylation but are fundamental for the subsequent Swi2-dependent nucleosome remodelling and consequent transcriptional activation. We show that the Gcn5 bromodomain stabilizes the Swi/Snf complex on this promoter.

Acquisition of a potential marker for insect transformation: isolation of a novel alcohol dehydrogenase gene from Bactrocera oleae by functional complementation in yeast.

The alcohol dehydrogenase genes make up one of the best studied gene families in Drosophila, both in terms of expression and evolution. Moreover, alcohol dehydrogenase genes constitute potential versatile markers in insect transformation experiments. However, due to their rapid evolution, these genes cannot be cloned from other insect genera by DNA hybridization or PCR-based strategies. We have therefore explored an alternative strategy: cloning by functional complementation of appropriate yeast mutants. Here we report that two alcohol dehydrogenase genes from the medfly Ceratitis capitata can functionally replace the yeast enzymes, even though the medfly and yeast genes have evolved independently, acquiring their enzymatic function convergently. Using this method, we have cloned an alcohol dehydrogenase gene from the olive pest Bactrocera oleae. We conclude that functional complementation in yeast can be used to clone alcohol dehydrogenase genes that are unrelated in sequence to those of yeast, thus providing a powerful tool for isolation of dominant insect transformation marker genes.

The Gcn5.Ada complex potentiates the histone acetyltransferase activity of Gcn5.

The Gcn5 histone acetyltransferase (HAT) is part of a large multimeric complex that is required for transcriptional activation in yeast. This complex can acetylate in vitro and in a Gcn5-dependent manner both nucleosomal and free core histones. For this reason it is believed that part of the function of the Gcn5.Ada complex is chromatin remodeling effected by histone acetylation. The roles of the other subunits of this complex are not yet known. We have generated mutated Gcn5 proteins with severely attenuated in vitro HAT activities. Despite their apparent loss in HAT activity, these GCN5 derivatives complemented all the defects of a gcn5 strain. We have shown that when these mutated proteins were produced in yeast cells in the absence of another component of the complex, Ada2, their activity was still compromised. By contrast, when produced in the wild type context, they were partially capable of acetylating free histones and were even more active when nucleosomal arrays were used as substrates. Kinetic enzymatic analyses showed that the rate of catalysis by Gcn5 was enhanced when the mutated proteins were produced in yeast in the presence of Ada2. Because Ada2 is required for the assembly of Gcn5, we conclude that one role for components of the Gcn5.Ada complex is the potentiation of its HAT activity.

The DNA target sequence influences the dependence of the yeast transcriptional activator Gcn4 on co-factors.

The yeast transcriptional activator Gcn4 requires the Ada2/Gcn5/Ada3 co-activator complex to exert part of its activation potential. Here we show that the sequence of the DNA target modulates the function of Gcn4 by modifying this requirement. Promoter configurations were generated that rendered Gcn4-induced transcription either completely dependent or completely independent of the Ada2/Gcn5/Ada3 complex. The topological constraints imposed by these configurations suggest that Gcn4 makes multiple contacts with the basic transcription machinery that are subject to modification by the incident DNA target. We propose that these modifications further determine the direction on the chromosome in which an otherwise symmetric, dimeric transcription factor will activate.

Two sporulation-specific chitin deacetylase-encoding genes are required for the ascospore wall rigidity of Saccharomyces cerevisiae.

The formation of the ascospore wall of Saccharomyces cerevisiae requires the coordinate activity of enzymes involved in the biosynthesis of its components such as chitosan, the deacetylated form of chitin. We have cloned the CDA1 and CDA2 genes which together account for the total chitin deacetylase activity of the organism. We have shown that expression of these genes is restricted to a distinct time period during sporulation. The two genes are functionally redundant, each contributing equally to the total chitin deacetylase activity. Diploids disrupted for both genes sporulate as efficiently as wild type cells, and the resulting mutant spores are viable under standard laboratory conditions. However, they fail to emit the natural fluorescence of yeast spores imparted by the dityrosine residues of the outermost ascospore wall layer. Moreover, mutant spores are relatively sensitive to hydrolytic enzymes, ether, and heat shock, a fact that underscores the importance of the CDA genes for the proper formation of the ascospore wall.

Gene overexpression reveals alternative mechanisms that induce GCN4 mRNA translation.

The Saccharomyces cerevisiae GCN4 gene which encodes the transcription activator Gcn4, is under translational regulation. Derepression of GCN4 mRNA translation is mediated by the Gcn2 protein kinase which phosphorylates the alpha subunit of eIF-2, upon amino-acid starvation. Here, we report that overexpression of certain Saccharomyces cerevisiae genes generates intracellular conditions that alleviate the requirement for a functional Gcn2 kinase to induce GCN4 mRNA translation. Our findings, combined with the fact that Gcn2 kinase is dispensable during the initiation phase of the cellular response to amino-acid limitation, provide the grounds to further elucidate the mechanisms underlying the physiology of this homeostatic response.

Genetic evidence for functional specificity of the yeast GCN2 kinase.

