Differential Display Methodology Research Articles

AraC transcriptional regulator, aspartate semialdehyde dehydrogenase and acyltransferase: Three putative genes in phenol catabolic pathway of Acinetobacter sp. Strain DF4

The objective of this study was to identify genes associated with the biodegradation of phenol by Acinetobacter sp. strain DF4 through the use of differential display (DD) methodology. The bacteria were grown in YEPG medium, and total RNA was extracted and analyzed using labeled primers to detect gene expression differences. Three distinctively expressed cDNA bands (ph1, ph2, and ph3) were identified, cloned, and sequenced. DNA analysis involved searching for open reading frames (ORFs), verifying results with the NCBI database, predicting promoter regions, and constructing phylogenetic trees using bioinformatics tools. The ph1 gene displayed a 97% identity with the AraC transcriptional regulator, suggesting its potential role in regulating the ortho-catabolic pathway of phenol. The ph2 gene showed a 98% identity with aspartate semialdehyde dehydrogenase, which is involved in phenol degradation. The ph3 gene had a 93% identity with acetyltransferase. Essential transcription factors, such as TATA, GTGTGT, CACA, and CTTTT, were detected, and the three genes promoter regions were predicted. This study successfully identified functional genes involved in the metabolism of cyclic chemicals, particularly phenol, using the DD technique. These findings provide insights into the biodegradation pathways of phenol by Acinetobacter sp. Strain DF4 and may contribute to the development of more efficient bioremediation strategies for phenol-contaminated environments.

Identification of Differentially Expressed Genes in BALB/c Mouse Liver upon Primary Infection with DENV1 and Sequential Heterologous Infection with DENV2.

Dengue virus (DENV) results in 100 million cases of infections and 22,000 deaths per year. Liver involvement, thrombocytopenia, haemorrhage and plasma leakage are characteristic manifestations of severe forms of DENV infection. However, the molecular pathways of DENV infection have not been comprehensively studied compared to the host immunological responses. We performed an in vivo study using the BALB/c mouse model with a modified mRNA differential display methodology (GeneFishingTM) using the annealing control primer (ACP) system to capture differentially expressed genes (DEGs) in mice liver upon primary infection with DENV1 and sequential heterologous infection with DENV2. Secondary heterologous infection with DENV2 was carried out at Immunoglobulin IgM and IgG peaks following the primary DENV1 infection with the hope of determining any potential effect antibodies IgM and IgG may have on sequential heterologous infection. 30 DEGs were identified and sequenced across all three treatment groups and they belong to a variety of important pathways such as apoptosis, innate immune response, inflammatory response, metabolic processes and oxidative stress. Analysis of differentially expressed genes in response to viral infection offers valuable knowledge about the dynamic and complex association between host cell and the virus. Furthermore, some DEGs identified support DENV induced liver damage.

Molecular analysis of the adaptive response in Salmonella Typhimurium after starvation in salty conditions.

The pathogenic bacterium Salmonella enterica serovar Typhimurium elicits a variety of genetic programs to adapt to stress conditions encountered within hostile environments such as host phagocytes and preserved food. In this work, differential display (DD) methodology was used to investigate the effect of one month starvation in a salty microcosm (0.5 M NaCl) on transcript profiling in a Salmonella Typhimurium LT2 strain. cDNA fragments resulting from differentially expressed mRNA were eluted from the gel, re-amplified, cloned, and then sequenced. A total of 21 differentially expressed bands were detected by DD reverse transcription-polymerase chain reaction (RT-PCR). However, only 12 of them were successfully identified as upregulated genes in stressed cells. Based on the sequencing data and BLAST analysis, these genes were sopA, ssaD, yhhK, gmK, cspC, uspA, ompR, phoP, stcC, fimA, acrA,and yehZ. As a confirmation of the differential expression, RT-PCR was carried out using a set of specific primers. Remarkably, the expression levels of these genes were significantly increased in starved bacteria compared to standard laboratory conditions. Our results indicate that the starvation of Salmonella Typhimurium over one month in a salty microcosm changes the expression of stress proteins, response regulator in a two-component system, outer membrane proteins, effector proteins translocated by Salmonella pathogenicity island SPI1 and SPI2 type III secretion systems (TTSS), several metabolic enzymes, efflux pumps, and transport proteins. This suggests that the expression of the identified genes is important for the response of this pathogen to starvation in salt.

