ABSTRACT
Cytoplasmic mRNA localization provides a means of generating cell asymmetry and segregating protein activity. Previous studies have identified two mRNAs that localize to the bud tips of the yeast Saccharomyces cerevisiae. To identify additional localized mRNAs, we immunoprecipitated the RNA transport components She2p, She3p, and Myo4p and performed DNA microarray analysis of their associated RNAs. A secondary screen, using a GFP-tagged RNA reporter assay, identified 22 mRNAs that are localized to bud tips. These messages encode a wide variety of proteins, including several involved in stress responses and cell wall maintenance. Many of these proteins are asymmetrically localized to buds. However, asymmetric localization also occurs in the absence of RNA transport, suggesting the existence of redundant protein localization mechanisms. In contrast to findings in metazoans, the untranslated regions are dispensable for mRNA localization in yeast. This study reveals an unanticipated widespread use of RNA transport in budding yeast.
Subject(s)
Cytoplasm/metabolism , Oligonucleotide Array Sequence Analysis , RNA, Messenger/metabolism , Biological Transport , Precipitin TestsABSTRACT
The most common human leukemia is B cell chronic lymphocytic leukemia (CLL), a malignancy of mature B cells with a characteristic clinical presentation but a variable clinical course. The rearranged immunoglobulin (Ig) genes of CLL cells may be either germ-line in sequence or somatically mutated. Lack of Ig mutations defined a distinctly worse prognostic group of CLL patients raising the possibility that CLL comprises two distinct diseases. Using genomic-scale gene expression profiling, we show that CLL is characterized by a common gene expression "signature," irrespective of Ig mutational status, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Nonetheless, the expression of hundreds of other genes correlated with the Ig mutational status, including many genes that are modulated in expression during mitogenic B cell receptor signaling. These genes were used to build a CLL subtype predictor that may help in the clinical classification of patients with this disease.
Subject(s)
Gene Expression , Immunoglobulins/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Mutation , Genotype , Humans , ImmunophenotypingABSTRACT
Iron homeostasis in the yeast Saccharomyces cerevisiae is regulated at the transcriptional level by Aft1p, which activates the expression of its target genes in response to low-iron conditions. The yeast genome contains a paralog of AFT1, which has been designated AFT2. To establish whether AFT1 and AFT2 have overlapping functions, a mutant containing a double aft1Deltaaft2Delta deletion was generated. Growth assays established that the single aft2Delta strain exhibited no iron-dependent phenotype. However, the double-mutant aft1Deltaaft2Delta strain was more sensitive to low-iron growth conditions than the single-mutant aft1Delta strain. A mutant allele of AFT2 (AFT2-1(up)), or overexpression of the wild-type AFT2 gene, led to partial complementation of the respiratory-deficient phenotype of the aft1Delta strain. The AFT2-1(up) allele also increased the uptake of (59)Fe in an aft1Delta strain. DNA microarrays were used to identify genes regulated by AFT2. Some of the AFT2-regulated genes are known to be regulated by Aft1p; however, AFT2-1(up)-dependent activation was independent of Aft1p. The kinetics of induction of two genes activated by the AFT2-1(up) allele are consistent with Aft2p acting as a direct transcriptional factor. Truncated forms of Aft1p and Aft2p bound to a DNA duplex containing the Aft1p binding site in vitro. The wild-type allele of AFT2 activated transcription in response to growth under low-iron conditions. Together, these data suggest that yeast has a second regulatory pathway for the iron regulon, with AFT1 and AFT2 playing partially redundant roles.
