An ultrasensitive fluorescence method suitable for quantitative analysis of mung bean nuclease and inhibitor screening in vitro and vivo.

Mung bean nuclease is a single stranded specific DNA and RNA endonuclease purified from mung bean sprouts. It yields 5'-phosphate terminated mono- and oligonucleotides. The activity level of this nuclease can act as a marker to monitor the developmental process of mung bean sprouts. In order to facilitate the activity and physiological analysis of this nuclease, we have developed a biosensing assay system based on the mung bean nuclease-induced single-stranded DNA scission and the affinity difference of graphene oxide for single-stranded DNA containing different numbers of bases. This end-point measurement method can detect mung bean nuclease in a range of 2×10(-4) to 4×10(-2) with a detection limit of 1×10(-4) unit/mL. In addition, we demonstrate the utility of the assay for screening chemical antibiotics and metal ions, resulting in the identification of several inhibitors of this enzyme in vitro. Furthermore, we firstly report that inhibiting mung bean nuclease by gentamycin sulfate and kanamycin in vivo can suppress mung bean sprouts growth. In summary, this method provides an alternative tool for the biochemical analysis for mung bean nuclease and indicates the feasibility of high-throughput screening specific inhibitors of this nuclease in vitro and in vivo.

A sensitive fluorescence turn-on assay of bleomycin and nuclease using WS2 nanosheet as an effective sensing platform.

As an important antitumor drug, bleomycin (BLM) is widely used in the treatment of a variety of cancers. In addition, nucleases play a crucial role in DNA replication, recombination and repair which are associated with cancer development. Thus, the development of BLM and nuclease detection methods is of great significance in cancer therapy and related biological mechanism research. Here, a WS2 nanosheet-based turn-on fluorescent sensing platform for simple, fast and sensitive detection of BLM and nuclease was reported. WS2 nanosheet exhibits different affinity toward ssDNA with different length and excellent fluorescence quenching ability. A fluorescein (FAM)-labeled long ssDNA could be adsorbed on the surface of WS2 nanosheet and the fluorescence was therefore quenched. In the presence of BLM·Fe(II) or S1 nuclease (a ssDNA-specific nuclease which was used as a model enzyme), an irreversible scission of long ssDNA was underwent through the BLM-induced oxidation cleavage or S1 nuclease-induced enzymatic hydrolysis. Short FAM-linked oligonucleotide fragments which could not be adsorbed on the nanosheet surface were then produced, resulting in a weak fluorescence quenching after mixing WS2 nanosheets. Thus, the fluorescence signal was restored. The proposed sensor displays a wide linear range and a high sensitivity with a detection limit of 0.3 nM for BLM and 0.01 U mL(-1) for S1 nuclease. It also exhibits a good performance in complex biological samples. This method not only provides a strategy for BLM or S1 nuclease assay but also offers a potential application in biomedical and clinical study.

Label-free fluorometric detection of S1 nuclease activity by using polycytosine oligonucleotide-templated silver nanoclusters.

S1 nuclease has an important function in DNA transcription, replication, recombination, and repair. A label-free fluorescent method for the detection of S1 nuclease activity has been developed using polycytosine oligonucleotide-templated silver nanoclusters (dC12-Ag NCs). In this assay, dC12 can function as both the template for the stabilization of Ag NCs and the substrate of the S1 nuclease. Fluorescent Ag NCs could be effectively formed using dC12 as the template without S1 nuclease. In the presence of S1 nuclease, dC12 is degraded to mono- or oligonucleotide fragments, thereby resulting in a reduction in fluorescence. S1 nuclease with an activity as low as 5×10(-8)Uµl(-1) (signal/noise=3) can be determined with a linear range of 5×10(-7) to 1×10(-3)Uµl(-1). The promising application of the proposed method in S1 nuclease inhibitor screening has been demonstrated using pyrophosphate as the model inhibitor. Furthermore, the S1 nuclease concentrations in RPMI 1640 cell medium were validated. The developed method for S1 nuclease is sensitive and facile because its operation does not require any complicated DNA labeling or laborious fluorescent dye synthesis.

