Just Justesen

Full genome gene expression analysis of the heat stress response in Drosophila melanogaster

Abstract The availability of full genome sequences has allowed the construction of microarrays, with which screening of the full genome for changes in gene expression is possible. This method can provide a wealth of information about biology at the level of gene expression and is a powerful method to identify genes and pathways involved in various processes. In this study, we report a detailed analysis of the full heat stress response in Drosophila melanogaster females, using whole genome gene expression arrays (Affymetrix Inc, Santa Clara, CA, USA). The study focuses on up- as well as downregulation of genes from just before and at 8 time points after an application of short heat hardening (36°C for 1 hour). The expression changes were followed up to 64 hours after the heat stress, using 4 biological replicates. This study describes in detail the dramatic change in gene expression over time induced by a short-term heat treatment. We found both known stress responding genes and new candidate genes, and processes to be involved in the stress response. We identified 3 main groups of stress responsive genes that were early–upregulated, early– downregulated, and late–upregulated, respectively, among 1222 differentially expressed genes in the data set. Comparisons with stress sensitive genes identified by studies of responses to other types of stress allow the discussion of heat-specific and general stress responses in Drosophila. Several unexpected features were revealed by this analysis, which suggests that novel pathways and mechanisms are involved in the responses to heat stress and to stress in general. The majority of stress responsive genes identified in this and other studies were downregulated, and the degree of overlap among downregulated genes was relatively high, whereas genes responding by upregulation to heat and other stress factors were more specific to the stress applied or to the conditions of the particular study. As an expected exception, heat shock genes were generally found to be upregulated by stress in general.

Gene structure and function of the 2-5-oligoadenylate synthetase family

Abstract. 2′-5′-Oligoadenylate synthetase was among the first interferon-induced antiviral enzymes to be discovered. This family of enzymes plays an important role in the mechanisms of action of interferon antiviral activity, but is also involved in other cellular processes such as apoptosis and growth control. We have reviewed the function and genomic structure of this class of at least nine proteins. By studying the recently available data in the human genome database and the human Expressed Sequence Tag database, we have been able to build a comprehensive picture of the 2′-5′-oligoadenylate synthetase gene family and its precise location on chromosome 12. Chromosomal localization as well as the intron/exon structure of all four genes has been established and an overview of the splice variant forms of the 2′-5′-oligoadenylate synthetases arising from expression of the four genes is presented. Alignments of the human 2′-5′-oligoadenylate synthetase sequences with non-human 2′-5′-oligoadenylate synthetase sequences suggest that the exon structure and several amino acid sequence motifs have been conserved during evolution.

Diadenosine oligophosphates (ApnA), a novel class of signalling molecules?

The diadenosine oligophosphates (Ap n A) were discovered in the mid‐sixties in the course of studies on aminoacyl‐tRNA synthetases (aaRS). Now, more than 30 years later, about 300 papers have been published around these substances in attempt to decipher their role in cells. Recently, Ap n A have emerged as intracellular and extracellular signalling molecules implicated in the maintenance and regulation of vital cellular functions and become considered as second messengers. Great variety of physiological and pathological effects in mammalian cells was found to be associated with alterations of Ap n A levels (n from 2 to 6) and Ap3A/Ap4A ratio. Cell differentiation and apoptosis have substantial and opposite effects on Ap3A/Ap4A ratio in cultured cells. A human Ap3A hydrolase, Fhit, appeared to be involved in protection of cells against tumourigenesis. Ap3A is synthesised by mammalian u synthetase (TrpRS) which in contrast to most other aaRS is unable to synthesise Ap4A and is an interferon‐inducible protein. Moreover, Ap3A appeared to be a preferred substrate for 2‐5A synthetase, also interferon‐inducible, priming the synthesis of 2′ adenylated derivatives of Ap3A, which in turn may serve as substrates of Fhit. Tumour suppressor activity of Fhit is assumed to be associated with involvement of the Fhit·Ap3A complex in cytokine signalling pathway(s) controlling cell proliferation. The Ap n A family is potentially a novel class of signal‐transducing molecules whose functions are yet to be determined.

