Novel transcribed regions in the human genome.

We have used genomic tiling arrays to identify transcribed regions throughout the human genome. Analysis of the mapping results of RNA isolated from five cell/tissue types, NB4 cells, NB4 cells treated with retinoic acid (RA), NB4 cells treated with 12-O-tetradecanoylphorbol-13 acetate (TPA), neutrophils, and placenta, throughout the ENCODE region reveals a large number of novel transcribed regions. Interestingly, neutrophils exhibit a great deal of novel expression in several intronic regions. Comparison of the hybridization results of NB4 cells treated with different stimuli relative to untreated cells reveals that many new regions are expressed upon cell differentiation. One such region is the Hox locus, which contains a large number of novel regions expressed in a number of cell types. Analysis of the trinucleotide composition of the novel transcribed regions reveals that it is similar to that of known exons. These results suggest that many of the novel transcribed regions may have a functional role.

Transcriptional landscape of the human and fly genomes: nonlinear and multifunctional modular model of transcriptomes.

Regions of the genome not coding for proteins or not involved in cis-acting regulatory activities are frequently viewed as lacking in functional value. However, a number of recent large-scale studies have revealed significant regulated transcription of unannotated portions of a variety of plant and animal genomes, allowing a new appreciation of the widespread transcription of large portions of the genome. High-resolution mapping of the sites of transcription of the human and fly genomes has provided an alternative picture of the extent and organization of transcription and has offered insights for biological functions of some of the newly identified unannotated transcripts. Considerable portions of the unannotated transcription observed are developmental or cell-type-specific parts of protein-coding transcripts, often serving as novel, alternative 5' transcriptional start sites. These distal 5' portions are often situated at significant distances from the annotated gene and alternatively join with or ignore portions of other intervening genes to comprise novel unannotated protein-coding transcripts. These data support an interlaced model of the genome in which many regions serve multifunctional purposes and are highly modular in their utilization. This model illustrates the underappreciated organizational complexity of the genome and one of the functional roles of transcription from unannotated portions of the genome.

Prokaryotic RNA preparation methods useful for high density array analysis: comparison of two approaches.

High density oligonucleotide arrays have been used extensively for expression studies of eukaryotic organisms. We have designed a prokaryotic high density oligonucleotide array using the complete Escherichia coli genome sequence to monitor expression levels of all genes and intergenic regions in the genome. Because previously described methods for preparing labeled target nucleic acids are not useful for prokaryotic cell analysis using such arrays, a mRNA enrichment and direct labeling protocol was developed together with a cDNA synthesis protocol. The reproducibility of each labeling method was determined using high density oligonucleotide probe arrays as a read-out methodology and the expression results from direct labeling were compared to the expression results from the cDNA synthesis. About 50% of all annotated E.coli open reading frames are observed to be transcribed, as measured by both protocols, when the cells were grown in rich LB medium. Each labeling method individually showed a high degree of concordance in replica experiments (95 and 99%, respectively), but when each sample preparation method was compared to the other, approximately 32% of the genes observed to be expressed were discordant. However, both labeling methods can detect the same relative gene expression changes when RNA from IPTG-induced cells was labeled and compared to RNA from uninduced E.coli cells.

Reprogramming of the macrophage transcriptome in response to interferon-gamma and Mycobacterium tuberculosis: signaling roles of nitric oxide synthase-2 and phagocyte oxidase.

Macrophage activation determines the outcome of infection by Mycobacterium tuberculosis (Mtb). Interferon-gamma (IFN-gamma) activates macrophages by driving Janus tyrosine kinase (JAK)/signal transducer and activator of transcription-dependent induction of transcription and PKR-dependent suppression of translation. Microarray-based experiments reported here enlarge this picture. Exposure to IFN-gamma and/or Mtb led to altered expression of 25% of the monitored genome in macrophages. The number of genes suppressed by IFN-gamma exceeded the number of genes induced, and much of the suppression was transcriptional. Five times as many genes related to immunity and inflammation were induced than suppressed. Mtb mimicked or synergized with IFN-gamma more than antagonized its actions. Phagocytosis of nonviable Mtb or polystyrene beads affected many genes, but the transcriptional signature of macrophages infected with viable Mtb was distinct. Studies involving macrophages deficient in inducible nitric oxide synthase and/or phagocyte oxidase revealed that these two antimicrobial enzymes help orchestrate the profound transcriptional remodeling that underlies macrophage activation.

