As we bring demethylating drugs to the clinic, we better know the DICE being cast.

In this issue, Weber and coworkers report that DNA-demethylating drugs alter the transcriptional expression of the cMet proto-oncogene. Abnormal transcription is driven by the antisense promoter of a Line-1 repetitive element present within an intron. The element is a recent addition to the genome and is absent in animal models of cancer.

Hepatic gene expression patterns in thyroid hormone-treated hypothyroid rats.

Thyroid hormone (T3) is essential for normal development, differentiation and metabolic balance. We have performed DNA microarray experiments using hepatic RNA from hypothyroid and T3-treated hypothyroid rats in order to characterize T3-induced gene expression patterns after various time points (6, 24 and 48 h after the administration of the hormone). Sixty-two of 4608 different genes displayed a reproducible T3-response, and cluster analysis divided these differentially regulated genes into six expression patterns. Thirty-six genes were not significantly regulated within the first 24 h. Transient transfection experiments of eight late-induced gene promoters failed to detect a thyroid hormone response element within their regulatory elements, suggesting an indirect activation mechanism(s). In search for an intermediate factor of T3 action, we examined whether various rather ubiquitous transcription factors, peroxisome proliferator-activated receptors (PPARs) and coactivators of the PPARgamma coactivator 1 family (PGC-1) are regulated by T3. Only PPARgamma and PERC/PGC-1beta exhibit a significant T3-response within the first 6 h after treatment, identifying these factors as candidate components for mediating the late-induced expression pattern. Regulation of early-induced genes within the first 6 h after administration of T3 on transcript levels correlates with altered protein levels after 24 and 48 h in vivo.

In situ detection of messenger RNA using digoxigenin-labeled oligonucleotides and rolling circle amplification.

The detection of specific RNA molecules in situ is routinely performed using haptenated probes, which are detected by either enzymatic amplification or direct fluorescence. A drawback of fluorescence labeling has been the reduced sensitivity relative to that of methods that use enzymes as signal generators. Reliable fluorescence detection methods often require the use of multiple oligonucleotide probes for each gene target. Here, we demonstrate that single haptenated DNA probes specific for actin mRNA may be detected in situ using antibody-coupled rolling circle amplification (immuno-RCA). This fluorescence-based detection method offers remarkable sensitivity due to the use of signal amplification and yet retains the ability to count hybridization signals as discrete objects. We demonstrate the detection of actin-specific immuno-RCA signals in the cytoplasm and use 3D image deconvolution of multiple z axis sections to show that there are hundreds of signals per cell. With some modifications, this method may be adaptable to the simultaneous detection of several RNA species, including low-copy-number mRNA.

Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibers using rolling circle DNA amplification.

Rolling circle amplification (RCA) is a surface-anchored DNA replication reaction that can be exploited to visualize single molecular recognition events. Here we report the use of RCA to visualize target DNA sequences as small as 50 nts in peripheral blood lymphocytes or in stretched DNA fibers. Three unique target sequences within the cystic fibrosis transmembrane conductance regulator gene could be detected simultaneously in interphase nuclei, and could be ordered in a linear map in stretched DNA. Allele-discriminating oligonucleotide probes in conjunction with RCA also were used to discriminate wild-type and mutant alleles in the cystic fibrosis transmembrane conductance regulator, p53, BRCA-1, and Gorlin syndrome genes in the nuclei of cultured cells or in DNA fibers. These observations demonstrate that signal amplification by RCA can be coupled to nucleic acid hybridization and multicolor fluorescence imaging to detect single nucleotide changes in DNA within a cytological context or in single DNA molecules. This provides a means for direct physical haplotyping and the analysis of somatic mutations on a cell-by-cell basis.

Immunoassays with rolling circle DNA amplification: a versatile platform for ultrasensitive antigen detection.

We describe an adaptation of the rolling circle amplification (RCA) reporter system for the detection of protein Ags, termed "immunoRCA. " In immunoRCA, an oligonucleotide primer is covalently attached to an Ab; thus, in the presence of circular DNA, DNA polymerase, and nucleotides, amplification results in a long DNA molecule containing hundreds of copies of the circular DNA sequence that remain attached to the Ab and that can be detected in a variety of ways. Using immunoRCA, analytes were detected at sensitivities exceeding those of conventional enzyme immunoassays in ELISA and microparticle formats. The signal amplification afforded by immunoRCA also enabled immunoassays to be carried out in microspot and microarray formats with exquisite sensitivity. When Ags are present at concentrations down to fM levels, specifically bound Abs can be scored by counting discrete fluorescent signals arising from individual Ag-Ab complexes. Multiplex immunoRCA also was demonstrated by accurately quantifying Ags mixed in different ratios in a two-color, single-molecule-counting assay on a glass slide. ImmunoRCA thus combines high sensitivity and a very wide dynamic range with an unprecedented capability for single molecule detection. This Ag-detection method is of general applicability and is extendable to multiplexed immunoassays that employ a battery of different Abs, each labeled with a unique oligonucleotide primer, that can be discriminated by a color-coded visualization system. ImmunoRCA-profiling based on the simultaneous quantitation of multiple Ags should expand the power of immunoassays by exploiting the increased information content of ratio-based expression analysis.

PNA molecular beacons for rapid detection of PCR amplicons.

The authors have developed a method for rapid detection of polymerase chain reaction (PCR) amplicons based on surface immobilized PNA-DNA hybrid probes ('molecular beacons') that undergo a fluorescent-linked conformational change in the presence of a complementary DNA target. Amplicons can be detected by simply adding a PCR reaction to a microtitre-well containing the previously immobilized probe, and reading the generated fluorescence. No further transfers or washing steps are involved. The authors demonstrate the specificity of the method for the detection of ribosomal DNA from Entamoeba histolytica.

