Trans-ethnic fine-mapping of genetic loci for body mass index in the diverse ancestral populations of the Population Architecture using Genomics and Epidemiology (PAGE) Study reveals evidence for multiple signals at established loci.

Most body mass index (BMI) genetic loci have been identified in studies of primarily European ancestries. The effect of these loci in other racial/ethnic groups is less clear. Thus, we aimed to characterize the generalizability of 170 established BMI variants, or their proxies, to diverse US populations and trans-ethnically fine-map 36 BMI loci using a sample of >102,000 adults of African, Hispanic/Latino, Asian, European and American Indian/Alaskan Native descent from the Population Architecture using Genomics and Epidemiology Study. We performed linear regression of the natural log of BMI (18.5-70 kg/m2) on the additive single nucleotide polymorphisms (SNPs) at BMI loci on the MetaboChip (Illumina, Inc.), adjusting for age, sex, population stratification, study site, or relatedness. We then performed fixed-effect meta-analyses and a Bayesian trans-ethnic meta-analysis to empirically cluster by allele frequency differences. Finally, we approximated conditional and joint associations to test for the presence of secondary signals. We noted directional consistency with the previously reported risk alleles beyond what would have been expected by chance (binomial p < 0.05). Nearly, a quarter of the previously described BMI index SNPs and 29 of 36 densely-genotyped BMI loci on the MetaboChip replicated/generalized in trans-ethnic analyses. We observed multiple signals at nine loci, including the description of seven loci with novel multiple signals. This study supports the generalization of most common genetic loci to diverse ancestral populations and emphasizes the importance of dense multiethnic genomic data in refining the functional variation at genetic loci of interest and describing several loci with multiple underlying genetic variants.

Detection of the chemical warfare agents bis-(2-chloroethyl)ethylamine (HN-1) and tris-(2-chloroethyl)amine (HN-3) in air.

This paper describes the method development and validation for detection of the chemical warfare agents HN-1 and HN-3 in air using C8 solid-phase extraction disks followed by liquid desorption and analysis by gas chromatography. The method is contrasted to the standard approach which uses solid sorbent tubes followed by thermal desorption and analysis by gas chromatography.

Oligozymes. Oligonucleotide-enzyme conjugates.

The simple use of nonisotopic hybridization probes to detect complementary sequences provides valuable information in a large number of research and commercial applications. In hybridization assays, the four "S's (speed, simplicity, sensitivity, and specificity) are important criteria for determining the choice of probe and label. The direct chemical combination of synthetic oligonucleotide probes and enzyme labels offer advantages unmatched by other approaches, with the oligonucleotide providing rapid hybridization and high specificity, and the direct enzyme label providing simple and sensitive detection. Such oligonucleotide-enzyme conjugates ("oligozymes") can be used in a variety of hybridization and detection formats, including dot blots, Southern/northern blots, in situ, and solution hybridization/capture schemes. The practical synthesis and use of such oligozymes are summarized.

The 'individualization' of large North American mammals.

The enforcement of wildlife laws and the captive breeding of threatened/endangered species requires the ability to identify individual animals. DNA profiles of a variety of large North American mammals, birds, and fish were generated using ten different oligonucleotide probes. The probes tested were four multilocus probes [33.6, 33.15, JE46, and (TGTC)5] and six 'human unilocus' probes [MS1 (D1S7), CMM101 (D14S13), YNH24 (D2S44), EFD52 (D17S26), TBQ7 (D10S28), and MS43 (D12S11). Each of the probes was chemically synthesized, and labeled by the attachment of alkaline phosphatase; after hybridization, the probes were detected by chemiluminescence catalyzed by the enzyme. Initial screening against zoo blots including samples of bear, wolf, large cat, wild sheep, deer, birds, marine mammals, and fish indicated that three multilocus probes [33.15, 33.6, (TGTC)5] gave informative patterns containing 15-40 bands for most or all of the animals tested, as did two of the 'human unilocus' probes (MS1 and CMM101). The other five probes appeared informative only in some species (for example, YNH24 against canids). Subsequent screenings of populations within species were used to determine genetic diversity by analysis of observed bandsharing (S). Large heterologous populations, such as white-tailed deer, exhibited highly diverse band patterns (S < or = 0.2). Geographically isolated and/or genetically constricted animals, such as endangered Mexican wolves, Tule elk, and Columbian white-tailed deer, exhibited much higher frequencies of bandsharing (0.6 < or = S < or = 0.95).(ABSTRACT TRUNCATED AT 250 WORDS)

Differential expression of growth-associated protein (GAP-43) mRNA in rat primary sensory neurons after peripheral nerve lesion: a non-radioactive in situ hybridisation study.