Subject(s)
Fungal Proteins/metabolism , Iron/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Transcription Factors/metabolism , Alleles , Amino Acid Sequence , Base Sequence , Binding Sites/genetics , DNA, Fungal/genetics , Fungal Proteins/genetics , Gene Deletion , Gene Expression , Genes, Fungal , Homeostasis , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Phenotype , Promoter Regions, Genetic , Protein Binding , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Sequence Homology, Amino Acid , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/geneticsABSTRACT
The envelope (Env) protein of Moloney murine leukemia virus is the primary mediator of viral entry. We constructed a large pool of insertion mutations in the env gene and analyzed the fitness of each mutant in completing two critical steps in the virus life cycle: (i) the expression and delivery of the Env protein to the cell surface during virion assembly and (ii) the infectivity of virions displaying the mutant proteins. The majority of the mutants were poorly expressed at the producer cell surface, suggesting folding defects due to the presence of the inserted residues. The mutants with residual infectivity had insertions either in the amino-terminal signal sequence region, two disulfide-bonded loops in the receptor binding domain, discrete regions of the carboxy-terminal region of the surface subunit (SU), or the cytoplasmic tail. Insertions that allowed the mutants to reach the cell surface but not to mediate detectable infection were located within the amino-terminal sequence of the mature Env, within the SU carboxy-terminal region, near putative receptor binding residues, and throughout the fusion peptide. Independent analysis of select mutants in this group allowed more precise identification of the defect in Env function. Mapping of mutant phenotypes to a structural model of the receptor-binding domain provides insights into the protein's functional organization. The high-resolution functional map reported here will be valuable for the engineering of the Env protein for a variety of uses, including gene therapy.
Subject(s)
Gene Products, env/physiology , Genes, env , Moloney murine leukemia virus/physiology , Amino Acid Sequence , Animals , Base Sequence , Cell Line , DNA Primers , Gene Products, env/chemistry , Gene Products, env/genetics , Humans , Models, Molecular , Molecular Sequence Data , Moloney murine leukemia virus/genetics , Mutagenesis , Phenotype , Protein Conformation , Protein Sorting SignalsABSTRACT
The global gene expression profiles for 67 human lung tumors representing 56 patients were examined by using 24,000-element cDNA microarrays. Subdivision of the tumors based on gene expression patterns faithfully recapitulated morphological classification of the tumors into squamous, large cell, small cell, and adenocarcinoma. The gene expression patterns made possible the subclassification of adenocarcinoma into subgroups that correlated with the degree of tumor differentiation as well as patient survival. Gene expression analysis thus promises to extend and refine standard pathologic analysis.
Subject(s)
Adenocarcinoma/genetics , Gene Expression , Genetic Variation , Lung Neoplasms/genetics , Adenocarcinoma/classification , Adenocarcinoma/mortality , Adenocarcinoma/pathology , Carcinoma, Large Cell/classification , Carcinoma, Large Cell/genetics , Carcinoma, Small Cell/classification , Carcinoma, Small Cell/genetics , Carcinoma, Squamous Cell/classification , Carcinoma, Squamous Cell/genetics , Gene Expression Profiling , Humans , Lung Neoplasms/classification , Lung Neoplasms/mortality , Lung Neoplasms/pathology , Survival Rate/trendsABSTRACT
Eukaryotic cells respond to DNA damage by arresting the cell cycle and modulating gene expression to ensure efficient DNA repair. The human ATR kinase and its homolog in yeast, MEC1, play central roles in transducing the damage signal. To characterize the role of the Mec1 pathway in modulating the cellular response to DNA damage, we used DNA microarrays to observe genomic expression in Saccharomyces cerevisiae responding to two different DNA-damaging agents. We compared the genome-wide expression patterns of wild-type cells and mutants defective in Mec1 signaling, including mec1, dun1, and crt1 mutants, under normal growth conditions and in response to the methylating-agent methylmethane sulfonate (MMS) and ionizing radiation. Here, we present a comparative analysis of wild-type and mutant cells responding to these DNA-damaging agents, and identify specific features of the gene expression responses that are dependent on the Mec1 pathway. Among the hundreds of genes whose expression was affected by Mec1p, one set of genes appears to represent an MEC1-dependent expression signature of DNA damage. Other aspects of the genomic responses were independent of Mec1p, and likely independent of DNA damage, suggesting the pleiotropic effects of MMS and ionizing radiation. The complete data set as well as supplemental materials is available at http://www-genome.stanford.edu/mec1.