Small ISGs coming forward.

Interferons (IFNs) were first characterized as antiviral proteins. Since then, IFNs have proved to be involved in malignant, angiogenic, inflammatory, immune, and fibrous diseases and, thus, possess a broad spectrum of pathophysiologic properties. IFNs activate a cascade of intracellular signaling pathways leading to upregulation of more than 1000 IFN-stimulated genes (ISGs) within the cell. The function of some of the IFN-induced proteins is well described, whereas that of many others remain poorly characterized. This review focuses on three families of small intracellular and intrinsically nonsecreted proteins (10-20 kDa) separated into groups according to their amino acid sequence similarity: the ISG12 group (6-16, ISG12, and ISG12-S), the 1-8 group (9-27/Leu13, 1-8U, and 1-8D), and the ISG15 group (ISG15/UCRP). These IFN-induced genes are abundantly and widely expressed and mainly induced by type I IFN. ISG15 is very well described and is a member of the ubiquitin-like group of proteins. 9-27/Leu-13 associates with CD81/TAPA-1 and plays a role in B cell development. The functions of 1-8U, 1-8D, 6-16, ISG12, and ISG12-S proteins are unknown at present.

Invariant amino acids essential for decoding function of polypeptide release factor eRF1

In eukaryotic ribosome, the N domain of polypeptide release factor eRF1 is involved in decoding stop signals in mRNAs. However, structure of the decoding site remains obscure. Here, we specifically altered the stop codon recognition pattern of human eRF1 by point mutagenesis of the invariant Glu55 and Tyr125 residues in the N domain. The 3D structure of generated eRF1 mutants was not destabilized as demonstrated by calorimetric measurements and calculated free energy perturbations. In mutants, the UAG response was most profoundly and selectively affected. Surprisingly, Glu55Arg mutant completely retained its release activity. Substitution of the aromatic ring in position 125 reduced response toward all stop codons. This result demonstrates the critical importance of Tyr125 for maintenance of the intact structure of the eRF1 decoding site. The results also suggest that Tyr125 is implicated in recognition of the 3d stop codon position and probably forms an H-bond with Glu55. The data point to a pivotal role played by the YxCxxxF motif (positions 125–131) in purine discrimination of the stop codons. We speculate that eRF1 decoding site is formed by a 3D network of amino acids side chains.

Mapping and identification of interferon gamma-regulated HeLa cell proteins separated by immobilized pH gradient two-dimensional gel electrophoresis.

Interferon gamma (IFN‐γ) is a potent immunomodulatory lymphokine, secreted by activated T‐lymphocytes and NK‐cells during the cellular immune response. Actions of IFN‐γ are mediated through binding to the IFN‐γ‐receptor, present on most cells, and the subsequent activation of a great magnitude of IFN‐γ responsive genes has been reported previously. Our goal is to identify and map IFN‐γ‐regulated HeLa cell proteins to the two‐dimensional polyacrylamide gel electrophoresis with the immobilized pH gradient (IPG) two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) system. A semiconfluent layer of HeLa cells was grown on tissue culture plates, and changes in protein expression due to 100 U/mL IFN‐γ were investigated at different periods after treatment, using pulse labeling with [35S]methionine/cysteine in combination with 2‐D PAGE (IPG). The identity of eight protein spots was elucidated by matrix‐assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS), and several variants of the IFN‐γ‐inducible tryptophanyl‐tRNA synthetase (hWRS) were detected by immunoblotting.

Gene structure of the murine 2'-5'-oligoadenylate synthetase family.

Abstract: The 2′-5′-oligoadenylate synthetases (OASs) are members of a family of interferon-induced proteins playing an important role in the antiviral effect of interferons as well as being involved in apoptosis and control of cellular growth. Based on sequence data from the murine BAC clone (RP23-39M18), and a number of EST and IMAGE clones and the Celera Mouse database, we identified twelve Oas genes in the mouse genome, all localized to the chromosome 5F region. In contrast to the single OAS1 gene found in humans, we identified eight closely linked Oas1 genes in the murine genome, together with the genes of Oas2 and Oas3. Compared to the single OASL gene found in humans, two genes of OAS-like proteins, Oasl1 and Oasl2, were identified. All the putative genes seem to be transcribed.¶The exon/intron structures of the murine Oas genes were found to be identical to those of the human genes.