SAW1 is required for SDSA double-strand break repair in S. cerevisiae.

SAW1, coding for Saw1, is required for single-strand annealing (SSA) DNA double-strand break (DSB) repair in Saccharomycescerevisiae. Saw1 physically associates with Rad1 and Rad52 and recruits the Rad1-Rad10 endonuclease. Herein we show by fluorescence microscopy that SAW1 is similarly required for recruitment of Rad10 to sites of Synthesis-Dependent Strand Annealing (SDSA) and associates with sites of SDSA repair in a manner temporally overlapped with Rad10. The magnitude of induction of colocalized Saw1-CFP/Rad10-YFP/DSB-RFP foci in SDSA is more dramatic in S and G2 phase cells than in M phase, consistent with the known mechanism of SDSA. We observed a substantial fraction of foci in which Rad10 was localized to the repair site without Saw1, but few DSB sites that contained Saw1 without Rad10. Together these data are consistent with a model in which Saw1 recruits Rad1-Rad10 to SDSA sites, possibly even binding as a protein-protein complex, but departs the repair site in advance of Rad1-Rad10.

Fluorescence turn-on detection of DNA and label-free fluorescence nuclease assay based on the aggregation-induced emission of silole.

By making use of the aggregation-induced emission feature of silole, compound 1 with an ammonium group is designed and synthesized with a view to developing a new optical probe for fluorescence turn-on detection of DNA and label-free fluorescence nuclease assay. The fluorescence of 1 increases largely upon mixing with DNA, in particular for long DNA, indicating that 1 can be used for fluorescence turn-on detection of DNA. More interestingly, 1 can be employed to follow the DNA cleavage process by nuclease. Therefore, a label-free fluorescence nuclease assay method is successfully established with 1. Furthermore, this label-free fluorescence assay can also be used for inhibitor screening of nucleases. Given its simplicity, easy operation, sensitivity and cost-effectiveness, this method can be extended to other nuclease assays and high-throughput screening of nuclease inhibitors.

An electrochemical study of enzymatic oligonucleotide digestion.

This paper describes the synthesis and application of a novel ferrocene (Fc) label that can be efficiently attached to oligonucleotides. We demonstrate how pulse electrochemical methods can be used to measure very low concentrations of ferrocene label and, importantly, show good electroanalytical discrimination between a labelled oligonucleotide and an enzyme digested labelled oligonucleotide, in which the ferrocene label nucleotide conjugate has been released. Real time in situ analysis gives a much greater understanding of the process. Potential applications include the detection of specific nucleic acid sequences and measurement of nuclease activity.

Development of nonradioactive microtiter plate assays for nuclease activity.

We have developed two microtiter plate assays for the detection of DNA cleavage by nucleases, using 3'-biotinylated oligonucleotide substrates. In the covalently linked oligonucleotide nuclease assay (CLONA), the biotinylated substrates are phosphorylated at the 5' end to facilitate their covalent immobilization on CovaLink NH plates. The cleavage of the covalently immobilized substrate by nucleases results in biotin release. The uncleaved substrate molecules are detected with an enzyme-avidin conjugate. The affinity-linked oligonucleotide nuclease assay (ALONA) makes use of substrates with a digoxigenin on the 5' end of the 3'-biotinylated DNA strand. The substrate binds specifically to the wells of streptavidin-coated microtiter plates, in which the nuclease reaction takes place. Uncleaved substrate retains the digoxigenin label, which is detected with an enzyme-labeled anti-digoxigenin antibody. We assessed the efficiency of these two assays by measuring S1 nuclease and DNase I activities, and the inhibitory effect of EDTA and aurintricarboxylic acid on the reaction. Both methods are more convenient than the standard radioactive nuclease assay and are suitable for high-throughput screening of potential nuclease inhibitors, nucleases, and catalytic antibodies. The ALONA assay was found to be more sensitive than the CLONA assay, with a performance similar to that of the standard nuclease assay.