Cover Picture: Proteomics 22'09

AbstractShigellosis, dysentery — by whatever name — Don't kiss the chimpsWith a 10–15% mortality rate when untreated in young children and immunedeficient patients, over one million deaths per year are attributable to this primate/human‐only disease. Furthermore, the Shigella spp. have developed resistance to b‐lactam, tetracycline, and aminoglycoside antibiotics very rapidly. A non‐primate model of the disease is based on gnotobiotic piglets. Aerobically grown bacteria shift to anaerobic metabolism when introduced into the GI tract of the piglets. Pieper et al. examined the aerobic and anaerobic proteomes using differential display methodology on 2‐D gels. Over 1050 separate gene products were identified. Bacterial defense genes and secretion system genes were among the types found. Novel proteins that might be targets for vaccines or drugs were also identified (OmpA, HtpG, and OspC2).Pieper, R. et al., Proteomics 2009, 9, 5029–5045.Form follow/leads functions? Essentiality, centrality and network topologyWho ever thought eight or ten years ago that we would be learning network‐speak to keep up with contemporary molecular and cell biology? Collaborating with mathematicians and physicists? Raise your hand. As I thought, not many. With the completion of the human genome DNA sequence it became clear that the sequence did not code for enough proteins to account for the complexity of life. At least two sources of complexity have been discovered to date: post‐translational modification and protein–protein interaction networks (PPINs). The structure of PPINs is examined in this work from Park et al., who raise the question (in Facebook terms): does popularity correlate with importance? They used two yeast PPINs to see how well centrality correlated with essentiality and found that, using 40 measures of centrality, the relationship was close for path‐based localized information centrality and gene essentiality. They found that random forest classifiers can work, too.Park, K. et al., Proteomics 2009, 9, 5143–5154.Standing on the shoulders of the great: Telomerase proteomicsIn those early days of molecular biology, when all “model replication systems” (bacteria and phages) went through circular intermediates, we did not need to worry about linear ends. Then we came to the end of the world, the end of the linear chromosome. Elizabeth Blackburn, Carol Greider, and Jack Szostak worked out the mechanics and enzymology of faithful replication of those linear ends and for that work received the 2009 Nobel Prize in physiology and medicine. Now researchers such as Zimmermann et al. can apply the tools of proteomics to tease apart the subtleties of telomere control of and control by other proteins. These researchers demonstrate the utility of SELDI MS/MS to examine the changes in specific cell fractions upon up‐ or down‐expression of telomere regulatory components. They confirmed several observations and found a new role for S100A6 (AKA “calcyclin”) in cell response to telomere dysfunction.Zimmermann, S. et al., Proteomics 2009, 9, 5175–5187.

Characterization of 14-3-3β Expression during Differentiation of the Megakaryocytic Cell Line MEG-01

