Showing papers by "Applied Biosystems published in 1996"

Mutations in GPC3, a glypican gene, cause the Simpson-Golabi-Behmel overgrowth syndrome

Abstract: Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked condition characterized by pre- and postnatal overgrowth with visceral and skeletal anomalies. To identify the causative gene, breakpoints in two female patients with X;autosome translocations were identified. The breakpoints occur near the 5' and 3' ends of a gene, GPC3, that spans more than 500 kilobases in Xq26; in three families, different microdeletions encompassing exons cosegregate with SGBS. GPC3 encodes a putative extracellular proteoglycan, glypican 3, that is inferred to play an important role in growth control in embryonic mesodermal tissues in which it is selectively expressed. Initial western- and ligand-blotting experiments suggest that glypican 3 forms a complex with insulin-like growth factor 2 (IGF2), and might thereby modulate IGF2 action.

X-linked anhidrotic (hypohidrotic) ectodermal dysplasia is caused by mutation in a novel transmembrane protein.

Abstract: Ectodermal dysplasias comprise over 150 syndromes of unknown pathogenesis. X-linked anhidrotic ectodermal dysplasia (EDA) is characterized by abnormal hair, teeth and sweat glands. We now describe the positional cloning of the gene mutated in EDA. Two exons, separated by a 200-kilobase intron, encode a predicted 135-residue transmembrane protein. The gene is disrupted in six patients with X;autosome translocations or submicroscopic deletions; nine patients had point mutations. The gene is expressed in keratinocytes, hair follicles, and sweat glands, and in other adult and fetal tissues. The predicted EDA protein may belong to a novel class with a role in epithelial-mesenchymal signalling.

New insights into the composition, molecular mass and stoichiometry of the protein complexes of plant mitochondria

Abstract: Recently a powerful electrophoresis method for the native preparation and characterization of the respiratory protein complexes of mitochondria from fungi and mammals has been developed, which employs Coomassie dyes to introduce charge shifts on proteins (Schagger and von Jagow (1991) Anal Biochem 199, 223–231) The procedure, which is called ‘blue native-polyacrylamide gel electrophoresis’ (BN-PAGE), was modified and introduced for the analysis of mitochondria from higher plants BN-PAGE of mitochondrial protein from potato allows the separation of nine distinct protein complexes between 100 and 1000 kDa and reveals novel results for their composition, molecular mass and stoichiometry For the first time soluble mitochondrial protein complexes, like the HSP60 complex (750 kDa) and a complex of 200 kDa, which includes a formate dehydrogenase, are analysed by BN-PAGE Complex I from potato (1000 kDa) is about 100 kDa larger than the corresponding enzyme from beef and can be resolved into more than 30 different subunits on a second gel dimension The F1F0 ATP synthase (580 kDa) and the cytochrome c oxidase (160 kDa) from potato seem to contain more subunits than hitherto reported Direct sequencing of subunits revealed that the F1 part of the F1F0 ATP synthase lacks the oligomycin sensitivity conferring protein (OSCP), which was reported to be present in F1 parts of dicotyledonous plants, but contains the ATPase inhibitory protein N-terminal sequences of 16 mitochondrial proteins were obtained, several of which are presented for the first time from a plant source BN-PAGE allows the preparation of mitochondrial protein complexes from gram amounts of plant tissue, as the procedure only requires milligram amounts of organelles This potential of BN-PAGE is demonstrated by the separation and characterization of the mitochondrial enzyme complexes from Arabidopsis thaliana Further analysis of organellar protein complexes by BN-PAGE will allow the generation of ‘protein maps’ from different tissues and developmental stages or from mutant plants

Cold responses of Arabidopsis mutants impaired in freezing tolerance

Abstract: Mutants of Arabidopsis thaliana L. (Heynh), characterized as deficient in their freezing tolerance after cold acclimation, were surveyed for some of the normal responses to cold exposure. In foliar tissue, the cold-inducibility of three proteins, the levels of sucrose and glucose, the fatty acyl composition of lipids, and the accumulation of anthocyanin was examined. Four mutations (sfr3, sfr4, sfr6, and sfr7) reduced or eliminated the accumulation of anthocyanin during cold acclimation. One mutation (sfr4) prevented the normally cold-induced elevation of sucrose and glucose levels ; both sfr4 and another mutation (sfr7) affected fatty acid composition after (and only after) cold acclimation. On the other hand mutations sfr1, sfr2 and sfr5 did not differ significantly from the wild type in any of the parameters tested, suggesting that they have other, perhaps highly specific, effects on low-temperature responses.