Mutation detection and single-molecule counting using isothermal rolling-circle amplification.

Rolling-circle amplification (RCA) driven by DNA polymerase can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. In the presence of two primers, one hybridizing to the + strand, and the other, to the - strand of DNA, a complex pattern of DNA strand displacement ensues that generates 10(9) or more copies of each circle in 90 minutes, enabling detection of point mutations in human genomic DNA. Using a single primer, RCA generates hundreds of tandemly linked copies of a covalently closed circle in a few minutes. If matrix-associated, the DNA product remains bound at the site of synthesis, where it may be tagged, condensed and imaged as a point light source. Linear oligonucleotide probes bound covalently on a glass surface can generate RCA signals, the colour of which indicates the allele status of the target, depending on the outcome of specific, target-directed ligation events. As RCA permits millions of individual probe molecules to be counted and sorted using colour codes, it is particularly amenable for the analysis of rare somatic mutations. RCA also shows promise for the detection of padlock probes bound to single-copy genes in cytological preparations.

Entamoeba histolytica contains a gene encoding a homologue to the 54 kDa subunit of the signal recognition particle.

We have determined the nucleotide sequence and predicted amino acid sequence of the 54 kDa subunit of the signal recognition particle (SRP54) from the amitochondrial protist Entamoeba histolytica. The SRP54 gene was isolated from a genomic library using a polymerase chain reaction (PCR) probe. Nucleotide sequence analysis of a 2.3 kb fragment, derived from a 7 kb genomic clone, revealed an open reading frame encoding a protein of 487 amino acids (MW 53.8 kDa). The identities of the predicted amino acid sequence with its homologues from other species were between 24 and 47%. Functional domains previously defined for the SRP54-type proteins were present in the entamoebal sequence, such as the amino-terminal GTP binding domain (G domain) and the carboxy-terminal methionine rich domain (M domain). SRP54 mRNA contains an extra G residue at the 5' end, suggesting that capping of poly-A(+) transcripts is present in E. histolytica. Evolutionary analysis of the SRP54 based on phylogenetic inference placed the E. histolytica sequence as an early divergence of the eukaryotic tree. Although the function of the entamoebal homologue remains to be elucidated, the identification of the SRP54 gene constitutes the first evidence for SRP related proteins in protozoans.

Identification and analysis of the u6 small nuclear RNA gene from Entamoeba histolytica.

Among the small nuclear RNAs (snRNAs) involved in the spliceosomal processing of pre-mRNA, U6 is the most conserved. As a first evidence for the presence of the splicing machinery in the amitochondrial protozoan Entamoeba histolytica (Eh), we have cloned the u6 snRNA gene. We find that in this organism u6 is a single copy gene that is transcribed as a poly(A)- RNA molecule of approximately 105 nucleotides. We have mapped the 5' end of the U6 snRNA transcript, and identified typical elements of a putative polymerase III promoter. This is the first snRNA gene reported in Eh. Sequence analysis indicates that this gene contains all the conserved nucleotides known to be important for U6 snRNA function. These results, in conjunction with the earlier finding of genes that contain pre-mRNA introns, suggest that Eh has a functional spliceosomal complex.

Sequence-specific detection of PCR-amplified DNA by restriction enzyme release of hybrids.

We have developed a method to detect PCR amplicons that contain a restriction enzyme site. The method is called Polymerase Chain Reaction-Hybridization and Enzymatic Release Assay (PCR-HERA). The PCR product is hybridized to a synthetic oligonucleotide that is immobilized on a microtitre plate. Specific cleavage of the resulting hybrid by a restriction endonuclease liberates a fluorescein labeled arm of the immobilized DNA. We demonstrate the use of PCR-HERA to detect species-specific sequences of extra-chromosomal, ribosomal DNA, of Entamoeba histolytica. This highly specific assay does not require any washing steps and can be easily automated.

Extremely sensitive, background-free gene detection using binary probes and beta replicase.

We have developed a specific and sensitive nucleic acid amplification assay that is suitable for routine gene detection. The assay is based on a novel molecular genetic strategy in which two different RNA probes are hybridized to adjacent positions on a target nucleic acid and then ligated to form an amplifiable reporter RNA. The reporter RNA is then replicated up to a hundred billion-fold in a 30-min isothermal reaction that signals the presence of the target. The assay can detect fewer than 100 nucleic acid molecules; it provides quantitative results over a wide range of target concentrations and it employs a universal format that can detect any infectious agent.

Identification and analysis of the start site of ribosomal RNA transcription of Entamoeba histolytica.

In this article we report the identification of the start site of ribosomal RNA transcription unit of the enteric parasite E. histolytica. We cloned the upstream region of the ribosomal RNA and we defined the 5' boundary of the transcription unit with nuclear run-on assays. We report that ribosomal transcription starts 2447 bp upstream the SSU ribosomal gene, at an adenosine residue. This data was supported both by S1 mapping and by primer extension analysis; that the mapped site was indeed the transcription start point was demonstrated by RNAse protection of the in vitro capped RNA. Our sequence data around the transcription start point shows two different tandem repeat clusters immediately downstream from the transcription start point.

An allosteric hammerhead ribozyme.

We have constructed an RNA molecule containing a hammerhead ribozyme that is under allosteric control. In the inactive state, the RNA enzyme is unable to cleave a suitable substrate. The formation of the active state of the ribozyme is triggered by a specific interaction with a DNA oligonucleotide effector that is complementary to a single-stranded loop in the RNA enzyme molecule. Other DNA or RNA molecules containing unrelated nucleotide sequences do not function as allosteric effectors. This work demonstrates the feasibility of designing RNA enzymes that are specifically activated in response to an artificially designed molecular recognition event. Such enzymes may have practical applications.