In yeast the GCN2 kinase mediates translational control of GCN4 by phosphorylating the alpha subunit of eIF-2 in response to extracellular amino acid limitation. Although phosphorylation of eIF-2 alpha has been shown to inhibit global protein synthesis, amino acid starvation results in a specific activation effect on GCN4 mRNA translation. Under the same conditions, translation of other mRNAs appears only slightly affected. The mechanism responsible for the observed selectivity of the GCN2 kinase is not clear. Here, we present genetic evidence that suggests that locally restricted action of the GCN2 kinase facilitates GCN4-specific translational regulation.

A transient GCN4 mRNA destabilization follows GCN4 translational derepression.

Studies based on experimental strategies that utilized either inhibitors or structural alterations point to the existence of an inverse relationship between translation and stability of a given mRNA. In this study we have investigated the potential link between translation and stability of the yeast GCN4 mRNA whose translational rates change with respect to amino acid availability. We observed that under conditions favoring its translation, the steady state levels of the GCN4 mRNA were decreased, but this was not due to a measurable alternation in its decay rate. We have demonstrated that an extensive destabilization of this message is intimately coupled with its increased access to heavy polysomes, which occurs transiently in the process of translational derepression. This transient change in the stability is what readjusts the steady state levels of the GCN4 mRNA. This study demonstrates in vivo the existence of a mechanism of mRNA degradation that is coupled with the process of translation.

Transcriptional interference caused by GCN4 overexpression reveals multiple interactions mediating transcriptional activation.

Overproduction of Gcn4p in yeast cells resulted in the inhibition of transcription from promoters controlled by the GAL4 or dA:dT elements. We have demonstrated that this effect is mediated through the activation domain of Gcn4p and that the function of the transcriptional activator at the affected promoter is impaired. The inhibitory effect of Gcn4p and that the function of the transcriptional activator at the affected promoter is impaired. The inhibitory effect of Gcn4p on these promoters persisted in yeast strains disrupted for the ADA2 and/or GCN5 genes, whose products are required for only part of the transcriptional activation capacity of Gcn4p and other activators, but was alleviated by overexpression of gamma TFIIB. These results support the hypothesis that general transcription factors become unavailable at certain promoters when an activator is overexpressed and strongly imply the existence of an Ada2p/Gcn5p-independent pathway of communication between acidic activators and the basic transcription machinery. In a genetic screen, we have isolated a mutation which neutralises the squelching effects of Gcn4p. This AFR1-1 (activation function reduced) mutation is dominant, it affects the transcriptional activation properties of a number of activators and results in lethality when combined with a gcn5 disruption. Our results suggest that the AFR1 gene product is involved in the mediation of transcriptional activation.

Genetic evidence for the interaction of the yeast transcriptional co-activator proteins GCN5 and ADA2.

The GCN5 and ADA2 proteins are required for the activation function of a number of transcriptional activators in the yeast Saccharomyces cerevisiae. By using appropriate LexA fusion proteins we demonstrated that both proteins are required for part of the function of the GCN4, GAL4 and the VP16 transcriptional activation domains. Analysis of a gcn5 ada2 double disruption mutant did not reveal any additive effects, suggesting that the two proteins act in the same pathway. The GCN5 and ADA2 proteins can each activate transcription when directed to the promoter region of a reporter gene, but only in the presence of a wild-type ADA2 or GCN5 gene, respectively. The activation capacity is enhanced when the corresponding endogenous gene copy is disrupted. Taken together, these genetic data suggest that the two proteins interact and define one complex that mediates transcriptional activation. The function of this complex requires the bromodomain region of the GCN5 protein.

Yap1p, a yeast transcriptional activator that mediates multidrug resistance, regulates the metabolic stress response.

Overexpression of the YAP1 transcriptional activator renders yeast cells resistant to multiple metabolic inhibitors. In an effort to identify other gene products required for this phenotype we have isolated genomic mutations which neutralize this effect. One such mutation was further characterized and the affected gene was shown to be identical to TPS2 which encodes trehalose phosphate phosphatase, an enzyme catalysing the second step in trehalose biosynthesis. We have analysed the transcriptional regulation of the TPS2 gene and have shown that its transcription is induced by a variety of stressful conditions caused by metabolic inhibitors, osmotic shock and heat shock. This transcriptional activation is mediated by multiple stress promoter elements (C4T) and requires the function of Yap1p as well as reduced activity of the cAMP-regulated protein kinase. Using an appropriate reporter gene we have shown that Yap1p is generally required for transcriptional regulation through the C4T stress element. These results show that the YAP1 protein has a pivotal role in the metabolic stress response and the acquisition of stress tolerance.

Genetic and biochemical evidence for yeast GCN2 protein kinase polymerization.

The GCN2 (general control kinase 2) protein is an eIF2-alpha (eukaryotic initiation factor alpha) kinase which mediates translational derepression of the yeast general control transcriptional activator, GCN4, upon amino-acid starvation. We isolated and characterized GCN2 mutations differentially affecting GCN2 function. Mutations mapping in, or close to, the ATP-binding site of the kinase moiety result in constitutively activated GCN2 molecules. A C-terminal regulatory mutation dramatically affects translation initiation rates resulting in pleiotropic phenotypes. The effect of mutations in both regions were found to depend on eIF2-alpha phosphorylation. We have demonstrated that GCN2 mutants have altered autophosphorylation activities in vitro, depending on the presence or absence of a wild-type GCN2 gene and that GCN2 elutes in gel-filtration chromatography fractions with high apparent molecular mass. Both these genetic and biochemical findings suggest that GCN2 functioning might involve polymerization to form dimers or tetramers.