Subject(s)
Cell Cycle Proteins/genetics , DNA Repair/physiology , DNA, Fungal/metabolism , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal/physiology , Radiation, Ionizing , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Cell Cycle Proteins/metabolism , DNA Damage/drug effects , DNA Damage/radiation effects , Fungal Proteins/genetics , Gene Expression Regulation, Fungal/drug effects , Gene Expression Regulation, Fungal/radiation effects , Intracellular Signaling Peptides and Proteins , Methyl Methanesulfonate/pharmacology , Mutation/genetics , Oligonucleotide Array Sequence Analysis , Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Sequence Homology , Signal Transduction/physiologyABSTRACT
Analysis of iron-regulated gene expression in Saccharomyces cerevisiae using cDNA microarrays has identified three putative cell wall proteins that are directly regulated by Aft1p, the major iron-dependent transcription factor in yeast. FIT1, FIT2, and FIT3 (for facilitator of iron transport) were more highly expressed in strains grown in low concentrations of iron and in strains in which AFT1-1(up), a constitutively active allele of AFT1, was expressed. Northern blot analysis confirmed that FIT1, FIT2, and FIT3 mRNA transcript levels were increased 60-230-fold in response to iron deprivation in an Aft1p-dependent manner. Fit1p was localized exclusively to the cell wall by indirect immunofluorescence. Deletion of the FIT genes, individually or in combination, resulted in diminished uptake of iron bound to the siderophores ferrioxamine B and ferrichrome, without diminishing the uptake of ferric iron salts, or the siderophores triacetylfusarinine C and enterobactin. FIT-deletion strains exhibited increased expression of Aft1p target genes as measured by a FET3-lacZ reporter gene or by Arn1p Western blotting, indicating that cells respond to the absence of FIT genes by up-regulating systems of iron uptake. Aft1p activation in FIT-deleted strains occurred when either ferrichrome or ferric salts were used as sources of iron during growth, suggesting that the FIT genes enhance uptake of iron from both sources. Enzymatic digestion of the cell wall resulted in the release of significant amounts of iron from cells, and the relative quantity of iron released was reduced in FIT-deletion strains. Fit1p, Fit2p, and Fit3p may function by increasing the amount of iron associated with the cell wall and periplasmic space.
Subject(s)
Cation Transport Proteins , Cell Wall/chemistry , Fungal Proteins/metabolism , Glycoproteins/metabolism , Iron/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Biological Transport , Cell Wall/metabolism , Deferoxamine/metabolism , Ferric Compounds/metabolism , Ferrichrome/metabolism , Fungal Proteins/genetics , Gene Expression Profiling , Glycoproteins/chemistry , Glycoproteins/genetics , Iron Chelating Agents/metabolism , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/geneticsABSTRACT
Using microarray analysis, we identified a unique ras superfamily gene, termed RERG (ras-related and estrogen-regulated growth inhibitor), whose expression was decreased or lost in a significant percentage of primary human breast tumors that show a poor clinical prognosis. Importantly, high RERG expression correlated with expression of a set of genes that define a breast tumor subtype that is estrogen receptor-positive and associated with a slow rate of tumor cell proliferation and a favorable prognosis for these cancer patients. RERG mRNA expression was induced rapidly in MCF-7 cells stimulated by beta-estradiol and repressed by tamoxifen treatment. Like Ras, RERG protein exhibited intrinsic GDP/GTP binding and GTP hydrolysis activity. Unlike Ras proteins, RERG lacks a known recognition signal for COOH-terminal prenylation and was localized primarily in the cytoplasm. Expression of RERG protein in MCF-7 breast carcinoma cells resulted in a significant inhibition of both anchorage-dependent and anchorage-independent growth in vitro and inhibited tumor formation in nude mice. These features of RERG are strikingly different from most Ras superfamily GTP-binding pro-teins and suggest that the loss of RERG expression may contribute to breast tumorigenesis.
