Extremely halophilic archaea contain retinal-binding integral membrane proteins called bacteriorhodopsins that function as light-driven proton pumps. So far, bacteriorhodopsins capable of generating a chemiosmotic membrane potential in response to light have been demonstrated only in halophilic archaea. We describe here a type of rhodopsin derived from bacteria that was discovered through genomic analyses of naturally occuring marine bacterioplankton. The bacterial rhodopsin was encoded in the genome of an uncultivated gamma-proteobacterium and shared highest amino acid sequence similarity with archaeal rhodopsins. The protein was functionally expressed in Escherichia coli and bound retinal to form an active, light-driven proton pump. The new rhodopsin exhibited a photochemical reaction cycle with intermediates and kinetics characteristic of archaeal proton-pumping rhodopsins. Our results demonstrate that archaeal-like rhodopsins are broadly distributed among different taxa, including members of the domain Bacteria. Our data also indicate that a previously unsuspected mode of bacterially mediated light-driven energy generation may commonly occur in oceanic surface waters worldwide.

An automated enzyme assay was performed within a microfabricated channel network. Precise concentrations of substrate, enzyme, and inhibitor were mixed in nanoliter volumes using electrokinetic flow. Reagent dilution and mixing were controlled by regulating the applied potential at the terminus of each channel, using voltages derived from an equivalent circuit model of the microchip. The enzyme beta-galactosidase (beta-Gal) was assayed using resorufin beta-D-galactopyranoside (RBG), a substrate that is hydrolyzed to resorufin, a fluorescent product. Reaction kinetics were obtained by varying the concentration of substrate on-chip and monitoring the production of resorufin using laser-induced fluorescence. Derived Michaelis--Menten constants compared well between an on-chip and a conventional enzyme assay. Bias in the derived K(m) and kcat was primarily due to the limited solubility of RBG and the associated lack of measurements at substrate concentrations exceeding the K(m). A Ki of 8 microM for the inhibitor phenylethyl beta-D-thiogalactoside (PETG) was determined from plots of initial rate versus substrate concentration obtained at three concentrations of PETG. The relative inhibition of beta-Gal by lactose, p-hydroxymercuribenzoic acid, and PETG was determined by varying the inhibitor concentration with constant enzyme and substrate concentration. An enzyme assay performed on the microchip within a 20-min period required only 120 pg of enzyme and 7.5 ng of substrate, reducing the amount of reagent consumed by 4 orders of magnitude over a conventional assay.

BACKGROUND Fragile X Syndrome (FXS) is a trinucleotide repeat disease that is caused by the expansion of CGG sequences in the 5’ untranslated region of the FMR1 gene. Molecular diagnoses of FXS and other emerging FMR1 disorders typically rely on two tests, PCR and Southern blotting. However, performance or throughput limitations in these methods currently constrain routine testing. METHODS We evaluated a novel FMR1 gene-specific PCR technology with 20 cell line DNA templates and 146 blinded clinical specimens. The CGG repeat number was determined by fragment sizing of PCR amplicons using capillary electrophoresis and compared with the results of FMR1 Southern blotting performed with the same samples. RESULTS The FMR1 PCR accurately detected full mutation alleles up to at least 1300 CGG repeats and comprising >99% GC character. All categories of alleles detected by Southern blot, including 66 specimens with full mutations, were also identified by FMR1 PCR for each of 146 clinical specimens. Since all full mutation alleles in heterozygous female samples were detected by PCR, allele zygosity was reconciled in every case. The PCR reagents also detected a 1% mass fraction of a 940 CGG allele in a background of 99% 23 CGG allele—roughly 5-fold greater sensitivity than Southern blotting. CONCLUSIONS The novel PCR technology can accurately categorize the spectrum of FMR1 alleles, including alleles previously considered too large to amplify, reproducibly detect low abundance full mutation alleles, and correctly infer homozygosity in female specimens, thus greatly reducing the need for sample reflexing to Southern blot.

(CGG) n repeat expansion in the FMR1 gene is associated with fragile X syndrome and other disorders. Current methods for FMR1 molecular testing rely on Southern blot analysis to detect expanded alleles too large to be PCR-amplified and to identify female homozygous alleles that often confound interpretations of PCR data. A novel , single-tube CGG repeat primed FMR1 PCR technology was designed with two genespecific primers that flank the triplet repeat region, as well as a third primer that is complementary to the (CGG) n repeat. This PCR was evaluated with 171 unique DNA samples , including a blinded set of 146 clinical specimens. The method detected all alleles reported by Southern blot analysis , including full mutations in 66 clinical samples and comprised up to 1300 CGG. Furthermore , a blinded cohort of 42 female homozygous and heterozygous specimens, including 21 with full mutation alleles , was resolved with 100% accuracy. Last , AGG interrupter sequences, which may influence the risk of (CGG) n expansion in the children of some carriers , were each correctly identified in 14 male and female clinical samples as referenced to DNA sequencing. As a result , this PCR provides robust detection of expanded alleles and resolves allele zygosity , thus minimizing the number of samples that require Southern blot analysis and producing more comprehensive FMR1 genotyping data than other methods. Expansion of cytosine-guanine-guanine (CGG) triplet repeats in the 5Ј-untranslated region of the fragile X mental retardation 1 (FMR1, NM_002024.4) gene is associated with several disorders, including fragile X syndrome, fragile X-associated tremor/ataxia syndrome, and fragile X-associated primary ovarian insufficiency. [1][2][3][4] Patients with the FMR1 full mutation (Ͼ200 CGG repeats) may be affected by a range of neurological, psychiatric, or emotional challenges, including mental retardation and/or autism.5 Deficits in development and particularly in attention and social communication have also been noted for many children with the FMR1 premutation. Moreover, premutation carriers (55 to 200 CGG repeats) are known to be at risk for fragile X-associated primary ovarian insufficiency and fragile X-associated tremor/ataxia syndrome, and some of these individuals may present additional complications, such as hypothyroidism and fibromyalgia.6 As a result, FMR1 disorders are linked to a range of clinical conditions, necessitating testing patients at different times during their life span. 7Fragile X syndrome molecular diagnosis is usually based on quantification of the (CGG) n repeat elements and the assessment of the methylation state of expanded alleles.5 Although PCR is the preferred approach to determine the (CGG) n repeat length of FMR1 alleles, typically only alleles with less than 100 to 150 CGG have

A microfabricated device for flow injection analysis and electrophoretic separation of acetylcholinesterase (AChE) inhibitors is described. Solutions of inhibitor, enzyme, substrate, and derivitizing agent were mixed within the channels of the microchip using computer-controlled electrokinetic transport. AChE-catalyzed hydrolysis of acetylthiocholine to thiocholine was measured in an on-chip reaction of thiocholine with coumarinylphenylmaleimide, and the resulting thioether was detected by laser-induced fluorescence. Inhibitors reduced the fluorescence signal and produced a negative peak diagnostic for the type of inhibition. A Gaussian peak was observed for competitive inhibitors, whereas a broad negative peak was observed for irreversible inhibitors. From a microchip assay for tacrine, an inhibition constant, K i, of 1.5 ± 0.2 nM was derived, which compared well with a standard cuvette assay. A flow injection assay of two irreversible inhibitors, carbofuran and eserine, was performed. With a 5-min stopped-flow reaction time, a detection limit of 10 nM carbofuran was obtained. As a potential multiplex screening device, a mixture of four cationic inhibitors, tacrine, edrophonium, and tetramethyl- and tetraethylammonium chloride, was separated and detected within 70 s.