Heterozygote and Mutation Detection by Direct Automated Fluorescent DNA Sequencing Using a Mutant Taq DNA Polymerase

Abstract: We describe a method for direct cycle sequencing of PCR fragments amplified from genomic DNA or cDNA. DNA sequencing template is amplified using PCR and oligonucleotide primers flanking the region of interest. The amplified fragment is directly cycle sequenced using fluorescent sequencing primers, Sanger dideoxy sequencing chemistry and an enzyme mixture of a mutant Taq DNA polymerase and thermostable pyrophosphatase. The sequence ladders produced are analyzed on a real-time, automated four-color sequencing system. The method produces sequence ladders from unpurified PCR fragments of sufficiently high quality such that heterozygotes can be reproducibly detected and identified by software that recognizes signal-strength patterns indicative of mixed-base positions.

Oligonucleotide ligation assay (OLA) for the diagnosis of familial hypercholesterolemia

Abstract: More than half of all deaths in Western society are related to arteriosclerotic cardiovascular diseases. Inherited disturbances in the low-density-lipoprotein (LDL) receptor and similar lipid-related defects account for the majority of these deaths. Testing procedures thus far rely on total cholesterol, LDL cholesterol, high-density-lipoprotein cholesterol, and triglyceride determinations. These tests are not able to provide any genetic information. We have developed an oligonucleotide ligation assay (OLA) that enables us to screen for high-risk individuals by testing for 19 common mutations in the LDL receptor and the apolipoprotein B genes using an automated genotyping-based two-step protocol. The novel OLA uses oligomeric pentaethyleneoxide mobility modifiers. The automated test will be useful in screening large populations for genetic data to distinguish relative from absolute risk, as well as for cost-effective familial analysis.

Long-Range Sequence Analysis in Xq28: Thirteen Known and Six Candidate Genes in 219.4 kb of High GC DNA Between the RCP/GCP and G6PD Loci

Abstract: DNA comprising 219 447 bp was sequenced in nine cosmids and verified at >99.9% precision. Of the standard repetitive elements, 187 Alus make up 20.6% of the sequence, but there were only 27 MERs (2.9%) and 17 L1 fragments (1.6%). This may be characteristic of such high GC (57%) regions. The sequence also includes an 11.3 kb tract duplicated with 99.2% identity at a distance of 38 kb. The region is 80-90% transcribed and 12.5% translated. Thirteen known genes and their exon-intron borders are all accurately predicted at least in part by GRAIL programs, as are six additional genes. From centromere to telomere, the orientation of transcription varies among the first eight genes, then runs centromeric to telomeric for the next five, and is in the opposite sense for the last six. Eighteen of the 19 genes are associated with CpG islands. Two islands are exact copies in the 11.3 kb repeat units, and could thus give rise to double dosage levels of an X-linked gene. Another island is associated with two genes transcribed in opposite directions. From the sequence data, three genes and their exon structure are inferred. One of them, previously associated with HEX2, is shown to be a different gene unrelated to hexokinases ; a second gene, previously known by an EST, is plexin, from its 65.5% identity with the Xenopus analog ; and a third is a subunit of a vacuolar H-ATPase, and is named VATPS1.

Flowable networks as DNA sequencing media in capillary columns

Abstract: A novel class of materials that self-assemble in water into equilibrium network structures with a well-defined mesh size consist of polyethylene glycols (PEG's) end-capped with micelle-forming fluorocarbon tails. These micellar systems form flowable aqueous gel-like networks that permit electrophoretic DNA sequencing in capillary columns. The gels have unusual rheological properties, including network breakdown under shear, resulting in plug flow that allows columns refill with complete ejection of byproducts of the previous sequencing analysis. In this system, DNA fragment electrophoretic mobilities are unaffected by the hydrophobicity of the polymer tails. Low molecular weight (M) PEG chains (M 8000) show catastrophic resolution loss for DNA fragments larger than 100 bases due to band broadening. For a longer PEG segment (M 35000) separating the end groups, band broadening occurs for DNA fragments larger than 300 bases, implying that the PEG segment length controls the mesh size in the equilibrium network structure. Optimum sequencing results were obtained from a 6% solution of a 1:1 mixture of C6F13 end-capped- and C8F17 end-capped PEG 35,000. The resolution limit of fluorescent-dye-labeled sequencing products in this formulation was 450 bases in 75 microns capillaries at 200 V/cm.