An alkaline phosphatase-labelled anti-sense oligodeoxynucleotide probe specific for growth-associated protein messenger RNA (GAP-43 mRNA) was used for non-radioactive in situ hybridisation histochemistry to follow relative changes in GAP-43 mRNA content in lumbar primary sensory neurons (L4-6) after unilateral ligation of the sciatic nerve. In normal dorsal root ganglia (DRG) 16% of neurons expressed GAP-43 mRNA, and these cells belonged to a sub-group of intermediate-sized (32-50 microns diameter) and large (> 50 microns) neurons. The hybridisation signal detected in these cells was weak to moderate. One day after nerve ligature a significant increase in the number of GAP-43 mRNA expressing neurons in the ipsilateral DRG was detected involving particularly the very small (12-20 microns) cells, and small cell population (20-32 microns), though the hybridisation signal was less pronounced in this latter cell group. A significant increase in the cellular content of GAP-43 mRNA was detected in both cell groups when compared to the normal DRG by 2 days after the lesion. At later times (4, 7, and 10 days postinjury) the intermediate-sized and large cell subpopulations also showed an increase in the number of GAP-43 mRNA positive neurons, followed by a significant rise in their content of GAP-43 mRNA. However, they did not reach the same intensity of hybridisation signal as seen in the small and very small neurons. All DRG neurons showed a maximum of GAP-43 mRNA expression by 10 days postsurgery. At longer times there was a slight decrease in the content of GAP-43 mRNA towards 14 days postinjury, but mRNA levels remained elevated up to 28 days after nerve ligature, the longest time point examined in this study. The different onset and levels of GAP-43 gene expression in the rat primary sensory neurons after lesion of their peripheral branch axons further characterize the different subclasses of these cells and may reflect their different involvement in the plastic changes following peripheral nerve injury.

Nonradioactive detection of vasopressin mRNA with in situ hybridization histochemistry.

In situ hybridization histochemistry has been used successfully by many laboratories to detect mRNAs within single cells in the CNS. The detection of these hybrids in CNS tissue sections has been accomplished mainly with radioactive probes. However, radiolabeled probes have intrinsic limitations, including the long exposure time required for high resolution autoradiography and the inability to detect multiple RNA species within the same neuron. Here we report a new method to detect mRNA in situ using a synthetic DNA probe conjugated to alkaline phosphatase (AP). The probe was synthesized to be complementary to the glycoprotein coding region of vasopressin mRNA. Under normal hybridization conditions high resolution detection of vasopressin mRNA within individual neurons was routinely obtained within 8 h. The distribution of hybridization signal obtained with the AP-conjugated probe was identical to that observed with the same probe radiolabeled with [35S]dATP. Hybridization-positive neurons were found in all regions of the CNS that have been previously reported to synthesize vasopressin, including magnocellular neurons in the paraventricular nucleus (PVN) and the supraoptic nucleus. Small diameter neurons were also observed in the suprachiasmatic nucleus, the accessory PVN, the bed nucleus of the stria terminalis, and a cell group along the ventral surface of the optic tract. These results suggest that nonradioactive detection of neuropeptide mRNA in situ can be easily accomplished within 24 h. Furthermore, the improved resolution with AP-conjugated oligonucleotide probes should enhance efforts to study the regulation of gene expression in the nervous system.

Simultaneous Visualization of Vasopressin and Oxytocin mRNA- Containing Neurons in the Hypothalamus Using Non-Radioactive in situ Hybridization Histochemistry.

In situ hybridization histochemistry (ISHH) using synthetic oligodeoxynucleotide probes has been used to demonstrate the sites of expression of mRNA for vasopressin (AVP) and oxytocin (OXT) in the rat brain. ISHH was performed with two types of non-radioactive probes, labelled with either alkaline phosphatase or 5'-biotin. Simultaneous detection of the mRNAs for both AVP and OXT was achieved using an alkaline phosphatase substrate for the AVP probe and an anti-biotin monoclonal (mouse) antibody for the OXT probe. These probes revealed two non-overlapping populations of AVP and OXT neurons on the same section.