Comparative analysis of follistatin-, activin beta A- and activin beta B-mRNA steady-state levels in diverse porcine tissues by multiplex S1 nuclease analysis.

OBJECTIVE: The relation of activins (dimers of the beta-subunits of inhibin) and follistatin (FS) (their binding protein) affect the growth and differentiation of many cell types. Activin- and FS-mRNAs show a widespread co-expression throughout the organism, indicating an essential role for the FS/activin system in diverse physiological processes. The present study was performed to investigate FS-, activin betaA-, and activin beta B-mRNA expression in porcine tissues and to compare the relative mRNA tissue distribution by a newly developed multiplex S1 nuclease protection assay. METHODS: Twenty micrograms total RNA from different porcine tissues were subjected to multiplex S1 analysis. Specific mRNA expression was determined by measurements of optical densities on autoradiographs. RESULTS: Activin beta A-mRNA expression was abundant in the ovary, adrenal gland, fat, vein, artery and uterus, activin beta B-mRNA was highly expressed in the ovary, pituitary, uterus, placenta, aorta and cerebellum. FS-mRNA showed a widespread expression with high levels in ovary, uterus, cerebellum, placenta and fat. The comparison of relative activin beta A-, activin beta B- and FS-mRNA expression within a certain tissue showed a predominance of activin beta A-mRNA in the adrenal gland, fat, artery, spinal cord, cerebrum and colon and of activin beta B-m RNA in pituitary, testis and placenta, while FS-mRNA levels exceeded those of activin subunits in epididymis, liver, lymphoid tissue, muscle, intestine, cerebellum, ovary and uterus. CONCLUSIONS: The presented data provide an overview of FS-, activin beta A-, and activin beta B-mRNA steady state levels in porcine tissues.

Recharacterization of the start sites for the major human transforming growth factor-beta 1 mRNA.

The start sites for the major human TGF-beta 1 transcripts have been reexamined. A comparison of ribonuclease and S1 nuclease protection analyses on native TGF-beta 1 mRNA and in vitro transcribed human TGF-beta 1 transcripts of defined sizes places the most 5' start site for the native TGF-beta 1 message approx. 50 nucleotides upstream from the previously published start site at base +1. Furthermore, the same techniques indicate that the apparent downstream start site at base +271 is an artefact due to the presence of an A + T-rich island in the middle of an otherwise highly G + C-rich sequence. This is not apparent if S1 nuclease protection is used alone, which emphasizes the importance of using the two techniques in combination for this type of analysis. Thus the major 2.5 kb TGF-beta 1 band seen on Northern blots comprises only mRNA transcribed from the more upstream of the two previously characterized promoters. This has important implications both for the transcriptional and translational regulation of this growth factor.

DnaA protein stimulates polA gene expression in Escherichia coli.

The polA gene of Escherichia coli encodes DNA polymerase I that is involved in DNA replication and repair. Despite the wide knowledge about structure and function of DNA polymerase I, there is little insight into the regulatory mechanisms involved in polA expression. DnaA is the initiator protein for DNA replication in E. coli. There are two putative DnaA-binding sites within the extended promoter region of polA. In this work we studied the influence of altered levels of DnaA protein on polA expression. We found that DnaA overproduction increases polA expression in stationary-phase cultures. The stimulation effect was independent of rpoS, which encodes the sigma factor for stationary-phase-inducible genes. However, it was modulated by ppGpp. Comparative S1 analyses revealed that the induction was based on transcriptional stimulation. Footprinting experiments demonstrated that DnaA binds only to the proximal DnaA box near the polA promoter. These results suggest an additional role for DnaA as transcriptional activator of polA at least under certain physiological conditions.

Hybridization histochemical and immunohistochemical localization of inhibin/activin subunits and messenger ribonucleic acids in the rat brain.

