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In this report, we describe the development of a mini-array system suitable for high-throughput quantification of proteins. This mini-array is a multiplexed, sandwich-type ELISA that measures the concentration of seven different human cytokines--TNF-alpha, IFN alpha, IFN gamma, IL-1 alpha, IL-1 beta, IL-6, and IL-10--from a single sample in each well of a 96-well plate. The mini-array is produced by spotting monoclonal antibodies (mAbs) in a 3 x 3 pattern in the bottom of the wells of 96-well polystyrene plates. Cytokines that are captured by the arrayed mAbs are detected by using biotinylated mAbs, followed by the addition of a streptavidin-horseradish peroxidase (HRP) conjugate and a chemiluminescent substrate. The light produced from the HRP-catalyzed oxidation of the substrate is measured at each spot in the array by imaging the entire plate with a commercially available CCD camera. Here, we demonstrate that these 96-well-plate format mini-arrays have performance characteristics that make them suitable for the high-throughput screening of anti-inflammatory compounds.

We found that the genes for human U2 small nuclear RNA (snRNA) are organized as a nearly perfect tandem array of 10 to 20 copies per haploid genome. Although the coding region for the mature form of U2 RNA was only 188 base pairs (bp) long, the basic repeating unit of the tandem array was 6 kilobase pairs in length. Comparison of DNA sequences immediately upstream from human Ul and U2 genes revealed two regions of strong homology: region I (15 bp long) lay upstream of region II (20 bp long) and was separated from it by about the same distance in Ul genes (25 bp) as in U2 genes (21 bp); however, region I and region II were located 174 bp further upstream from the 5' end of the snRNA coding sequence in Ul genes than in U2 genes. Homologs of region II were also found upstream of the snRNA coding region in a mouse U2 gene and two rat Ul genes. Murphy et al. (Cell 29:265-274, 1982) U2 RNA is an abundant small nuclear RNA (snRNA) found in both plant and animal cells (5). The primary sequence of this snRNA species has been highly conserved through evolution as judged by nucleotide sequence analysis of U2 RNA from rat (30), chicken, pheasant (4), and wheat embryo (J. M. Skuzeski, personal communication), fingerprint analysis of U2 RNA from mouse and man (17,26), and DNA sequence analysis of a gene for mouse (27) and frog U2 snRNA (20). U2, like Ul, has a 5' terminal 2,2,7-trimethylguanosine cap structure, and both Ul and U2 appear to be transcribed by RNA polymerase 11 (5, 25). U2 RNA, as well as the snRNAs Ul, U4, U5, and U6 exist as components of small nuclear ribonucleoprotein particles (13,14,17). Indirect evidence suggests that Ul small nuclear ribonucleoproteins play a role in the nuclear splicing of mRNA precursors (16,23), and a similar role for U2 small nuclear ribonucleoproteins has been proposed (28; but see reference 4).The initial characterization of the multigene families for human Ul, U2, U3, U4, and U6 snRNAs has established that most of the human chromosomal loci complementary to these snRNAs are actually defective gene copies, or pseudogenes, which appear to be dispersed in the genome (2,8,9,11,12,19,22,36,41 Fig. 2A and C) was labeled in vitro with [c-32P]dATP by primed synthesis of the complementary strand.Blotting procedures. Genomic blots were prepared by the method of Southern (34) except that the DNA was depurinated by soaking the gel in 0.25 N HCl before denaturation (37).on May 10, 2018 by guest