Nonisotopic oligomeric probes for the human enteroviruses.

Oligonucleotide probes were prepared from highly conserved regions of human enteroviral genomes. These reagents were labeled with either 32P or alkaline phosphatase and were successfully used in blot assays to detect a wide variety of human enteroviruses, proving the potential utility of oligomeric sequences as pan-enteroviral probes. The availability of nonisotopic probes will ultimately make a hybridization assay for enteroviruses easier, shorter, and more adaptable for routine diagnostic laboratories.

Continuous, on-line DNA sequencing using oligodeoxynucleotide primers with multiple fluorophores.

A method for sequencing DNA by using a difluoresceinated primer and laser excitation is described. Dideoxy protocols have been determined that provide sequences for 600 bases starting with base 1 with less than 1% error in a single load. Electrophoresis is at 20 W and the bands are detected 24 cm from the bottom of the loading well with a scanning fluorescence detector. Bands are imaged on a TV screen in two dimensions. The sequences can be read from the TV screen manually or semiautomatically by using a simple software program. The system allows more bases to be read with a lower error rate than any other reported automated sequencing method.

Assessment of a synthetic DNA probe for Plasmodium falciparum in African blood specimens.

Synthetic DNA oligomers homologous to 21-base long repetitive sequences of Plasmodium falciparum DNA were labeled with 32P using T4 kinase, and were hybridized with purified DNA and with processed blood samples from Africa. The sequence PFR1, its antiparallel oligomer PFR1R, and PFR1 covalently attached to biotin hybridized similarly to P. falciparum DNA. One-microliter aliquots of blood from Zaire spotted on prewet nylon filters and hybridized with PFR1 gave detectable autoradiogram signals from samples with parasitemias as low as 1,000 parasites/mm3. Blood lysis and protein digestion followed by alkylation allowed dot-blot processing of larger aliquots of blood. After hybridization with PFR 1 and autoradiography, 26 samples were scored positive visually, compared with 34 scored positive by microscopy. The effective sensitivity for processed 10-microliter samples was about 500 parasites/mm3. Signals from hybridized probes were quantitated by liquid scintillation counting and densitometry, and were proportional to the amounts of purified P. falciparum DNA applied to the filter. Autoradiogram signals also were roughly proportional (correlation coefficient, r = 0.77) to the number of parasites/mm3 of blood from field samples as determined by microscopic examination.

Use of enzyme-linked synthetic DNA in diagnosis of falciparum malaria.

Plasmodium falciparum contains a family of 21-base-long repetitive DNA sequences in its genome. A 21-base synthetic DNA oligomer, formerly labelled with phosphorus-32 for autoradiographic detection of P falciparum DNA, was covalently coupled to alkaline phosphatase for histochemical detection. The conjugate (PFR1-AP) detected purified P falciparum DNA with a sensitivity and specificity equal to that of 32P-labelled probes after 2-day exposures. PFR1-AP did not detect host DNA or DNA of other Plasmodium species. In African blood specimens PFR1-AP specifically detected P falciparum infections of 100 parasites/microliter. This sensitive, rapid, nonisotopic probe will allow more widespread use of DNA hybridisation in the diagnosis of malaria.

Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes.

Short synthetic oligonucleotides have been covalently cross-linked to alkaline phosphatase using the homobifunctional reagent disuccinimidyl suberate. The oligomers, twenty-one to twenty-six bases in length, are complementary to unique sequences found in herpes simplex virus, hepatitis B virus, Campylobacter jejuni and enterotoxigenic Escherichia coli. Each oligomer contains a single modified base with a 12-atom "linker arm" terminating in a reactive primary amine. Cross-linking through this amine results in oligomer-enzyme conjugates composed of one oligomer per enzyme molecule that have full alkaline phosphatase activity and can hybridize to target DNA fixed to nitrocellulose within 15 minutes. The hybrids are detected directly with a dye precipitation assay at a sensitivity of 10(6) molecules (2 X 10(-18) mol) of target DNA in 4 hours development time. The enzyme has no apparent effect on selectivity or kinetics of oligonucleotide hybridization and the conjugates can be hybridized and melted off in a conventional manner.

Typing of herpes simplex virus with synthetic DNA probes.