Subject(s)
Breast Neoplasms/genetics , Estrogens/pharmacology , GTP-Binding Proteins/genetics , Genes, ras , Growth Inhibitors/genetics , 3T3 Cells , Amino Acid Sequence , Animals , Breast Neoplasms/pathology , Female , GTP Phosphohydrolases/metabolism , Humans , Mice , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Tumor Cells, CulturedABSTRACT
The purpose of this study was to classify breast carcinomas based on variations in gene expression patterns derived from cDNA microarrays and to correlate tumor characteristics to clinical outcome. A total of 85 cDNA microarray experiments representing 78 cancers, three fibroadenomas, and four normal breast tissues were analyzed by hierarchical clustering. As reported previously, the cancers could be classified into a basal epithelial-like group, an ERBB2-overexpressing group and a normal breast-like group based on variations in gene expression. A novel finding was that the previously characterized luminal epithelial/estrogen receptor-positive group could be divided into at least two subgroups, each with a distinctive expression profile. These subtypes proved to be reasonably robust by clustering using two different gene sets: first, a set of 456 cDNA clones previously selected to reflect intrinsic properties of the tumors and, second, a gene set that highly correlated with patient outcome. Survival analyses on a subcohort of patients with locally advanced breast cancer uniformly treated in a prospective study showed significantly different outcomes for the patients belonging to the various groups, including a poor prognosis for the basal-like subtype and a significant difference in outcome for the two estrogen receptor-positive groups.
Subject(s)
Breast Neoplasms/genetics , Carcinoma in Situ/genetics , Carcinoma, Ductal, Breast/genetics , Carcinoma, Lobular/genetics , DNA, Neoplasm , Fibroadenoma/genetics , Gene Expression , Algorithms , Breast Neoplasms/classification , Carcinoma in Situ/classification , Carcinoma, Ductal, Breast/classification , Carcinoma, Lobular/classification , Female , Fibroadenoma/classification , Gene Expression Profiling , Humans , Oligonucleotide Array Sequence Analysis/methods , Tumor Suppressor Protein p53/geneticsABSTRACT
Current development in molecular techniques has extended the opportunities to explore genetic alterations in malignant tissue. There is a need to improve prognostication and, in particular, to understand the mechanisms of treatment resistance in different tumours. Gene analyses by microarrays allow concomitant analyses of several genes in concert, providing new opportunities for tumour classification and understanding of key biological disturbances. This paper outlines our continuing studies exploring prognostic and, we hope, predictive factors in breast cancer therapy.
Subject(s)
Breast Neoplasms/genetics , Gene Expression Profiling , Genes, erbB-2/physiology , Oligonucleotide Array Sequence Analysis , Breast Neoplasms/diagnosis , Breast Neoplasms/metabolism , Epithelial Cells/physiology , Female , Fluorescent Dyes , Humans , Prognosis , RNA, Neoplasm/biosynthesis , Reproducibility of Results , Tumor Cells, CulturedABSTRACT
The Saccharomyces cerevisiae genome contains a predicted gene, YPR008w, homologous to the gene encoding the copper-activated transcription factor Ace1. The product of the YPR008w gene, designated Haa1, regulates the transcription of a set of yeast genes, many of which encode membrane proteins. Two main target genes of Haa1 are the multidrug resistance gene YGR138c and the YRO2 homolog to the plasma membrane Hsp30. Haa1 is localized to the nucleus. Haa1-induced expression of YGR138c and YRO2 appears to be direct. Induction of HAA1 using a GAL1/HAA1 fusion gene resulted in rapid galactose-induced expression of both HAA1 and target genes. Although Haa1 has a sequence very similar to the Cu-activated DNA binding domain of Ace1, expression of Haa1 target genes was found to be independent of the copper status of cells. Haa1 does not exhibit metalloregulation in cells incubated with a range of transition metal salts. Haa1 does not exhibit any cross-talk with Ace1. Overexpression of Haa1 does not compensate for cells lacking a functional Ace1. The lack of metalloregulation of Haa1 despite the strong sequence similarity to the copper regulatory domain of Ace1 is discussed.