Flexible use of high-density oligonucleotide arrays for single-nucleotide polymorphism discovery and validation.

A method for identifying and validating single nucleotide polymorphisms (SNPs) with high-density oligonucleotide arrays without the need for locus-specific polymerase chain reactions (PCR) is described in this report. Genomic DNAs were divided into subsets with complexity of ~10 Mb by restriction enzyme digestion and gel-based fragment size resolution, ligated to a common adaptor, and amplified with one primer in a single PCR reaction. As a demonstration of this approach, a total of 124 SNPs were located in 190 kb of genomic sequences distributed across the entire human genome by hybridizing to high-density variant detection arrays (VDA). A set of independent validation experiments was conducted for these SNPs employing bead-based affinity selection followed by hybridization of the affinity-selected SNP-containing fragments to the same VDA that was used to identify the SNPs. A total of 98.7% (74/75) of these SNPs were confirmed using both DNA dideoxynucleotide sequencing and the VDA methodologies. With flexible sample preparation, high-density oligonucleotide arrays can be tailored for even larger scale genome-wide SNP discovery as well as validation.

Comparing genomes within the species Mycobacterium tuberculosis.

The study of genetic variability within natural populations of pathogens may provide insight into their evolution and pathogenesis. We used a Mycobacterium tuberculosis high-density oligonucleotide microarray to detect small-scale genomic deletions among 19 clinically and epidemiologically well-characterized isolates of M. tuberculosis. The pattern of deletions detected was identical within mycobacterial clones but differed between different clones, suggesting that this is a suitable genotyping system for epidemiologic studies. An analysis of genomic deletions among an extant population of pathogenic bacteria provided a novel perspective on genomic organization and evolution. Deletions are likely to contain ancestral genes whose functions are no longer essential for the organism's survival, whereas genes that are never deleted constitute the minimal mycobacterial genome. As the amount of genomic deletion increased, the likelihood that the bacteria will cause pulmonary cavitation decreased, suggesting that the accumulation of mutations tends to diminish their pathogenicity. Array-based comparative genomics is a promising approach to exploring molecular epidemiology, microbial evolution, and pathogenesis.

Detection of deleted genomic DNA using a semiautomated computational analysis of GeneChip data.

Genomic diversity within and between populations is caused by single nucleotide mutations, changes in repetitive DNA systems, recombination mechanisms, and insertion and deletion events. The contribution of these sources to diversity, whether purely genetic or of phenotypic consequence, can only be investigated if we have the means to quantitate and characterize diversity in many samples. With the advent of complete sequence characterization of representative genomes of different species, the possibility of developing protocols to screen for genetic polymorphism across entire genomes is actively being pursued. The large numbers of measurements such approaches yield demand that we pay careful attention to the numerical analysis of data. In this paper we present a novel application of an Affymetrix GeneChip to perform genome-wide screens for deletion polymorphism. A high-density oligonucleotide array formatted for mRNA expression and targeted at a fully sequenced 4.4-million-base pair Mycobacterium tuberculosis standard strain genome was adapted to compare genomic DNA. Hybridization intensities to 111,000 probe pairs (perfect complement and mismatch complement) were measured for genomic DNA from a clinical strain and from a vaccine organism. Because individual probe-pair hybridization intensities exhibit limited sensitivity/specificity characteristics to detect deletions, data-analytical methodology to exploit measurements from multiple probes in tandem locations across the genome was developed. The TSTEP (Tandem Set Terminal Extreme Probability) algorithm designed specifically to analyze the tandem hybridization measurements data was applied and shown to discover genomic deletions with high sensitivity. The TSTEP algorithm provides a foundation for similar efforts to characterize deletions in many hybridization measures in similar-sized and larger genomes. Issues relating to the design of genome content screening experiments and the implications of these methods for studying population genomics and the evolution of genomes are discussed.

Analysis of drug pharmacology towards predicting drug behavior by expression profiling using high-density oligonucleotide arrays.

