A universal primer mixture for sequence determination at the 3' ends of cDNAs

We have devised a universal primer which can be used to sequence the 3'-ends of cloned cDNAs containing a polyA tail. The primer consists of an equimolar mixture of three primers: 20 T nucleotides followed by either an A, C, or G nucleotide (5'----3'). With this primer mixture and the dideoxynucleotide chain termination method, we determined the 3'-terminal sequence of human beta-actin cDNA in an Okayama-Berg vector, in four parallel sets of reactions containing either a single primer (T20G, T20C, or T20A) or an equimolar mixture of all three primers. Priming with both T20A and the triple mixture gave clearly readable results that agree with the known sequence of the human beta-actin gene, and we have applied this method successfully to several other cDNAs in the Okayama-Berg expression vector. Use of this universal primer mixture facilitates determination of sequences at the 3'-ends of cDNAs while by-passing the polyA tail region.     

Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore

Multiplex quantitative PCR based on novel design of fluorescent primers is described. Fluorogenic primers are labeled with a single fluorophore on a base close to the 3′ end with no quencher required. A tail of 5–7 nt is added to the 5′ end of the primer to form a blunt-end hairpin when the primer is not incorporated into a PCR product. This design provides a low initial fluorescence of the primers that increases up to 8-fold upon formation of the PCR product. The hairpin oligonucleotides (ΔG from –1.6 to –5.8 kcal/mol) may be as efficient as linear primers and provide additional specificity to the PCR by preventing primer-dimers and mispriming. Multiple fluorogenic primers were designed by specialized software and used for real-time quantitation of c-myc and IL-4 cDNAs in the presence of reference genes such as β-actin, GAPDH and 18S rRNA. Targets of 10–10⁷ copies were detected with precision in PCR using FAM-labeled primers for variable genes and JOE-labeled primers for the reference genes. This method was also used to detect single nucleotide polymorphism of the human retinal degeneration gene by allele-specific PCR with end-point detection using a fluorescent plate reader or a UV-transilluminator. We conclude that fluorogenic mono-labeled primers are an efficient and cost-effective alternative to FRET-labeled oligonucleotides.     

Phenolic ring deiodination in cultured rat hepatoma cells, and subcellular localization of deiodinases in cultured rat hepatoma, monkey hepatocarcinoma cells and normal rat liver homogenates

The cultured rat hepatoma cell (R117-21B) homogenates metabolized 3,[3′,5′-¹²⁵I]triiodothyronine by phenolic ring deiodination and produced radioactive iodide and 3,3′-diiodothyronine. Thyroxine (T₄) was converted to 3,3′,5-triidothyronine (T₃). The production of ¹²⁵I⁻ presented the deiodinase activity. The optimal pH for phenolic ring deiodination was observed to be pH 6.0–7.0. This enzyme reaction was accelerated by dithiothreitol. Propylthiouracil strongly inhibited the phenolic ring deiodination at 0.1 mM, whereas an effect of 20 mM methylmercaptoimidazol on the deiodination was very weak or absent.Excess unlabeled iodothyronines (T₄, T₃ and 3,5-diiodo-l-thyronine inhibited the phenolic ring deiodination of labeled 3,3′,5′-triiodothyronine, althought their inhibitory effect was slightly different. Triiodothyroacetic acid was a better inhibitor than T₃. Diiodotyrosine did not affect phenolic ring deiodination in cultured rat hepatoma cell homogenates.Phenolic and nonphenolic ring deiodinase activities of cultured monkey hepatocarcinoma cell and rat liver homogenates were also studied by the use of 3,[3′,5′-¹²⁵I]triiodothyronine and [3,5-¹²⁵I]thyroxine, respectively. Both deiodinase activities were observed in particulate fractions (mitochondrial and microsomal) of cultured cell and rat liver homogenates.     