Evolution of the 2′-5′-Oligoadenylate Synthetase Family in Eukaryotes and Bacteria

The 2′-5′-oligoadenylate synthetase (OAS) belongs to a nucleotidyl transferase family that includes poly(A) polymerases and CCA-adding enzymes. In mammals and birds, the OAS functions in the interferon system but it is also present in an active form in sponges, which are devoid of the interferon system. In view of these observations, we have pursued the idea that OAS genes could be present in other metazoans and in unicellular organisms as well. We have identified a number of OAS1 genes in annelids, mollusks, a cnidarian, chordates, and unicellular eukaryotes and also found a family of proteins in bacteria that contains the five OAS-specific motifs. This indicates a specific relationship to OAS. The wide distribution of the OAS genes has made it possible to suggest how the OAS1 gene could have evolved from a common ancestor to choanoflagellates and metazoans. Furthermore, we suggest that the OASL may have evolved from an ancestor of cartilaginous fishes, and that the OAS2 and the OAS3 genes evolved from a mammalian ancestor. OAS proteins function in the interferon system in mammals. This system is only found in jawed vertebrates. We therefore suggest that the original function of OAS may differ from its function in the interferon system, and that this original function of OAS is preserved even in OAS genes that code for proteins, which do not have 2′-5′-oligoadenylate synthetase activity.

Phototransduction genes are up-regulated in a global gene expression study of Drosophila melanogaster selected for heat resistance

Abstract The genetic architecture underlying heat resistance remains partly unclear despite the well-documented involvement of heat shock proteins (Hsps). It was previously shown that factors besides Hsps are likely to play an important role for heat resistance. In this study, gene expression arrays were used to make replicate measurements of gene expression before and up to 64 hours after a mild heat stress treatment, in flies selected for heat resistance and unselected control flies, to identify genes differentially expressed in heat resistance–selected flies. We found 108 genes up-regulated and 10 down-regulated using the Affymetrix gene expression platform. Among the up-regulated genes, a substantial number are involved in the phototransduction process. Another group of genes up-regulated in selected flies is characterized by also responding to heat shock treatment several hours after peak induction of known Hsps revert to nonstress levels. These findings suggest phototransduction genes to be critically involved in heat resistance, and support a role for components of the phototransduction process in stress-sensing mechanisms. In addition, the results suggest yet-uncharacterized genes responding to heat stress several hours after treatment to be involved in heat stress resistance. These findings mark an important increase in the understanding of heat resistance.

Refocusing of B-cell responses following a single amino acid substitution in an antigen

Intranasal immunization of BALB/c strain mice was carried out using baculovirus‐derived human chorionic gonadotrophin (hCG) β‐chain, together with Escherichia coli heat‐labile enterotoxin. Gonadotrophin‐reactive immunoglobulin A (IgA) was induced in a remote mucosal site, the lung, in addition to a systemic IgG response. The extensive sequence homology with luteinizing hormone (LH) results in the production of LH cross‐reactive antibodies when holo‐hCG is used as an immunogen. In contrast to wild‐type hCGβ, a mutated hCGβ‐chain containing an arginine to glutamic acid substitution at position 68 did not induce the production of antibodies which cross‐react with LH. Furthermore, the epitopes utilized in the B‐cell response to the mutated hCGβ shifted away from the immunodominant region of the parent wild‐type molecule towards epitopes within the normally weakly immunogenic C terminus. This shift in epitope usage was also seen following intramuscular immunization of rabbits. Thus, a single amino acid change, which does not disrupt the overall structure of the molecule, refocuses the immune response away from a disadvantageous cross‐reactive epitope region and towards a normally weakly immunogenic but antigen‐unique area. Similar mutational strategies for epitope‐refocusing may be applicable to other vaccine candidate molecules.