An important aspect of the drug development process is prediction of efficacious and toxic side effects. Profiling of mRNA expression is a powerful approach to analyze the molecular phenotype of cells under various conditions, for example, in response to stimulation by compounds. We attempt to explore the approach of using expression profiling to identify patterns or fingerprints that are correlated with specific drug properties or behaviors. Identification of such expression patterns may also lead to revelation of the potential action mechanism of drugs and fingerprints indicative of certain drug efficacy or side effects. We describe here a strategy that was used to identify a set of genes whose differential expression pattern correlates with activation mode and target specificity of a specific group of drug compounds.

The transcriptional responses of respiratory epithelial cells to Bordetella pertussis reveal host defensive and pathogen counter-defensive strategies.

Bordetella pertussis, the causative agent of whooping cough, has many well-studied virulence factors and a characteristic clinical presentation. Despite this information, it is not clear how B. pertussis interaction with host cells leads to disease. In this study, we examined the interaction of B. pertussis with a human bronchial epithelial cell line (BEAS-2B) and measured host transcriptional profiles by using high-density DNA microarrays. The early transcriptional response to this pathogen is dominated by altered expression of cytokines, DNA-binding proteins, and NFkappaB-regulated genes. This previously unrecognized response to B. pertussis was modified in similar but nonidentical fashions by the antiinflammatory agents dexamethasone and sodium salicylate. Cytokine protein expression was confirmed, as was neutrophil chemoattraction. We show that B. pertussis induces mucin gene transcription by BEAS-2B cells then counters this defense by using mucin as a binding substrate. A set of genes is described for which the catalytic activity of pertussis toxin is both necessary and sufficient to regulate transcription. Host genomic transcriptional profiling, in combination with functional assays to evaluate subsequent biological events, provides insight into the complex interaction of host and pathogen.

Emergence of dual resistance to zidovudine and lamivudine in HIV-1-infected patients treated with zidovudine plus lamivudine as initial therapy.

Presence of mutations associated with resistance to zidovudine or lamivudine was determined in isolates of HIV-1 obtained after long-term follow-up of 64 infected individuals who received zidovudine, lamivudine, or both drugs as initial antiretroviral therapy. Zidovudine resistance mutations were less frequent in isolates from patients treated with combination lamivudine plus zidovudine compared with zidovudine alone, but these mutations accumulated over time. Phenotypic resistance to both drugs was found in isolates from 3 of 23 patients. In 3 other patients, lamivudine-resistant virus detected at week 12 was replaced by wild-type virus after longer follow-up, which correlated with a return to baseline levels of plasma HIV-1 RNA. These results show that dual resistance to zidovudine and lamivudine develops over time despite the delayed emergence of zidovudine-resistant mutations. These results also suggest a selective advantage in vivo for HIV-1 species that are wild-type at RT codon 184.

HIV-1 infectability of CD4+ lymphocytes with relation to beta-chemokines and the CCR5 coreceptor.

CD4+ lymphocytes exhibit variable permissiveness to the replication of HIV-1. A cohort of sexually-exposed-yet-uninfected individuals were previously shown to have CD4+ lymphocytes refractory for M-tropic viral replication. In particular, two individuals from this population whose CD4+ lymphocytes exhibited complete resistance to M-tropic viral replication were later shown to be homozygous for a 32bp (delta32) deletion in the gene encoding for CCR5. In screening diverse populations, HIV-1 infected individuals heterozygous for the delta32 allele were statistically favored in their disease course to harbor lower viral loads and exhibit slower rates of CD4+ cell loss when compared to control CCR5 wild-type individuals. Further comparative analysis between individuals in the exposed but uninfected cohort who demonstrated intermediate levels of in vitro viral replication and CD4+ lymphocytes isolated from uninfected delta32 heterozygous individuals indicate that reduced levels of in vitro M-tropic replication are a CCR5-related phenomenon: CD4+ lymphocytes from these individuals were more sensitive to the HIV-1 blocking effects of recombinant chemokines, displayed lower CCR5 cell surface expression levels and a proportionate increase in the production of RANTES when compared to CD4+ lymphocytes from control individuals. These results suggest that the CCR5 phenotype is important in determining the replicative capacity of M-tropic HIV-1 in vitro. The implications of these results with relation to HIV-1 transmission and disease progression are discussed.

High density synthetic oligonucleotide arrays.