The 14-3-3 family of proteins is highly conserved among mammalian species which consists of seven homologous isoforms (β,ε, γ, η, θ, σ and ζ). They are involved in the regulation of most cellular processes including several metabolic pathways, redox-regulation, transcription, RNA processing, protein synthesis, protein folding and degradation, cytoskeletal organization, apoptosis, intracellular trafficking/targeting, and cell cycle. In a previous study, we identified several novel genes associated with hematopoietic lineage commitment and/or differentiation using a hematopoietic liquid culture system and a differential display methodology. One of these, TP-3 (thrombopoietin-stimulated clone-3) is selectively expressed in normal human megakaryocytic lineage cells. cDNA sequences analysis revealed 100% identity with the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, beta polypeptide (YWHAB) gene that encodes for 14-3-3β protein. Accordingly, we set out to determine the role 14-3-3β may have in megakaryocyte proliferation and differentiation. For this purpose, we employed the megakaryocytic precursor cell line, MEG-01, that can be induce to differentiate by 12-0-tetradecanoylphorbol-13-acetate (TPA) treatment. After TPA challenge, we detected an increase in the 14-3-3β mRNA expression in MEG-01 cells. Similarly, the phosphorylated form of 14-3-3β protein also was induced during MEG-01 cells differentiation by TPA treatment, while the total 14-3-3β protein levels did not reveal any differences between MEG-01 cells treated with or without TPA. To confirm the correlation between 14-3-3β expression and the megakaryocyte differentiation, we compared its expression patterns to that of the platelet-specific antigens GP IIb/IIIa and GP Ib which are known to be markers of maturation within the megakaryocyte lineage using flow cytometry and immunofluorescent staining methods. After TPA treatment, the percentage of MEG-01 cells expressing GP IIb/IIIa and GP Ib significantly increased from 19% to 95% and from 0% to 27% (p<0.05) over the course of 8 days, respectively. In contrast, 14-3-3β siRNA knockdown resulted in a reduction of platelet-specific antigens GP IIb/IIIa and GP Ib in TPA-treated population from 41.6 ± 7.01 to 12.8 ± 5.47 and from 24.1 ± 2.32 to 7.9 ± 0.21 (p<0.05), respectively. To better understand potential targets of 14-3-3β effects in megakaryocytic differentiation and proliferation, we investigated whether the changes of cell-cycle-related gene expressions were related to the MEG-01 cells differentiation. We used semi-quantitative real-time polymerase chain reaction (RT-PCR) to examine expression pattern of important known genes involved in cell-cycle regulation. We found enhanced expression of p21 and cyclin D1 in TPA-treated MEG-01 cells, while such treatment was associated with a suppression of Cdc25A and Cdc25C. By Western blot analysis, we confirmed this differential effect on gene expression was associated with corresponding changes in protein expression. Taken together, our data provide the new insights into the 14-3-3 β its role in the megakaryocyte differentiation via the signal transduction pathways of cyclin D1 and p21 expression. We are current investigating whether 14-3-3β dysfunction may contribute to the pathogenesis of certain acquired disorders of megakaryopoiesis.

Recent advances in 2D electrophoresis: an array of possibilities

2D electrophoresis is currently the most widespread technique used for performing functional proteomics (i.e., the large-scale analysis of alterations in protein expression levels). Nevertheless, several limitations inherent to this technology have restricted the full potential of this protein differential display methodology for years. This has even led to the abandonment of 2D electrophoresis by several groups that switched to performing gel-free functional proteomics analyses based on liquid chromatography and mass spectrometry. Meanwhile, important recent advances in 2D electrophoresis, such as the introduction of fluorescent 2D difference gel electrophoresis and numerous protein prefractionation techniques, have thoroughly modernized 2D electrophoresis, making it again one of the preferred methods for the analysis of protein expression differences in many laboratories.

Suppression of ps20 Expression in the Rat Uterus by Tamoxifen and Estrogens

Using differential display methodology, we isolated a tamoxifen-regulated cDNA. This cDNA was identical to the ps20 cDNA isolated from urogenital sinus mesenchymal cells. ps20 expression was detected in various female rat tissues, with the highest expression in lung and heart. ps20 transcripts were low during estrus and proestrus, but high during the diestrous stage of the estrous cycle coincident with estrogen-induced uterine cell proliferation. Treatment of ovary-intact or ovariectomized rats with estrogens or tamoxifen resulted in increased uterine weight and decreased ps20 expression. Uterine involution associated with ovariectomy or antiestrogen treatment led to up-regulation of ps20. Antibody against rat ps20 recognized the native rat ps20 in conditioned medium of primary rat uterine cells and stable ps20-transfected MCF-7 cells with molecular masses of approximately 24, 27, and 29 kDa. In primary rat uterine cells, ps20 secretion was enhanced by ICI 182,780, but was inhibited by estrogens and tamoxifen. Immunohistochemistry revealed that ps20 was localized to smooth muscle and luminal epithelial cells as well as the glandular population of uterine tissue. Conditioned medium derived from ps20-transfected MCF-7 cells, but not Escherichia coli recombinant ps20, exhibited mild growth suppression on PC-3 cells. The data indicate that ps20 expression is negatively regulated by estrogens and tamoxifen and suggest that ps20 may function as a mediator of local growth.