Inhibin and activin are best known as gonadal glycoprotein hormones but have a broad anatomical distribution. We previously described the central distribution ofinhibin/activin beta A- and beta B-subunit proteins in some neuronal cell bodies, fibers, and nuclei of the rat brain and reported a possible role for central activin in suckling-induced oxytocin secretion and corticotropin releasing factor release. In the present report, we mapped the detailed immunohistochemical localization of inhibin/activin alpha-, beta A-, and beta B-subunits throughout the rat brain to further clarify their central distribution. In addition, the localization and distribution of their corresponding mRNAs was assessed. The results are summarized as follows: 1) Both beta A- and beta B-subunit immunoreactivity are found in neuronal cell bodies in the nucleus of the solitary tract and the dorsal and ventral medullary reticular nuclei, and in fibers and terminals of known projection sites for these nuclei. 2) beta B-subunit immunoreactivity is localized in a group of perifornical neurons in the hypothalamus. 3) beta A-subunit immunoreactivity is present in discrete populations of neuronal cell nuclei scattered throughout the CNS. 4) mRNAs encoding each of the inhibin/activin subunits are expressed in all major brain regions as determined by S1 nuclease assay and in a variety of specific neuroanatomical sites as shown by in situ hybridization. The results suggest that central inhibin and activin proteins are produced in the brain where they may potentially serve inter- and intracellular functions in multiple systems.

Cancer biomarkers in human atherosclerotic lesions: detection of DNA adducts.

Since somatic mutations are suspected to contribute to the pathogenesis not only of cancer but also of atherosclerotic plaques, we measured DNA adducts in the smooth muscle layer of atherosclerotic lesions in abdominal aorta specimens taken at surgery from seven patients. DNA adducts were evaluated in three laboratories by means of different molecular dosimetry methods, including: (a) HPLC/fluorescence, which specifically identifies the DNA adducts of the anti-benzo(a)pyrene (BPDE) isomer; (b) two- and three-dimensional synchronous fluorescence spectrophotometries, which detect DNA adducts of BPDE and other reactive metabolites of polycyclic aromatic hydrocarbons; and (c) 32P postlabeling, which reveals the presence of a variety of types of DNA adducts. The HPLC/fluorescence method provided for the first time evidence for the presence of BPDE-DNA specific adducts in three of six specimens tested. Synchronous fluorescence spectrophotometry displayed broad areas of fluorescence in all seven specimens, thereby suggesting the occurrence not only of BDPE-DNA but also of other DNA adducts with similar fluorescence characteristics. All specimens were also positive at 32P postlabeling, which revealed multiple spots detectable following enrichment either with nuclease P1 or butanol, indicative of the presence of different aromatic DNA adducts. Thus, the data obtained by applying typical cancer biomarkers provide further support to the hypothesis that there may be similarities between the carcinogenic and the atherogenic processes, and in particular that genetic alterations caused by DNA-binding agents in the artery wall may be detected in atherosclerotic lesions.

Heterologous expression of the cytochrome P450cam hydroxylase operon and the repressor gene of Pseudomonas putida in Escherichia coli.

The cytochrome P450cam hydroxylase operon (camDCAB) of Pseudomonas putida is negatively regulated by a repressor, CamR, which also represses its own gene. The expression in P. putida of both camR and camDCAB is derepressed in the presence of D-camphor. We examined the expression in Escherichia coli of camR and camDCAB by monitoring the enzyme activity of the camD gene product. In the presence or absence of D-camphor in the cell culture, the expression in E. coli of camD was significant and constitutive, suggesting no expression of camR. This lack of expression was confirmed by monitoring the beta-galactosidase activity of camR-lacZ translational fusions. However, S1 nuclease mapping revealed that synthesis of camR mRNA in E. coli was significant and constitutive, as observed in the case of camDCAB mRNA. Thus, it is likely that the expression of camR in E. coli is limited at the translational level.