Experimental genomics involves taking advantage of sequence information to investigate and understand the workings of genes, cells and organisms. We have developed an approach in which sequence information is used directly to design high-density, two-dimensional rays of synthetic oligonucleotides. The GeneChipe probe arrays are made using spatially patterned, light-directed combinatorial chemical synthesis and contain up to hundreds of thousands of different oligonucleotides on a small glass surface. The arrays have been designed and used for quantitative and highly parallel measurements of gene expression, to discover polymorphic loci and to detect the presence of thousands of alternative alleles. Here, we describe the fabrication of the arrays, their design and some specific applications to high-throughput genetic and cellular analysis.

Mycobacterium species identification and rifampin resistance testing with high-density DNA probe arrays.

Species identification within the genus Mycobacterium and subsequent antibiotic susceptibility testing still rely on time-consuming, culture-based methods. Despite the recent development of DNA probes, which greatly reduce assay time, there is a need for a single platform assay capable of answering the multitude of diagnostic questions associated with this genus. We describe the use of a DNA probe array based on two sequence databases: one for the species identification of mycobacteria (82 unique 16S rRNA sequences corresponding to 54 phenotypical species) and the other for detecting Mycobacterium tuberculosis rifampin resistance (rpoB alleles). Species identification or rifampin resistance was determined by hybridizing fluorescently labeled, amplified genetic material generated from bacterial colonies to the array. Seventy mycobacterial isolates from 27 different species and 15 rifampin-resistant M. tuberculosis strains were tested. A total of 26 of 27 species were correctly identified as well as all of the rpoB mutants. This parallel testing format opens new perspectives in terms of patient management for bacterial diseases by allowing a number of genetic tests to be simultaneously run.

Simultaneous genotyping and species identification using hybridization pattern recognition analysis of generic Mycobacterium DNA arrays.

High-density oligonucleotide arrays can be used to rapidly examine large amounts of DNA sequence in a high throughput manner. An array designed to determine the specific nucleotide sequence of 705 bp of the rpoB gene of Mycobacterium tuberculosis accurately detected rifampin resistance associated with mutations of 44 clinical isolates of M. tuberculosis. The nucleotide sequence diversity in 121 Mycobacterial isolates (comprised of 10 species) was examined by both conventional dideoxynucleotide sequencing of the rpoB and 16S genes and by analysis of the rpoB oligonucleotide array hybridization patterns. Species identification for each of the isolates was similar irrespective of whether 16S sequence, rpoB sequence, or the pattern of rpoB hybridization was used. However, for several species, the number of alleles in the 16S and rpoB gene sequences provided discordant estimates of the genetic diversity within a species. In addition to confirming the array's intended utility for sequencing the region of M. tuberculosis that confers rifampin resistance, this work demonstrates that this array can identify the species of nontuberculous Mycobacteria. This demonstrates the general point that DNA microarrays that sequence important genomic regions (such as drug resistance or pathogenicity islands) can simultaneously identify species and provide some insight into the organism's population structure.

Reduced HIV-1 infectability of CD4+ lymphocytes from exposed-uninfected individuals: association with low expression of CCR5 and high production of beta-chemokines.

We examined the human immunodeficiency virus type 1 infectability of CD4+ lymphocytes isolated from CCR5 wild-type individuals, individuals heterozygous for the delta32 allele of CCR5, and HIV-1-exposed but uninfected (EU) individuals who had CD4+ lymphocytes refractory to M-tropic viral replication. None of the EU individuals were found to be heterozygous for the delta32 allele. The CD4+ lymphocytes isolated from CCR5/delta32 and EU individuals were less infectable with an M-tropic viral isolate of HIV-1 than CCR5/CCR5 control individuals but were equally as infectable with a T-tropic viral isolate. The restriction to M-tropic viral isolate replication did not associate with any profound genotypic change in the CCR5 gene. CD4+ lymphocytes from CCR5/delta32 and CCR5/CCR5 EU individuals were more sensitive to the HIV-inhibitory effects of the recombinant beta-chemokines RANTES, MIP-1alpha, and MIP-1beta than were CD4+ lymphocytes from CCR5/CCR5 control individuals. CD4+ lymphocytes from EU individuals also showed increased sensitivity to recombinant beta-chemokines and low surface expression of CCR5. A phenotype of low CCR5 expression and high secretion of beta-chemokines is associated with reduced infectability of cells by M-tropic HIV-1. This phenotype may also be associated with protection against sexual transmission of HIV-1.