Methods and reagents for combined PCR amplification and hybridization probing

Abstract: An oligonucleotide probe is disclosed, the probe including an oligonucleotide, a fluorescer molecule attached to a first end of the oligonucleotide and a quencher molecule attached to the opposite end of the oligonucleotide. The probe is rendered impervious to digestion by the 5′→3′ exonuclease activity of a polymerase and the 5′→3′ extension of by a polymerase. The invention also includes methods for performing combined PCR amplification and hybridization probing, one such method including the steps of contacting a target nucleic acid sequence with PCR reagents and an oligonucleotide probe as described above, and subjecting these reagents to thermal cycling. One preferred refinement of the above method further includes the addition of a strand displacer to facilitate amplification. Additional similar combined PCR hybridization methods are disclosed, such methods not requiring probes having their 5′ ends protected, wherein (i) the polymerase lacks 5′→3′ exonuclease activity, (ii) a 5′→3′ exonuclease inhibitor is included, and (iii) an exonuclease deactivation step is performed.

An Optimized Protocol for In Situ Hybridization Using PCR-Generated 33P-Labeled Riboprobes

Abstract: This report describes in detail an in situ hybridization protocol that has been optimized to produce consistent, sensitive results across a wide range of probe constructs and tissue types. Specific advances incorporated into this protocol include (1) the use of PCR gene fragments to generate cRNA probes, (2) the use of 33P-labeled probes and (3) the use of ultrafiltration microconcentrators to purify the probe and eliminate background. The use of 33PCR to generate RNA probes and the use of 33P-labeled probes in in situ hybridization have been described previously; however, this protocol is unique because it combines these techniques in a simple, streamlined procedure that includes the use of microconcentrators to purify the probe. This protocol is particularly appropriate for high-volume in situ hybridization laboratories using a wide variety of probe constructs and tissues.

RNA facilitates RecA-mediated DNA pairing and strand transfer between molecules bearing limited regions of homology.

Abstract: The RecA protein ofEscherichia coli catalyzes homologous pairing and strand exchange between a wide range of molecules showing nucleotide sequence complementarity, including a linear duplex and a single-stranded DNA molecule. We demonstrate that RecA can promote formation of joint molecules when the duplex contains an RNA/DNA hairpin and a single-stranded circle serves as the pairing partner. A chimeric RNA/DNA hairpin can be used to form stable joint molecules with as little as 15 bases of shared homology as long as the RNA stretch contains complementarity to the circle. The joint molecule bears some resemblance to a triple helical structure composed of RNA residues surrounded by two DNA strands which are in a parallel orientation. Evidence is presented that supports the notion that short stretches of RNA can be used in homologous pairing reactions at lengths below that required for DNA-DNA heteroduplex formation.

NMR studies of the most conserved RNA domain of the mammalian signal recognition particle (SRP).

Abstract: Mammalian signal recognition particle (SRP) and its homologues exhibit a phylogenetically conserved RNA domain, whose predicted secondary structure exhibits a hairpin motif with two bulged regions. Two RNA fragments comprising one (24 nt) or two (43 nt) of the conserved bulges were studied. Each fragment binds specifically to the domain of the Escherichia coli homologue of the SRP54 protein, which is involved in signal sequence recognition. The SRP RNA fragments exhibited a pronounced structural stabilization in the presence of Mg2+. Assignments of all base, H1', H2', and most imino proton resonances in the presence of Mg2+ were obtained for the 24mer RNA via NOE spectroscopy and correlated homonuclear NMR methods. 2D NOE patterns permitted a coarse structural description, revealing a relatively compact A-type geometry for the 24mer without any indications of looped-out nucleotides, syn-oriented bases, or base triplets. The GGAA-loop is structurally very similar to that of the GCAA tetraloop [Heus HA, Pardi A, 1991, Science 253:191-194]. Mg2+ seems to stabilize the structure of the conserved bulged region, which involves G:A and C:A mismatch pairs. Deviations from ideal A-type helicity are found for a larger region than the predicted secondary structure implies. Although no explicit assignment effort has been dedicated to the 43mer yet, striking similarity in chemical shift changes upon addition of Mg2+ allowed some structural conclusions. The bulge present in both RNA fragments exhibits a similar, pronounced flexibility in the absence of Mg2+, indicating that the additional bulge in the 43mer does not stabilize the other bulge.