Quantitative multiplexing analysis of PCR-amplified ribosomal RNA genes by hierarchical oligonucleotide primer extension reaction

A method, termed hierarchical oligonucleotide primer extension (HOPE), is developed for quantitative, multiplexing detection of DNA targets present in PCR-amplified community 16S rRNA genes. It involves strand extension reaction and multiple oligonucleotide primers modified with different lengths of polyA at the 5′ end and targeting 16S rRNA genes at different phylogenetic specificities. On annealing to the targets, these primers are extended with a single fluorescently labeled dideoxynucleoside triphosphate or a dye-terminator. Using a DNA autosequencer, these extended primers are separated and identified by size and dye color, and quantified and normalized based on the fluorescence intensities and internal size standards. Using a primer-to-target ratio >1000, constant primer extension efficiencies can be obtained with individual primers to establish a ‘calibration factor’ between individual primers and a universal or domain-specific primer, providing the relative abundance of targeted rRNA genes with respect to total rRNA genes. HOPE up to 10-plexing is demonstrated to correctly identify 20 different bacterial strains, and quantify different Bacteroides spp. in 16S rRNA gene amplicons from different model bacteria mixtures and the influent and effluent of a wastewater treatment plant. Single mismatch discrimination with detection sensitivity of a target down to 0.01–0.05% of total DNA template is achieved.     

Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers

Genomes are becoming heavily annotated with important features. Analysis of these features often employs oligonucleotides that hybridize at defined locations. When the defined location lies in a poor sequence context, traditional design strategies may fail. Locked Nucleic Acid (LNA) can enhance oligonucleotide affinity and specificity. Though LNA has been used in many applications, formal design rules are still being defined. To further this effort we have investigated the effect of LNA on the performance of sequencing and PCR primers in AT-rich regions, where short primers yield poor sequencing reads or PCR yields. LNA was used in three positional patterns: near the 5′ end (LNA-5′), near the 3′ end (LNA-3′) and distributed throughout (LNA-Even). Quantitative measures of sequencing read length (Phred Q30 count) and real-time PCR signal (cycle threshold, C_T) were characterized using two-way ANOVA. LNA-5′ increased the average Phred Q30 score by 60% and it was never observed to decrease performance. LNA-5′ generated cycle thresholds in quantitative PCR that were comparable to high-yielding conventional primers. In contrast, LNA-3′ and LNA-Even did not improve read lengths or C_T. ANOVA demonstrated the statistical significance of these results and identified significant interaction between the positional design rule and primer sequence.     

Sequence tagged microsatellite profiling (STMP): improved isolation of DNA sequence flanking target SSRs

Sequence tagged microsatellite profiling (STMP) enables the rapid development of large numbers of co-dominant DNA markers, known as sequence tagged microsatellites (STMs). Each STM is amplified by PCR using a single primer specific to the conserved DNA sequence flanking the microsatellite repeat in combination with a universal primer that anchors to the 5′-ends of the microsatellites. It is also possible to convert STMs into conventional microsatellite, or simple sequence repeat (SSR), markers that are amplified using a pair of primers flanking the repeat sequence. Here, we describe a modification of the STMP procedure to significantly improve the capacity to convert STMs into conventional SSRs and, therefore, facilitate the development of highly specific DNA markers for purposes such as marker-assisted breeding. The usefulness of this technique was demonstrated in bread wheat.     

Thyroid hormone: Aldosterone antagonism in cultured epithelial cells

Thyroid hormone (T₃) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have exammined the effect of T₃ on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5·10⁻⁸ M T₃ to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T₃ completely inhibits the maximal increase in short-circuit current in response to 1·10⁻⁷ M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T₃ and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5·10⁻⁹ M T₃. T₃ has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T₃ on nuclear binding of [³H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were K′_d1 = (0.82 ± 0.36)·10⁻¹⁰ M and K′_d2 = (3.2±0.60)·10⁻⁸ M.The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1·10⁻⁸ M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5·10⁻⁸ M T₃ showed a K′_d1 = (0.15 ± 0.10)·10⁻¹⁰ M and K′_d2 = (3.5 ± 0.10)·10⁻⁸ M. We conclude that T₃ i action of aldosterone on sodium transport at a site after receptor binding in the nucleus.     