The primary structure of a fungal chitin deacetylase reveals the function for two bacterial gene products.

Chitin deacetylase (EC 3.5.1.41) hydrolyzes the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin. A cDNA to the Mucor rouxii mRNA encoding chitin deacetylase was isolated, characterized, and sequenced. Protein sequence comparisons revealed significant similarities of the fungal chitin deacetylase to rhizobial nodB proteins and to an uncharacterized protein encoded by a Bacillus stearothermophilus open reading frame. These data suggest the functional homology of these evolutionarily distant proteins. NodB is a chitooligosaccharide deacetylase essential for the biosynthesis of the bacterial nodulation signals, termed Nod factors. The observed similarity of chitin deacetylase to the B. stearothermophilus gene product suggests that this gene encodes a polysaccharide deacetylase.

Two distinct yeast transcriptional activators require the function of the GCN5 protein to promote normal levels of transcription.

When yeast cells are grown under conditions of amino acid limitation, transcription of amino acid biosynthetic genes is increased through the action of the GCN4 transcriptional regulator. gcn5 mutant strains exhibit poor growth under such conditions. We have established that GCN4 requires the function of GCN5 in order to promote normal levels of transcriptional activation. In addition, we have shown that GCN5 is also required for the activity of the HAP2--HAP3--HAP4 transcriptional activation complex, which mediates the transcription of genes involved in respiratory functions. Thus, GCN5 is a new member of the recently revealed general class of transcriptional regulators that collaborate with certain specific DNA binding activators to promote high levels of transcription. We have cloned and sequenced the GCN5 gene. The deduced GCN5 protein contains a region conserved in other yeast, Drosophila and human proteins, all members of this new class of transcriptional activators.

Molecular cloning of an essential yeast gene encoding a proteasomal subunit.

We present the cloning and sequence of a Saccharomyces cerevisiae gene, PUP2, which encodes for a proteasomal subunit. The PUP2 protein is similar to other proteasomal components from yeast, as well as from Drosophila and rat. Although not-properly-folded proteins have been implicated to constitute substrates of proteasomes, we show that the accumulation of such proteins does not induce expression of the PUP2 gene. Finally, gene disruption experiments demonstrate that PUP2 belongs to the class of yeast proteasomal subunits that are essential for cell viability.

Translational activation of GCN4 mRNA in a cell-free system is triggered by uncharged tRNAs.

Translation of GCN4 mRNA is activated when yeast cells are grown under conditions of amino acid limitation. In this study, we established the conditions through which translation of the GCN4 mRNA could be activated in a homologous in vitro system. This activation paralleled the in vivo situation: it required the small open reading frames located in the 5' untranslated region of the GCN4 mRNA, and it was coupled with reduced rates of 43S preinitiation complex formation. Translational derepression in vitro was triggered by uncharged tRNA molecules, demonstrating that deacylated tRNAs are more proximal signals for translational activation of the GCN4 mRNA.

Coupling of GCN4 mRNA translational activation with decreased rates of polypeptide chain initiation.

The steady-state translational activation of the GCN4 mRNA is based upon an increase in the rate of ribosome initiation at the protein coding AUG following translation of the 5' most proximal open reading frame located in its untranslated region. Such an increase is effected when the cellular amount of the GCN2 protein kinase is increased or when the function of the GCD1 gene product is defective. Here, we report conditions that result in a dramatic transient increase in the rate of GCN4 protein synthesis, which also requires the prior translation of the 5' most proximal open reading frame but is independent of the GCN2 protein. This activation of GCN4 mRNA translation coincides with a decrease in the rate of total cellular protein synthesis. We also observed low rates of protein synthesis in the gcd1 strain and in strains that overexpress the GCN2 protein kinase. The process in protein synthesis that is affected is formation of 43S preinitiation complexes. These results reveal the existence of a coupling between this process in translational initiation and the mechanism that activates translation of GCN4 mRNA.

Evidence that the GCN2 protein kinase regulates reinitiation by yeast ribosomes.

The yeast gene GCN4 produces an mRNA that has a long 5' 'untranslated' region containing four small open reading frames (ORFs) preceding the protein coding frame. This configuration suppresses the rate by which GCN4 protein is synthesized. However, translational derepression of the GCN4 mRNA occurs when yeast cells are grown under conditions of amino acid limitation. Such translational derepression requires the GCN2 protein kinase and the presence of the 5' most proximal ORF. In this study we show that a functional coupling between the translation of the first ORF and the amount of the GCN2 protein is responsible for the translational derepression of the GCN4 mRNA. Our evidence suggests that this coupling involves an increase in the ability of 40S ribosomal subunits that have translated the first frame to resume scanning and reinitiate translation at a downstream AUG independently of the base sequence in the intervening region.