Ordered Shotgun Sequencing of a 135 kb Xq25 YAC Containing ANT2 and Four Possible Genes, Including Three Confirmed by EST Matches

Abstract: Ordered shotgun sequencing (OSS) has been successfully carried out with an Xq25 YAC substrate. yWXD703 DNA was subcloned into lambda phage and sequences of insert ends of the lambda subclones were used to generate a map to select a minimum tiling path of clones to be completely sequenced. The sequence of 135 038 nt contains the entire ANT2 cDNA as well as four other candidates suggested by computer-assisted analyses. One of the putative genes is homologous to a gene implicated in Graves' disease and it, ANT2 and two others are confirmed by EST matches. The results suggest that OSS can be applied to YACs in accord with earlier simulations and further indicate that the sequence of the YAC accurately reflects the sequence of uncloned human DNA.

Semi-automated PCR method for quantitating MDR1 expression

Abstract: The MDR1 gene is involved in drug resistance in man y hematopoietic and solid tumors The Quantitative PCR System 5000 ™ (QPCR-5000; Perkin-Elmer) is a new instrument system tha t uses electrochemiluminescence to automatically quantitate pol y merase chain reaction (PCR) products A comparative study b e tween radioactively labeled PCR ( 3 2 P-PCR) and QPCR was pe r formed to analyze the MDR1 gene expression in the drug-resistan t (Doxorubicin) cell lines Dox40, Dox6, the parental cell line 8226/S , CEM Dox1 and three acute myeloid leukemia (AML) patient sam ples Using the Dox40 and Dox6 resistant cell lines, we compared the sensitivities of QPCR and 3 2 P-PCR A strong signal was ob tained from QPCR at 20 to 25 cycles (which is in the linear range fo r quantitation), while a weak signal was obtained using 3 2 P-PCR a t the same cycle number Dilution experiments gave better precision with the QPCR than with the radioactive method AML samples wer e studied with the MDR1-specific MAbs MRK16 and 4E3, and the e f flux function was analyzed using Rh-123 retention in the absence o r presence of verapamil The three samples showed high (D = 079) , medium (D = 052) and negative (D = 008) p-glycoprotein (P-gp ) levels and correlated with efflux function The MDR1 / β 2 -M mRNA ratios for 3 2 P-PCR were 041, 040 and 012, respectively, and wer e 0127, 0097 and 0028, respectively, for QPCR There were signif i cant differences between the samples with high and medium P-gp levels comparing the two methods Very low levels of MDR1 in CEM Dox1 cells could be detected only by QPCR In conclusion, QPCR was found to be more reproducible, accurate and sensitive than

Synthesis and purification in a single column on a high-throughput automated oligonucleotide production system.

Abstract: Oligonucleotides are synthesized and purified in a single column filled with a mixture of nucleoside-loaded and underivatized, high-cross link, non-swelling polystryene beads. A new oligonucleotide production system has been developed to completely automate-the entire process of sequence entry, phosphoramidite synthesis, cleavage, deprotection, purification, quantitation, and sample collection. Up to 48 primer-length, high-purity oligonucleotides can be produced in a 24 hour period of unattended automation on the ABI 3948 DNA Synthesizer. Synthesis, cleavage, and purification occur within the unique OneStepTM column. Analyses by MicroGel capillary electrophoresis, anion-exchange HPLC, PAGE, and enzymatic digestion/HPLC routinely exhibit > 90% product purity. Stringent PCR, automated fluorescent sequencing, and genetic analysis experiments show the positive effects of the total automation of purified oligonucleotide production.