Gene expression changes in rat white adipose tissue after a high-fat diet determined by differential display

The differences in gene expression pattern of visceral white adipose tissue between control and high-fat-fed rats were compared using the mRNA differential display methodology. The results, confirmed by Northern blot, showed eight genes upregulated: adiponectin, fibrillin-1, transferrin, Y-box binding protein-1, IgE receptor β chain (FcεRIβ), α-1 haemoglobin, and ribosomal proteins S10 and L7 and four genes downregulated: caveolin-2, lactate dehydrogenase-A, mitochondrial 16S rRNA, and mitochondrial cytochrome oxidase subunit I/serine tRNA. Two of these genes have been already related to obesity (adiponectin and caveolin-2) while the others are known to participate in metabolic, signalling or transcription regulation pathways that can be relevant in energy (lipid and/or carbohydrate) metabolism.

Fluorescent two-dimensional difference gel electrophoresis unveils the potential of gel-based proteomics.

Comparing different proteomes by classical two-dimensional electrophoresis is challenging and often complicated by substantial gel-to-gel variation. Separating two or more protein samples labelled with different fluorescent dyes in one single gel, as in two-dimensional difference gel electrophoresis, reduces this variability considerably. Recent technological innovations, specifically the introduction of a pooled internal standard, even further improve the quantification accuracy and statistical confidence of this method. In addition, decreasing the sample complexity by one of several protein or organelle fractionation procedures increases the number of spots investigated by this protein differential display methodology.

Viral haemorrhagic septicaemia virus induces vig -2, a new interferon-responsive gene in rainbow trout

An mRNA differential display methodology was used to study the rainbow trout response to viral infection. A new transcript (vig -2) induced by viral haemorrhagic septicaemia virus (VHSV) in rainbow trout leucocytes was identified from the head-kidney. vig -2 was also induced in vivo during experimental infection and following DNA immunisation with a plasmid containing a gene encoding the viral glycoprotein. Viral induction of vig -2 was blocked by cycloheximide (CHX), indicating its dependency on a newly synthesised intermediate protein. This intermediate protein is most probably related to interferon because treatment of cells with a conditioned medium displaying an interferon-like activity resulted in a strong vig -2 expression, which was not blocked by CHX treatment. The cDNA sequence of the vig -2 transcript displays several mRNA destabilisation motifs and two signals characteristic of immediate–early gene expression. Curiously,vig -2 has no evident encoding potential except for a small 51 amino acid putative polypeptide with no clear similarity to any sequence available in the databanks. Therefore, the complete vig -2 genomic sequence was determined from a lambda phage clone retrieved from a genomic DNA library of rainbow trout. The genomic organisation of vig -2 shows five exons delimited with typical splice acceptor and donor sites. A promoter with a canonical ISRE, confirming that vig -2 is an interferon-responsive gene, is also present 115nt upstream of the first exon.

Identification and partial characterization of differentially expressed mRNAs in normal human endometria and endometrial carcinomas by differential display RT-PCR.

Differential display methodology was employed to examine and compare the mRNA species derived from normal endometrial tissue and endometrial carcinoma (grade 3, stage III) tissue biopsies. Two cDNA sequences, one expressed in the tumour group only (T19) and the other expressed only in the normal group (N22), were selected for verification of differential expression by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The expression of N22 was restricted to the normal group, suggesting a possible tumour suppressing function. Sequence analysis of this fragment revealed a high degree of similarity to a human cDNA sequence of unknown function. The expression of T19 mRNA was observed in both normal and neoplastic tissues, however the relative abundance was significantly higher in endometrial carcinomas. Expression of T19 mRNA was further examined in a larger clinical sample set and was significantly increased in the tumours (n = 16), with a three-fold increase when compared with the normal endometria, n = 5 (Kruskal-Wallis analysis of variance, P<0.05). Subsequent sequence analysis of T19 revealed a high degree of similarity to the 3' untranslated region of a rat growth factor responsive gene, SM-20. Further characterization of these mRNA transcripts may lead to the identification of novel genes involved in endometrial tumourogenesis.

Rapid down-regulation of CD63 transcription by progesterone in human endometrial stromal cells.