Subject(s)
DNA-Binding Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/metabolism , Trans-Activators/chemistry , Transcription Factors/chemistry , Amino Acid Sequence , Cell Membrane/metabolism , Cell Nucleus/metabolism , Copper/metabolism , Cysteine/chemistry , DNA-Binding Proteins/metabolism , Diploidy , Galactose/metabolism , Genetic Vectors , Green Fluorescent Proteins , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Molecular Sequence Data , Mutation , Protein Binding , Protein Structure, Tertiary , RNA, Messenger/metabolism , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Sequence Homology, Amino Acid , Trans-Activators/metabolism , Transcription Factors/metabolism , Zinc/metabolismABSTRACT
The mammalian BAF complex regulates gene expression by modifying chromatin structure. In this report, we identify 80 genes activated and 2 genes repressed by the BAF complex in SW-13 cells. We find that prior binding of NFI/CTF to the NFI/CTF binding site in CSF1 promoter is required for the recruitment of the BAF complex and the BAF-dependent activation of the promoter. Furthermore, the activation of the CSF1 promoter requires Z-DNA-forming sequences that are converted to Z-DNA structure upon activation by the BAF complex. The BAF complex facilitates Z-DNA formation in a nucleosomal template in vitro. We propose a model in which the BAF complex promotes Z-DNA formation which, in turn, stabilizes the open chromatin structure at the CSF1 promoter.
Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation , Macrophage Colony-Stimulating Factor/genetics , Nuclear Proteins/metabolism , Promoter Regions, Genetic/genetics , Transcription Factors/metabolism , Base Sequence , CCAAT-Enhancer-Binding Proteins/metabolism , Chromatin/chemistry , Chromatin/genetics , Chromatin/metabolism , DNA Helicases , DNA, Superhelical/chemistry , DNA, Superhelical/genetics , DNA, Superhelical/metabolism , DNA-Binding Proteins/chemistry , Gene Expression Profiling , Gene Silencing , Humans , Macromolecular Substances , Models, Genetic , Molecular Sequence Data , NFI Transcription Factors , Nuclear Proteins/genetics , Nucleic Acid Conformation , Oligonucleotide Array Sequence Analysis , Plasmids/chemistry , Plasmids/genetics , Plasmids/metabolism , Protein Binding , Protein Subunits , RNA, Messenger/genetics , RNA, Messenger/metabolism , Response Elements/genetics , Templates, Genetic , Transcription Factors/chemistry , Transcription Factors/genetics , Transcriptional Activation , Tumor Cells, CulturedABSTRACT
One mechanism by which cells adapt to environmental changes is by altering gene expression. Here, we have used cDNA microarrays to identify genes whose expression is altered by exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The goal of our study was to enhance our understanding of toxicity mediated through the pathway by which TCDD stimulates gene expression. To model this toxicity response, we exposed human hepatoma (HepG2) cells to TCDD (10 nM for 18 h) and analyzed mRNA by two-color fluorescent hybridization to cDNA sequences immobilized on glass microscope slides (2.5 x 7.5 cm) covering a surface area of 2.25 cm(2). We analyzed approximately one-third of the genes expressed in HepG2 cells and found that TCDD up- or down-regulates 112 genes two-fold or more. Most changes are relatively subtle (two- to four-fold). We verified the regulation of protooncogene cot, XMP, and human enhancer of filamentation-1 (HEF1), genes involved in cellular proliferation, as well as metallothionein, plasminogen activator inhibitor (PAI1), and HM74, genes involved in cellular signaling and regeneration. To characterize the response in more detail, we performed time-course, dose-dependence studies, and cycloheximide experiments. We observed direct and indirect responses to TCDD implying that adaptation to TCDD (and other related environmental stimuli) is substantially more complex than we previously realized.