Extensive polymorphisms observed in HIV-1 clade B protease gene using high-density oligonucleotide arrays.

Naturally occurring mutations in HIV-1-infected patients have important implications for therapy and the outcome of clinical studies. However, little is known about the prevalence of mutations that confer resistance to HIV-1 protease inhibitors in isolates derived from patients naive for such inhibitors. In the first clinical application of high-density oligonucleotide array sequencing, the sequences of 167 viral isolates from 102 patients have been determined. The DNA sequence of USA HIV-1 clade B proteases was found to be extremely variable and 47.5% of the 99 amino acid positions varied. This level of amino acid diversity is greater than that previously known for all worldwide HIV-1 clades combined (40%). Many of the amino acid changes that are known to contribute to drug resistance occurred as natural polymorphisms in isolates from patients who had never received protease inhibitors.

Defining subsets of naive and memory B cells based on the ability of their progeny to somatically mutate in vitro.

The increased affinity of memory antibody responses is due largely to the generation and selection of memory B cells that accumulate somatic mutations after initial antigenic stimulation. Further affinity maturation and mutation also accompany subsequent immunizations. Previous studies have suggested that, like primary antibody-forming cell (AFC) clones, secondary AFC do not accumulate further mutations and, therefore, the origins of progressive affinity maturation remain controversial. Here, we report the generation of somatically mutated memory B cell clones in vitro. Our findings confirm the existence of a naive B cell subset whose progeny, rather than generating AFC, somatically mutate and respond to subsequent antigenic stimulation. Interestingly, upon stimulation, a subset of memory B cells also generates antigen-responsive cells that accumulate further somatic mutations.

Specific immunoglobulin cDNA clones produced from hybridoma cell lines and murine spleen fragment cultures by 3SR amplification.

The isothermal 3SR amplification method has been employed to assist in cloning the VL and VH genes from cells of hybridomas and splenic fragment cultures expressing antibodies for phosphorylcholine (PC) and estradiol (E2), respectively. As a first step, pools of degenerate primer pairs were identified complementary to immunoglobulin light and heavy chain variable (V) genes and capable of amplifying immunoglobulin RNA specifically at 42 degrees C. To evaluate the functionality of the 3SR-cloned immunoglobulin genes, anti-PC VH and VL cDNAs were joined together to form a single chain (sc) antibody construct and were expressed in Escherichia coli under the regulation of the alkaline phosphatase (phoA) promoter. Similarly, the combination of a murine spleen fragment and 3SR methodologies were employed to clone a selected pool of cDNAs for cultures producing anti-estradiol antibodies. This approach of using the murine spleen fragment and 3SR isothermal amplification offers the advantages of B-cell follicle architecture for antigen-driven B-cell maturation and proliferation and RNA-specific amplification, respectively. The potential utility of these advantages for the production of monoclonal antibodies and for providing the capability of studying memory B-cell development are discussed.

Use of self-sustained sequence replication amplification reaction to analyze and detect mutations in zidovudine-resistant human immunodeficiency virus.

Mutations at amino acid positions 67, 70, 215, and 219 in the human immunodeficiency virus type 1 (HIV-1) pol gene correlate with the emergence of resistance to zidovudine (AZT). These four positions were monitored in viral RNA extracted from infected peripheral blood mononuclear cells (PBMC) and viral stocks obtained after coculture with uninfected lymphocytes. Genotype determinations were made using the self-sustained sequence replication (3SR) and differential bead-based sandwich hybridization (BBSH) assay. The hybridization results obtained by 3SR and BBSH analyses were verified by dideoxynucleotide sequencing of the 3SR products. Correlation of 3SR and BBSH with polymerase chain reaction and Southern hybridization analyses of the PBMC and corresponding viral isolates indicated that PBMC and corresponding HIV-1 isolates may differ in their genotypes at the monitored amino acid positions, variations from the wild-type nucleotide sequence may occur proximal to the codons being monitored, and viral isolates possessing the same genotypes at the four monitored amino acid positions showed a threefold variation in their ID50 measurements.