Differentiation of endometrial stromal cells (decidualization) plays a crucial role in embryo implantation and maintenance of pregnancy. While progesterone is a key factor in regulating endometrial cell decidualization, the molecular mechanisms remain unclear. In the present study, we investigated the effect of gene transcription in human endometrial stromal cells (ESC) by progesterone, oestrogen or vehicle using the polymerase chain reaction-based differential display methodology. A transcript which is down-regulated by progesterone, but not by vehicle and oestrogen, was identified from a differential display band and the progesterone sensitivity of its expression was verified in Northern blot analysis. The level of the gene expression in progesterone-treated ESC was approximately 60% of that in the vehicle- and oestrogen-treated ESC. This cDNA was revealed to be human CD63 antigen, a recently identified member of the transmembrane 4 superfamily. The inhibitory effect of progesterone is observed within 30 min after hormone treatment. In human endometrium, CD63 mRNA levels were significantly decreased (P < 0.05) during the secretory phase compared with levels during the proliferative phase. This down-regulation of CD63 in vivo elevated levels of progesterone in the secretory phase. These results suggest that CD63 transcription is down-regulated by progesterone in human endometrium.

Identification and characterization of mRNAs differentially expressed in thyroid cells stimulated by a mitogenic treatment

The aim of our work is to identify new genes and proteins involved in the control of the proliferation of thyroidcells as putative protooncogenes and antioncogenes. Several strategies are discussed. A first study has allowed to identify three new genes. Further search will use the differential display and gene arrays methodology. The role of the identified proteins codedby the genes is studied in vitro by the search of partner proteins by the double hydrid method and in vivo by mice gene knockout technology.

Search for Genes Potentially Involved inMycobacterium tuberculosisVirulence by mRNA Differential Display

An mRNA differential display (DD) assay was developed to compare gene expression betweenMycobacterium tuberculosisH37Rv and its avirulent mutant H37Ra. The DD protocol made use of an oligo(dT) to prime reverse-transcriptase (RT)-dependent transcription of poly-A tailed mRNAs and a PCR amplification of the RT products by using ten 12-base arbitrary primers in all their pair combinations. This analysis yielded 745 and 708 bands, including 52 and 15 differentially generated bands, in the strains H37Rv and H37Ra, respectively. Six cDNAs that appeared to be expressed in H37Rv, but not in H37Ra, were reamplified and cloned and at least 10 inserts were sequenced for each cloned cDNA. After resolving discrepant results, 6 inserts were found highly homologous toM. tuberculosisH37Rv genes. Three of these,i.e.,genes Rv2770c, Rv1345, and Rv0288, coding respectively for a member of the PPE protein family, a probable polyketide synthase, and a member of the protein family containing ESAT-6, have been predictively associated to immunological or pathogenetic aspects ofM. tuberculosisinfection; the other genes,i.e.,Rv2336, Rv1320c, and Rv2819c, code for proteins with unknown functions. These results show that mRNA DD methodology can represent a potential tool for investigation ofM. tuberculosisgene expression.

Reprogramming of Fibroblast Nuclei after Transfer into Bovine Oocytes

Recent landmark achievements in animal cloning have demonstrated that the events of cell differentiation can, in principle, be reversed. This reversal necessarily requires large-scale genetic reprogramming, of which little is known. In the present study we characterized the extent to which blastocyst stage-specific mRNA expression would be conserved in bovine embryos produced by nuclear transfer (NT) using fetal fibroblasts as nuclei donors (FF NT). The mRNA pool of FF NT embryos was compared with that of NT embryos reconstructed from embryonic blastomeres (Emb NT), with embryos produced under in vivo or in vitro conditions, and finally with fibroblast cells. Embryo/cell-specific mRNA pools were contrasted using differential display methodology. Random oligonucleotide primer pair combinations were used to subfractionate mRNA populations and represent individual mRNAs as copy DNA (cDNA) bands ranging in size from 100 to 800 base pairs. Regardless of whether bovine blastocysts developed in vivo or in vitro, or were derived after nuclear transplantation with embryonic blastomeres or fetal fibroblasts, their mRNA profile was highly conserved and distinct from that of fetal fibroblast cells. There was approximately 95% conservation in cDNA banding patterns between FF NT, Emb NT, and in vivo derived blastocysts, when compared with in vitro derived blastocysts. In contrast, the cDNA banding in fibroblasts was only 67% conserved with in vitro derived blastocysts (p < 0.0001), indicating that dramatic changes in gene transcription are induced by nuclear transplantation. After nuclear transplantation, gene expression in fetal fibroblasts is reprogrammed so to mimic that of preimplantation embryo development. Future characterization of these changes will be invaluable for the identification of suitable cell types to serve as nuclear donors for embryo reconstruction and provide information that can be used to improve the efficiency of cloning animals by nuclear transplantation.