Subject(s)
Gene Expression Profiling , Gene Expression/drug effects , Liver/drug effects , Oligonucleotide Array Sequence Analysis , Polychlorinated Dibenzodioxins/toxicity , Carcinoma, Hepatocellular/genetics , Cell Line , Cycloheximide/pharmacology , Dose-Response Relationship, Drug , Humans , Liver Neoplasms/genetics , Molecular Sequence Data , Polychlorinated Dibenzodioxins/administration & dosage , Protein Synthesis Inhibitors/pharmacology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
We determined the distribution of repressor-activator protein 1 (Rap1) and the accessory silencing proteins Sir2, Sir3 and Sir4 in vivo on the entire yeast genome, at a resolution of 2 kb. Rap1 is central to the cellular economy during rapid growth, targeting 294 loci, about 5% of yeast genes, and participating in the activation of 37% of all RNA polymerase II initiation events in exponentially growing cells. Although the DNA sequence recognized by Rap1 is found in both coding and intergenic sequences, the binding of Rap1 to the genome was highly specific to intergenic regions with the potential to act as promoters. This global phenomenon, which may be a general characteristic of sequence-specific transcriptional factors, indicates the existence of a genome-wide molecular mechanism for marking promoter regions.
Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , Genome, Fungal , Physical Chromosome Mapping/methods , Promoter Regions, Genetic/physiology , Saccharomyces cerevisiae/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae , Binding Sites/genetics , DNA, Intergenic/metabolism , Fungal Proteins/metabolism , Gene Expression Regulation/physiology , Genes, Fungal/physiology , Glycolysis/genetics , Histone Deacetylases/metabolism , Open Reading Frames/physiology , Protein Binding/physiology , RNA Polymerase II/metabolism , Ribosomal Proteins/genetics , Saccharomyces cerevisiae/metabolism , Sirtuin 2 , Sirtuins , Telomere/metabolism , Trans-Activators/metabolismABSTRACT
The combination of genome-wide expression patterns and full genome sequences offers a great opportunity to further our understanding of the mechanisms and logic of transcriptional regulation. Many methods have been described that identify sequence motifs enriched in transcription control regions of genes that share similar gene expression patterns. Here we present an alternative approach that evaluates the transcriptional information contained by specific sequence motifs by computing for each motif the mean expression profile of all genes that contain the motif in their transcription control regions. These genome-mean expression profiles (GMEP's) are valuable for visualizing the relationship between genome sequences and gene expression data, and for characterizing the transcriptional importance of specific sequence motifs. Analysis of GMEP's calculated from a dataset of 519 whole-genome microarray experiments in Saccharomyces cerevisiae show a significant correlation between GMEP's of motifs that are reverse complements, a result that supports the relationship between GMEP's and transcriptional regulation. Hierarchical clustering of GMEP's identifies clusters of motifs that correspond to binding sites of well-characterized transcription factors. The GMEP's of these clustered motifs have patterns of variation across conditions that reflect the known activities of these transcription factors. Software that computed GMEP's from sequence and gene expression data is available under the terms of the Gnu Public License from http://rana.lbl.gov/.