Three single-stranded oligonucleotide probes, 22 bases long, homologous to unique regions of herpes simplex virus (HSV) types 1 (HSV-1) and 2 (HSV-2) and a region common to both were chemically synthesized with use of a modified phosphochloridite protocol. For hybridization experiments each probe was labeled with use of polynucleotide kinase and [gamma-32P] ATP to a specific activity of approximately 2 X 10(9) cpm/micrograms. Two hundred one clinical isolates of HSV (96 HSV-1 and 105 HSV-2) collected from vesicles in the mucocutaneous junction of the mouth or from the genital area were analyzed. There was a 99% (199 of 201) agreement between hybridization and monoclonal antibody typing; the two discrepant isolates of HSV-2 that were negative by monoclonal antibody typing were confirmed as HSV-2 by restriction endonuclease analysis. The probes detected between 10(4) and 10(5) HSV infectious units and from 150 to 600 HSV-infected Vero cells. No binding was detected between any of the three probes and isolates of cytomegalovirus, Epstein-Barr virus, and varicella-zoster virus.

Synthesis of (E)-5-(3,3,3-trifluoro-1-propenyl)-2'-deoxyuridine and related analogues: potent and unusually selective antiviral activity of (E)-5-(3,3,3-trifluoro-1-propenyl)-2'-deoxyuridine against herpes simplex virus type 1.

Syntheses of (E)-5-(3,3,3-trifluoro-1-propenyl)-2'-deoxyuridine (TFPe-dUrd) (1), 5-(3,3,3-trifluoro-1-propyl)-2'-deoxyuridine (11), 5-(3,3,3-trifluoro-1-methoxy-1-propyl)-2'-deoxyuridine (8), and 5-(3,3,3-trifluoro-1-hydroxy-1-propyl)-2'-deoxyuridine (10) from 5-chloromercuri-2'-deoxyuridine are described. The antiviral activity of TFPe-dUrd was determined in cell culture against herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and vaccinia virus and compared concurrently with 5-(1-propenyl)-2'-deoxyuridine, 5-(2-bromovinyl)-2'-deoxyuridine, 5-iodo-2'-deoxyuridine, and 5-(trifluoromethyl)-2'-deoxyuridine. TFPe-dUrd demonstrated a potent and unusually selective activity against HSV-1, with a 2-log reduction in virus yield at 0.03 micrograms/mL (0.09 microM); L-1210 cell growth was inhibited by 50% only at 290 micrograms/mL. Isopycnic centrifugation of 32P-labeled DNA indicated that if 0.5 or 2 microM TFPe-dUrd was present for 0-6 h postinfection, viral DNA synthesis was reduced by ca. 50 and 85%, respectively; concomitantly, a new DNA band appeared at lower density than normal cellular or viral DNA.

Selective antiviral agents. The metabolism of 5-propyl-2'-deoxyuridine and effects on DNA synthesis in herpes simplex virus type 1 infections.

The metabolism and disposition of 5-propyl-2'-deoxyuridine (Pr-dUrd) in herpes simplex virus type 1 infections were investigated in cell culture using [14C]Pr-dUrd, [32P]orthophosphate, and several methods including high pressure liquid chromatography and isopycnic centrifugation. Results in infected cells indicate Pr-dUrd 1) is taken up and phosphorylated to mono-, di-, and triphosphates; 2) is incorporated into DNA; 3) preferentially inhibits synthesis of viral DNA; 4) blocks re-initiation of viral DNA synthesis even after removal of the nucleoside from the culture; and 5) exerts these effects early in the course of infection (before 6 h postinfection). Pr-dUrd was not phosphorylated in uninfected cells, and had little or no effect on apparent cellular DNA synthesis in infected or uninfected cells. Present evidence suggests one possible antiviral event could be the lethal effect of Pr-dUrd after incorporation into viral DNA by alteration of DNA template-directed functions such as replication.

Differential effect of nucleoside analog triphosphates on ribonucleotide reductases from uninfected and herpes simplex virus-infected HeLa cells.

Effects of the triphosphates of eight pyrimidine nucleoside analogs (5-substituted, 2'-fluoroara-, and acyclonucleosides) and acycloguanosine were examined on the ribonucleotide reductases prepared from uninfected and herpes simplex virus (types 1 and 2)-infected HeLa cells. Of the analogs tested, E-5-propenyl- and E-5-(2-bromovinyl)-dUTP were more potent inhibitors than dTTP of the enzymes from virus-infected cells, whereas only the former compound showed this effect on the uninfected HeLa enzyme.