Vig-1, a new fish gene induced by the rhabdovirus glycoprotein, has a virus-induced homologue in humans and shares conserved motifs with the MoaA family.

We used mRNA differential display methodology to analyze the shift of transcription profile induced by the fish rhabdovirus, viral hemorrhagic septicemia virus (VHSV), in rainbow trout leukocytes. We identified and characterized a new gene which is directly induced by VHSV. This VHSV-induced gene (vig-1) encodes a 348-amino-acid protein. vig-1 is highly expressed during the experimental disease in lymphoid organs of the infected fish. Intramuscular injection of a plasmid vector expressing the viral glycoprotein results in vig-1 expression, showing that the external virus protein is sufficient for the induction. vig-1 expression is also obtained by a rainbow trout interferon-like factor, indicating that vig-1 can be induced through different pathways. Moreover, vig-1 is homologous to a recently described human cytomegalovirus-induced gene. Accordingly, vig-1 activation may represent a new virus-induced activation pathway highly conserved in vertebrates. The deduced amino acid sequence of vig-1 is significantly related to sequences required for the biosynthesis of metal cofactors. This suggests that the function of vig-1 may be involved in the nonspecific virus-induced synthesis of enzymatic cofactors of the nitric oxide pathway.

Interleukin‐10 gene transfer activates interferon‐γ and the interferon‐γ‐inducible genes Gbp1/Mag1 and Mig1 in mammary tumors

Expression of IL-10 as a transgene inhibits murine mammary tumor growth and metastasis. Using differential display methodology, we sought genes whose expression was modulated by IL-10. We compared mRNA isolated from parental murine mammary 66.1 tumors, as well as tumors derived from neor-transfected cells and 6 different IL-10-expressing cell lines. We identified 2 cDNA products that were up-regulated in all 6 IL-10-expressing tumors in comparison to parental and 66-neo tumors. One cDNA corresponds to the murine guanylate-binding protein gene Gbp-1/Mag-1. The other cDNA corresponds to the chemokine Mig-1 (monokine induced by IFN-γ). Both genes were originally identified in IFN-γ-activated macrophages or macrophage cell lines. We now report that cultured mammary epithelial tumor cell lines also express both genes in response to treatment with IFN-γ and LPS. Furthermore, IFN-γ mRNA is elevated in IL-10-expressing tumors in comparison with parental or neo-transfected tumors. Thus, high-level expression of IL-10 as a transgene results in activation rather than suppression of IFN-γ as well as 2 IFN-γ-inducible genes. Up-regulation of host IFN-γ is critical to anti-tumor activity since IL-10 no longer inhibits tumor growth in hosts with a deletion in the IFN-γ gene. Additionally, Gbp-1/Mag-1 and Mig-1 gene induction no longer occur in IFN-γ mutant mice. Int. J. Cancer 80:624–629, 1999. © 1999 Wiley-Liss, Inc.

Interleukin-10 gene transfer activates interferon-gamma and the interferon-gamma-inducible genes Gbp-1/Mag-1 and Mig-1 in mammary tumors.