Two types of neonatal-to-adult fast myosin heavy chain transitions in rat hindlimb muscle fibers.

Adult fast myosin heavy chain (MHC) isoforms are accumulated in fibers of rat hindlimb skeletal muscle which initially contain neonatal MHC at birth. The specific factors controlling these transitions are not known, but in rat and mouse muscle tissue the transition between the neonatal and adult fast MHC proteins does not appear to require continuous innervation. We have reinvestigated the role of innervation in the neonatal-to-adult fast MHC protein and mRNA transitions that occur in developing rat fast-twitch muscles using immunohistochemistry and S1 nuclease mapping. We find that neonatal MHC-containing developing fibers exhibit different responses after denervation at birth regarding the disappearance of neonatal MHC and the accumulation of adult fast MHC isoforms. Immunohistochemistry shows that one fiber population loses neonatal MHC and accumulates adult fast IIB (or possibly IIX) MHC over a period of 2-3 weeks, whereas in the other population neonatal MHC does not decrease nor does the adult fast IIA isoform accumulate to high levels. The results of S1 analysis of mRNAs show that the levels of neonatal MHC mRNA do not decrease in muscles denervated at birth. We also demonstrate that in young adult rats this mRNA is reexpressed in denervated or paralyzed muscles. Since the appearance of IIB mRNA has been previously shown to be nerve-independent (S.D. Russell, N. Cambon, B. Nadal-Ginard, and R.G. Whalen, 1988, J. Biol. Chem. 263, 6370-6374), these results suggest that fibers containing neonatal MHC in rat hindlimb muscles at birth are already differentiated (i.e., preprogrammed) to accumulate either the adult fast IIA or IIB MHC isoforms and that the neonatal-to-adult MHC transitions occurring in these two fiber populations are controlled by different mechanisms.

Regulation of elastin gene expression.

Recent isolation and characterization of cDNAs encompassing the full length of chicken, cow, and human elastin mRNA have led to the elucidation of the primary structure of the respective tropoelastins. Comparison of the tropoelastin from the different species has revealed that large segments of the sequence are conserved, but considerable variation also exists, ranging in extent from relatively small alterations, such as conservative amino acid substitutions, to large-scale deletions and insertions. Several distinct approaches have yielded compelling evidence of a single elastin gene per haploid genome. Analysis of the bovine and human elastin genes revealed that functionally distinct hydrophobic and cross-link domains of the protein are encoded in separate exons which alternate in the genes. The human gene contains 34 exons, the intron/exon ratio is unusually large (20:1), and the introns contain large amounts of repetitive sequences that may predispose to genetic instability. Comparison of the cDNA and genomic sequences has demonstrated that the primary transcript of both species is subject to considerable alternative splicing, which can account for the presence of multiple tropoelastin isoforms. It is likely that the conformation of elastin is, at least in part, that of a random coil, and therefore it might be expected that the stringency for conservation of the amino acid sequence would be less than that for other proteins with unique conformations. This suggests that functional elastin molecules that vary in their sequence and fitness may exist in the human population and be compatible with a normal life. Potentially though, these variations could have profound consequences on the properties of vital tissues found in the cardiovascular and pulmonary systems over the lifetime of the individual. Consequently, analysis of the structure of the elastin gene and its variation in what is regarded as the normal human population, rather than in those individuals with clearly heritable diseases, assumes greater importance. The 5'-flanking region of the gene is G + C rich and contains several SP-1 and AP2 binding sites, as well as putative glucocorticoid, cAMP, and TPA responsive elements, but no consensus TATA box or functional CAAT box. Primer extension and S1 mapping of the elastin mRNA indicated that transcription was initiated at multiple sites. Transfection experiments using promoter elements/reporter gene constructs demonstrated that the basic promoter element was found within region -128 to -1. In addition, three distinct up-regulatory and two down-regulatory regions were delineated. Taken together, these findings suggest that the regulation of elastin gene expression is complex and takes place at several levels.