Multicomponent non-covalent associations of β-cyclodextrin (β-CD)-drug inclusion complexes with diethanolamine (DEA). Detection and characterization of gaseous protonated 1 :1 :1 β-CD-drug-DEA adducts by ionspray ionization and tandem mass spectrometry

Abstract: The multicomponent non-covalent associations of β-cyclodextrin (β-CD)–drug host–guest complexes with appropriate molecules are of current interest to the pharmaceutical industry, as they can increase dramatically the solubility in water of scarcely soluble guest drugs. The present study by ionspray and tandem mass spectrometry (MS) of the multicomponent associations with diethanolamine (DEA) of the β-CD host–guest complexes of two drugs, namely glybenclamide and furosemide, allowed the detection and characterization of the gaseous protonated 1:1:1 drug–β-CD–DEA adducts. Their dissociation patterns upon collision activation provided interesting information on the relative strength of the interactions binding the components of such charged non-covalent associations in the gas phase. In particular, from the results above it clearly emerged that the stability of the 1:1 drug–β-CD inclusion complexes within such gaseous ternary associations was much weaker than that shown previously for the analogous 1:1 terfenadine (TFN)–β-CD complex originating by the tandem MS dissociation of the 1:1:1 TFN–β-CD–HA (HA = tartaric or citric acid) protonated adducts, generated by ionspray MS. Also, we noted that these findings appear consistent with the relative stability of the corresponding 1:1 drug–β-CD host–guest complexes in solution.

Purification of 5′-O-Trityl-on Oligoribonucleotides. Investigation of Phosphate Migration During Purification and Detritylation.

Abstract: Synthetic oligoribonucleotides (RNA) are efficiently prepared with 2′-O-tert-butyldimethylsilyl nucleoside 3′-O-phosphoramidites with labile base-protection; Admf or APac, Gdmf, Cibu, U. After cleavage from the polystyrene support, the exocyclic amine protecting groups are removed with conc. NH4OH: ethanol/3:1 by heating at 55°C for 3–5 h. The 2′-O- silyl protecting groups are removed with tetra-n-butylammonium fluoride in THF or more conveniently with neat triethylamine trihydrofluoride. To gain the advantages of increased capacity on reverse phase HPLC and the convenience of cartridge based purification (OPC, Oligonucleotide Purification Cartridge), the 5′ trityl was left on the RNA as the final protecting group to be removed. The mild conditions which are effective for trityl removal are shown to preserve 3′-5′ phosphate linkage integrity in RNA. The absence of phosphate migration is demonstrated by model studies, utilizing N4 -isobutyryl-5′-O-DMT-3′-O-TBDMS-2′-O-(2-cyanoethyl-N,N-diisopropylp...

Automated Chemical Synthesis of Biologically Active tRNA Having a Sequence Corresponding to Ascaris suum Mitochondrial tRNAMet toward NMR Measurements.

Abstract: RNA samples corresponding to Ascaris suum mitochondrial tRNA(Met) were chemically and automatically synthesized in amounts sufficient for NMR measurement. Conventional and rapid deprotection methods gave tRNA samples with the same amino acid-accepting activity as those prepared by other method; enzymatic synthesis, and enzymatic ligation of chemically synthesized fragments. The synthetic tRNA showed the same 1H-NMR spectrum in the iminoproton region as the ligated tRNA. This rapid and reliable preparation method thus provides biologically active tRNA for NMR measurement, and further, it is applicable for synthesis of other large synthetic RNAs, by combining the site-specific isotopic labeling method.

High performance liquid chromatographic analysis of sulphonamides and dihydrofolate reductase inhibitors. III. the effect of a competing base, and separations with an ion pairing agent

Abstract: The effect of tertiary butyl ammonium phosphate as a competing base has been investigated in the reverse phase separation of twenty-two sulphonamides (SFA) and three commonly used dihydrofolate reductase inhibitors (DHFR). At the concentrations of t-butyl ammonium phosphate examined, the retention of the DHFR was dramatically reduced, but did not aid the separation. The effects on the SFA were inconsistent with the known pKa, l data and suggested either more complex mechanisms of interaction with the stationary phase or some doubt regarding the pKa, l values.