Subject(s)
Algorithms , Gene Expression Profiling/statistics & numerical data , Genome , Base Sequence , Cluster Analysis , Computational Biology , DNA, Fungal/genetics , Genome, Fungal , Oligonucleotide Array Sequence Analysis/statistics & numerical data , Saccharomyces cerevisiae/genetics , SoftwareABSTRACT
The SOS response in UV-irradiated Escherichia coli includes the upregulation of several dozen genes that are negatively regulated by the LexA repressor. Using DNA microarrays containing amplified DNA fragments from 95.5% of all open reading frames identified on the E. coli chromosome, we have examined the changes in gene expression following UV exposure in both wild-type cells and lexA1 mutants, which are unable to induce genes under LexA control. We report here the time courses of expression of the genes surrounding the 26 documented lexA-regulated regions on the E. coli chromosome. We observed 17 additional sites that responded in a lexA-dependent manner and a large number of genes that were upregulated in a lexA-independent manner although upregulation in this manner was generally not more than twofold. In addition, several transcripts were either downregulated or degraded following UV irradiation. These newly identified UV-responsive genes are discussed with respect to their possible roles in cellular recovery following exposure to UV irradiation.
Subject(s)
Escherichia coli/genetics , Escherichia coli/radiation effects , Gene Expression Profiling , SOS Response, Genetics , Ultraviolet Rays , Bacterial Proteins/genetics , Base Sequence , DNA, Bacterial , Oligonucleotide Array Sequence Analysis , Serine Endopeptidases/genetics , Transcription, GeneticABSTRACT
Using a cDNA microarray, we compared the expression of approximately 8000 genes between two unique, clonally related T cell lines derived from different stages of a progressive T cell lymphoma involving skin. A total of 180 genes was found to be differentially expressed at the RNA level by a factor of fivefold or greater. Compared with the cells from the earlier, clinically indolent stage of the lymphoma, 56 genes were up-regulated, whereas 124 genes were down-regulated in the cells from the advanced, clinically aggressive stage lymphoma. The functions of approximately 65% of these genes are currently unknown. The 22 genes with a known function that were up-regulated in the advanced lymphoma cells included several genes involved in promotion of cell proliferation and survival as well as drug resistance. The 42 functionally characterized genes that were down-regulated in the advanced lymphoma cells included negative regulators of cell activation and cell cycle, and mediators of cell adhesion, apoptosis, and genome integrity. The differential expression identified by the cDNA microarray analysis was confirmed for selected genes by reverse transcription-polymerase chain reaction and Northern blotting. The identified differences in gene expression may be related to the differences in behavior between the early and advanced stages of the T cell lymphoma and point to directions for further investigations into mechanisms of lymphoma progression.
Subject(s)
DNA, Complementary/genetics , Gene Expression Profiling , Lymphoma, T-Cell/genetics , Oligonucleotide Array Sequence Analysis , Blotting, Northern , Disease Progression , Humans , Lymphoma, T-Cell/pathology , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, CulturedABSTRACT
BACKGROUND: We have developed and tested a method for printing protein microarrays and using these microarrays in a comparative fluorescence assay to measure the abundance of many specific proteins in complex solutions. A robotic device was used to print hundreds of specific antibody or antigen solutions in an array on the surface of derivatized microscope slides. Two complex protein samples, one serving as a standard for comparative quantitation, the other representing an experimental sample in which the protein quantities were to be measured, were labeled by covalent attachment of spectrally resolvable fluorescent dyes. RESULTS: Specific antibody-antigen interactions localized specific components of the complex mixtures to defined cognate spots in the array, where the relative intensity of the fluorescent signal representing the experimental sample and the reference standard provided a measure of each protein's abundance in the experimental sample. To test the specificity, sensitivity and accuracy of this assay, we analyzed the performance of 115 antibody/antigen pairs. 50% of the arrayed antigens and 20% of the arrayed antibodies provided specific and accurate measurements of their cognate ligands at or below concentrations of 0.34 microg/ml and 1.6 microg/ml, respectively. Some of the antibody/antigen pairs allowed detection of the cognate ligands at absolute concentrations below 1 ng/ml, and partial concentrations of 1 part in 106, sensitivities sufficient for measurement of many clinically important proteins in patient blood samples. CONCLUSIONS: These results suggest that protein microarrays can provide a practical means to characterize patterns of variation in hundreds of thousands of different proteins in clinical or research applications.