Escherichia coli were separated from a mixture containing human blood cells by means of dielectrophoresis and then subjected to electronic lysis followed by proteolytic digestion on a single microfabricated bioelectronic chip. An alternating current electric field was used to direct the bacteria to 25 microlocations above individually addressable platinum microelectrodes. The platinum electrodes were 80 microns in diameter and had center-to-center spacings of 200 microns. After the isolation, the bacteria were lysed by a series of high-voltage pulses. The lysate contained a spectrum of nucleic acids including RNA, plasmid DNA, and genomic DNA. The lysate was further examined by electronically enhanced hybridization on separate bioelectronic chips. Dielectrophoretic separation of cells followed by electronic lysis and digestion on an electronically active chip may have potential as a sample preparation process for chip-based hybridization assays in an integrated DNA/RNA analysis system.

The structurally diverse peroxisome proliferators ciprofibrate, clofibrate, and bis(2-ethylhexyl) phthalate [(EtHX)2>Pht] increase the activities of hepatic catalase and peroxisomal fatty acid 3-oxidation enzymes in conjunction with profound proliferation of peroxisomes in hepatocytes. In order to delineate the level at which these enzymes are induced in the liver, the transcriptional activity of specific genes for fatty acyl-CoA oxidase (FAOxase) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (PBE), the first two enzymes of the peroxisomal f-oxidation system, and for catalase were measured in isolated hepatocyte nuclei obtained from male rats following a single intragastric dose of ciprofibrate, clofibrate, or (EtHx)2>Pht. All three peroxisome proliferators rapidly increased the rate of FAOxase and PBE gene transcription in liver, with near maximal rates (9-15 times control) reached by 1 hr and persisting until at least 16 hr after administration of the compound. FAOxase and PBE mRNA levels, measured by blot-hybridization analysis and FAOxase and PBE protein content, analyzed by immunoblotting, increased concurrently up to at least 16 hr following a single dose of peroxisome proliferator. The catalase mRNA level increased about 1.4-fold, but the transcription rate of the catalase gene was not sign tiy affected. The results show that the peroxisome proliferators clofibrate, ciprofibrate, and (EtHx)2>Pht selectively increase the rate of transcription of peroxisomal fatty add P-oxidation enzyme genes. Whether the tnscriptional effects are mediated by peroxisome proliferatorreceptor complexes remains to be elucidated.

DNA amplification systems are powerful technologies with the potential to impact a wide range of diagnostic applications. In this study we explored the feasibility and limitations of a modified ligase chain reaction (Gap-LCR) in detection and discrimination of DNAs that differ by a single base. LCR is a DNA amplification technology based on the ligation of two pairs of synthetic oligonucleotides which hybridize at adjacent positions to complementary strands of a target DNA. Multiple rounds of denaturation, annealing and ligation with a thermostable ligase result in the exponential amplification of the target DNA. A modification of LCR, Gap-LCR was developed to reduce the background generated by target-independent, blunt-end ligation. In Gap-LCR, DNA polymerase fills in a gap between annealed probes which are subsequently joined by DNA ligase. We have designed synthetic DNA targets with single base pair differences and analyzed them in a system where three common probes plus an allele-specific probe were used. A single base mismatch either at the ultimate 3' end or penultimate 3' end of the allele specific probe was sufficient for discrimination, though better discrimination was obtained with a mismatch at the penultimate 3' position. Comparison of Gap-LCR to allele-specific PCR (ASPCR) suggested that Gap-LCR has the advantage of having the additive effect of polymerase and ligase on specificity. As a model system, Gap-LCR was tested on a mutation in the reverse transcriptase gene of HIV, specifically, one of the mutations that confers AZT resistance. Mutant DNA could be detected and discriminated in the presence of up to 10,000-fold excess of wild-type DNA.

Philadelphia chromosome-positive acute lymphoblastic leukemia occurs in two molecular forms, those with and those without rearrangement of the breakpoint cluster region on chromosome 22. The molecular abnormality in the former group is similar to that found in chronic myelogenous leukemia. To characterize the abnormality in the breakpoint cluster region-unrearranged form, we have mapped a 9;22 translocation from the Philadelphia chromosome-positive acute lymphoblastic leukemia cell line SUP-B13 by using pulsed-field gel electrophoresis and have cloned the DNA at the translocation junctions. We demonstrate a BCR-ABL fusion gene on the Philadelphia chromosome. The breakpoint on chromosome 9 is within ABL between exons Ia and II, and the breakpoint on chromosome 22 is :50 kilobases upstream of a breakpoint cluster region in an intron of the BCR gene. This upstream BCR breakpoint leads to inclusion of fewer BCR sequences in the fusion gene, compared with the BCR-ABL fusion gene of chronic myelogenous leukemia. Consequently, the associated mRNA and protein are smaller. The exons from ABL are the same. Analysis of leukemic cells from four other patients with breakpoint cluster regionunrearranged Philadelphia chromosome-positive acute lymphoblastic leukemia revealed a rearrangement on chromosome 22 close to the breakpoint in SUP-B13 in only one patient. These data indicate that breakpoints do not cluster tightly in this region but are scattered, possibly in a large intron. Given the large size of BCR and the heterogeneity in breakpoint location, detection of BCR rearrangement by standard Southern blot analysis is difficult. Pulsed-field gel electrophoresis should allow detection at the DNA level in every patient and thus will permit clinical correlation of the breakpoint location with prognosis.A Philadelphia chromosome (Ph') resulting from a 9;22 translocation, t(9;22)(q34;qll), characterizes the malignant cells in virtually all patients with chronic myelogenous leukemia (CML) (1, 2), in 10% of those with acute lymphoblastic leukemia (ALL) (3), and in rare cases of acute myelogenous leukemia (4). The t(9;22) is the most common chromosomal abnormality in adults with ALL (3); it is found in one of five of these patients and in well over half of those with specific chromosomal translocations. In children with ALL, -6% are found to have the t(9;22). In CML and in a subgroup of Ph'-positive ALL, the breakpoints of the translocation occur in a 200-kilobase (kb) area within the 5' introns of the ABL gene (5) and in the 5.8-kb breakpoint cluster region (bcr) of the BCR gene (6, 7) on chromosomes 9 and 22, respectively. This results in a 5' BCR-ABL 3' fusion gene on the Ph' (8,9) that is expressed as an 8.5-kb hybrid BCR-ABL mRNA. The translation product of the hybrid message is a 210-kDa tyrosine kinase (P210BCRABL) (10). The P210 is thought to play a central role in the malignant process (11). In contrast, in many cases of Ph'-positive ALL, the breakpoint on chromosome 22 is not in a bcr (12)(13)(14), and the 8.5-kb ...

Abstract. In naturally synchronous plasmodia of Physarum polycephalum, both tubulin and histone gene transcription define periodic cell cycle-regulated events. Using a slot-blot hybridization assay and Northern blot analysis, we have demonstrated that a major peak of accumulation of both a-tubulin and histone H4 transcripts occurs in late G2 phase. Nuclear transcription assays indicate that both genes are transcriptionally activated at the same point in the cell cycle: mid G2 phase. While the rate of tubulin gene transcription drops sharply at the M/S-phase boundary, the rate of histone gene transcription remains high through most of S phase. We conclude that the cell cycle regulation of tubulin expression occurs primarily at the level of transcription, while histone regulation involves both transcriptional and posttranscriptional controls. It is possible that the periodic expression of both histone and tubulin genes is triggered by a common cell cycle regulatory mechanism.