We have cloned and partially characterized 24 loci from the human genome which are complementary to U1, U2, or U3, the three major species of small nuclear RNA (snRNA) in HeLa cells. When compared to the known Ul (human) and U2 (rat) snRNA sequences, the DNA sequences we report here for the complementary regions from two of the clones, Ul Among the various classes of molecules found in all eukaryotic cells, the small nuclear RNAs (snRNAs) have received relatively little attention until recently. The sequences of eight of these small homogeneous RNA species from mammalian cells (Ul, U2, U3A, U3B, U4, U5, U6, and 4.5S RNA) have been determined (1-6). U3 snRNA is found in the nucleolus, and other snRNA species are associated with either the nucleoskeleton or the nucleoplasm (7). At least three of the snRNAs are subject to considerable evolutionary conservation: Ul and U2 snRNA from chicken, rat, mouse, and human cells yield identical ribonuclease Ti fingerprints (1,8,9), and the RNA sequences of U1 from HeLa cells and Ula from rat Novikoff hepatoma differ in only 2 of 165 positions (1). We have also shown that a snRNA from the lower eukaryotic cellular slime mold Dictyostelium discoideum is over 40% homologous to U3 snRNA from the rat (10).Two recent developments have prompted new interest in the intracellular packaging and function of the snRNAs. First, antibodies produced by patients with the autoimmune disease systemic lupus erythematosus have been shown to recognize discrete cellular components that contain snRNAs complexed with a defined set of proteins (8), thus opening the way for detailed structural studies of small nuclear ribonucleoprotein particles. Second, Lerner et al. (9) (kb) (17).We originally decided to clone the genes encoding snRNAs in the hope that a systematic investigation of their organization and expression would also open up new avenues for understanding the function of the snRNAs themselves. We report here the surprising result that most, if not all, of the genomic loci complementary to snRNAs U1, U2, and U3 that we have examined contain divergent, and in some cases truncated, gene copies when compared with the corresponding HeLa cell snRNA species. MATERIALS AND METHODSA library of 15-kb partial EcoRI fragments of human placental DNA in the A vector Charon 4A (18) was kindly supplied by A. Biro, P. V. Choudary, J. T. Elder, and S. M. Weissman. Plaques were screened by the method of Benton and Davis (19); we used as probes U1, U2, and U3 snRNAs isolated from HeLa cells and labeled in vitro at the 3' end with 5'-32P-labeled pCp and T4 RNA ligase as described (14). As little as 106 cpm of snRNA at a specific activity of 106 cpm/,ug was sufficient to screen six 140-mm nitrocellulose filters, each bearing 8000 plaques. With each probe, 0. 1% of the plaques were scored as positive and repurified for further study. Small quantities of recombinant DNA were prepared from 4-ml NZY cultures (20); larger quantities were prepared from recombinant phage grown and purified as described (18)....

A genomic clone (XScG7) from Saccharomyces cerevisiae encoded a 650-nucleotide poly(A)-containing [poly(A)+] RNA that was about 50 times more abundant in MATa cells that had been exposed to the peptide pheromone a-factor than in untreated cells. This RNA was transcribed from a cluster of repetitive sequences: both intact and truncated delta and sigma elements adjacent to a tRNATTrP gene. Strand-specific probes indicated that this RNA initiated within an intact sigma element and contained sigma sequences at its 5' end. MATa cells produced two other prominent poly(A)+ RNAs (500 and 5,300 bases) in response to a-factor that were homologous to the same strand of sigma but transcribed from other locations in the genome. Induction of the sigma-related transcripts was rapid, was not blocked by inhibition of protein synthesis, required a functional receptor (STE2 gene product), and hence appeared to be a primary response to pheromone. Pulse-labeling confirmed that accumulation of sigma RNA following a-factor administration was accounted for by an increase in its rate of transcription. The sigma RNAs also were induced in MATa cells that had been treated with a-factor, but were not present at significant levels in MATaIMATa diploids. In MATa cells transformed with a plasmid in which the XScG7 sigma element was inserted just upstream of a gene coding for the intracellular form of invertase (SUC2) lacking its own promoter, a new poly(A)+ RNA (2.2 kilobases) appeared in response to a-factor that hybridized to both sigma and SUC2 probes, and intracellular invertase activity was elevated about 10-fold within 30 min. Primer extension showed that transcription from the hybrid gene initiated exclusively within the sigma sequence (117 nucleotides from the 3' end of the element).Regulation of cellular physiology and development in multicellular organisms is a complex process that often involves modulation of gene expression by peptides, growth factors, and other hormones (35,41,47). In the unicellular eucaryote Saccharomyces cerevisiae, mating between haploids of opposite cell type (MATa cells and MATa cells) is initiated by their exposure to mating pheromones. The mating pheromones are oligopeptides (MATa cells secrete a-factor and MATa cells produce a-factor) that elicit specific physiological changes in their target cells (including arrest of the cell cycle in the Gl phase, synthesis of cell surface agglutinins, and characteristic changes in cell shape) and are required for the mating response (for a review, see references 39 and 44). In addition to the genes encoding the pheromone precursors and the MAT locus which regulates their expression, other genetic loci have been identified that are required for mating competence. Two of these genes, STE2 and STE3, probably encode cell surface receptors for a-factor and a-factor, respectively (27,29).We and others have demonstrated that the events of the mating process are preceded by rapid and dramatic changes in gene expression (28, 40). We report here our discovery that transcripts f...