Expression of IL-10 as a transgene inhibits murine mammary tumor growth and metastasis. Using differential display methodology, we sought genes whose expression was modulated by IL-10. We compared mRNA isolated from parental murine mammary 66.1 tumors, as well as tumors derived from neo(r)-transfected cells and 6 different IL-10-expressing cell lines. We identified 2 cDNA products that were up-regulated in all 6 IL-10-expressing tumors in comparison to parental and 66-neo tumors. One cDNA corresponds to the murine guanylate-binding protein gene Gbp-1/Mag-1. The other cDNA corresponds to the chemokine Mig-1 (monokine induced by IFN-gamma). Both genes were originally identified in IFN-gamma-activated macrophages or macrophage cell lines. We now report that cultured mammary epithelial tumor cell lines also express both genes in response to treatment with IFN-gamma and LPS. Furthermore, IFN-gamma mRNA is elevated in IL-10-expressing tumors in comparison with parental or neo-transfected tumors. Thus, high-level expression of IL-10 as a transgene results in activation rather than suppression of IFN-gamma as well as 2 IFN-gamma-inducible genes. Up-regulation of host IFN-gamma is critical to anti-tumor activity since IL-10 no longer inhibits tumor growth in hosts with a deletion in the IFN-gamma gene. Additionally, Gbp-1/Mag-1 and Mig-1 gene induction no longer occur in IFN-gamma mutant mice.

28] Solid-phase differential display and bacterial expression systems in selection and functional analysis of cDNAs

Differential gene expression can be expected during activation and differentiation of cells as well as during pathological conditions, such as cancer. A number of strategies have been described to identify and understand isolated differentially expressed genes. The differential display methodology has rapidly become a widely used technique to identify differentially expressed mRNAs. In this chapter we described a variant of the differential display method based on solid-phase technology. The solid-phase procedure offers an attractive alternative to solution-based differential display because minute amounts of sample can be analyzed in considerably less time than previously. The employed solid support, monodisperse super paramagnetic beads, which circumvents precipitation and centrifugations steps, has also allowed for optimization of the critical enzymatic and preparative steps in the differential display methodology. We also described how bacterial expression can be used as a means to elucidate gene function. An efficient dual-expression system was presented, together with a basic concept describing how parallel expression of selected portions of cDNAs can be used for production of cDNA-encoded proteins as parts of affinity-tagged fusion proteins. The fusion proteins are suitable both for the generation of antibodies reactive to the target cDNA-encoded protein and for the subsequent affinity enrichment of such antibodies. Affinity-enriched antibodies have proved to be valuable tools in various assays, including immunoblotting and immunocytochemical staining, and can thus be used to localize the target cDNA-encoded protein to certain cells in a tissue section or even to a specific cell compartment or organelle within a cell. High-resolution localization of a cDNA-encoded protein would provide valuable information toward the understanding of protein function.

CDNA Cloning of a Novel, Developmentally Regulated Immediate Early Gene Activated by Fibroblast Growth Factor and Encoding a Nuclear Protein

We have utilized the polymerase chain reaction (PCR)-based differential display methodology (Liang, P., and Pardee, A. B. (1992) Science 257, 967-969) to identify a novel transcript whose expression levels increased in Xenopus embryo explants during mesoderm induction by fibroblast growth factor. The PCR product was used to clone a 2.3-kilobase pair cDNA representing this transcript, which we have named er1 (early response 1). The er1 cDNA contained a single open reading frame predicted to encode a protein of 493 amino acid residues. A data base homology search revealed that the predicted ER1 amino acid sequence contains three regions of similarity to the rat and human proteins encoded by the metastasis-associated gene, mta1, and two regions of similarity to the Caenorhabditis elegans sequence that is similar to mta1. The fibroblast growth factor-induced increase in er1 steady-state levels was not dependent on de novo protein synthesis, demonstrating that er1 is an immediate-early gene. Northern blot analysis revealed a single 2.8-kilobase pair mRNA that was observed predominantly during the initial cleavage and blastula stages of Xenopus development, with little or no detectable mRNA during subsequent development. Quantitative PCR analysis of early developmental stages showed that er1 peaked during late blastula. Computer-assisted analysis of the predicted ER1 amino acid sequence revealed two putative nuclear localization signals, four highly acidic regions clustered at the N terminus and a proline-rich region located near the C terminus. Subcellular localization by immunocytochemistry revealed that the ER1 protein was targeted exclusively to the nucleus. Transactivation assays using various regions of ER1 fused to the DNA binding domain of GAL4 demonstrated that the N-terminal acidic region is a potent transactivator. These data suggest that ER1 may function as a transcription factor.