Purification of chemically synthesised dinucleoside(5′,5′) polyphosphates by displacement chromatography

Dinucleoside(5′,5′) polyphosphates (Ap_nA, Ap_nG, Gp_nG, n=3–6) are new group of hormones controlling important biological processes. Because some of the dinucleoside(5′,5′) polyphosphates are commercially not available purification of chemical synthesised dinucleoside(5′,5′) polyphosphates became necessary in order to test their physiological and pharmacological properties. It was the aim of this study to find a method which allows purification of 0.1–0.2 g quantities of dinucleoside polyphosphates by analytical HPLC columns yielding products with impurities lower than 1.0%. Adenosine(5′)-polyphospho-(5′)guanosines were synthesised by mixing the corresponding mononucleotides. The reaction results in a complex mixture of Ap_nA, Ap_nG and Gp_nG (with n=3–6 in all cases). The reaction mixture was concentrated on a preparative C₁₈ reversed-phase column. The concentrate was displaced on a reversed-phase stationary. As a result of displacement chromatography, anion-exchange chromatography in gradient modus yielded baseline separated dinucleoside polyphosphates (homogeneity of the fractions>99%). The identity of the substances were determined by matrix assisted laser desorption ionisation mass spectrometry.     

DNA Sequence-Specific Ligands: XI.* The Synthesis and Binding to DNA of bis-Netropsins with the C-Ends of Their Netropsin Fragments Tethered by Tetra- or Pentamethylene Linkers

Bis-Netropsins with the C-ends of their netropsin fragments tethered via tetra- or pentamethylene linkers and with Gly or L-Lys-Gly residues on their N-ends were synthesized. The footprinting technique was used to study the specificity of bis-netropsin binding to the specially constructed DNA fragments containing various clusters of A · T pairs. It was found that the linker length affects the binding of bis-netropsins, with the tetramethylene linker providing better protection than the pentamethylene linker. It was shown that the newly synthesized bis-netropsins bind tighter to the 5"-A ₄ T ₄-3" sequence, whereas the bis-netropsin with a linker between the netropsin N-ends binds better to 5"-T ₄ A ₄-3" sequences.     

Characterisation of the beta-tubulin gene of Cylicocyclus nassatus

mRNA and genomic DNA were isolated from adult Cylicocyclus nassatus, and the mRNA was reverse transcribed. The cDNA was PCR amplified using degenerate primers designed according to the alignment of the β-tubulin amino acid sequences of other species. To complete the coding sequence, the 3′ end was amplified with the 3′-RACE, and for amplification of the 5′ end the SL1-primer was used. The cDNA of the β-tubulin gene of C. nassatus spans 1429 bp and encodes a protein of 448 amino acids. Specific primers were developed from the cDNA sequence to amplify the genomic DNA sequence and to analyse the genomic organisation of the β-tubulin gene. The complete sequence of the genomic DNA of the β-tubulin gene of C. nassatus has a size of 2652 bp and is organised into nine exons and eight introns. The identities with the exons of the gru-1 β-tubulin gene of Haemonchus contortus range between 79% and 97%.     

The effect of propylthiouracil on thyroid-stimulating hormone-induced alterations in iodothyronine secretion from perfused dog thyroids

The aim of this study was to see whether the inhibitory effect of propylthiouracil on thyroidal secretion of 3,5,3′-triiodothyronine (T₃) and 3,3′,5′-triiodothyronine (rT₃) could be reproduced in intensively stimulated thyroids, and to elucidate whether an increase in the fractional deiodination of thyroxine (T₄) to T₃ and rT₃ during iodothyronine secretion might be responsible for the transient fall in the T₄/T₃ and T₄/rT₃ ratios in thyroid secretion seen in the early phase after stimulation of thyroid secretion.For this purpose T₄, T₃ and rT₃ were measured in effluent from isolated dog thyroid lobes perfused in a non-recirculation system using a synthetic hormone free medium. 1 mmol/l propylthiouracil induced a significant reduction in thyroid-stimulating hormone (TSH) stimulated T₃ and rT₃ release while the release of T₄ was unaffected. This supports our previous conclusion that T₄ is partially monodeiodinated to T₃ and rT₃ during thyroid secretion. Infusion of 1 mmol/l propylthiouracil for 30 min or 3 mmol/l propylthiouracil for 120 min did not abolish the transient fall in effluent T₄/T₃ and T₄/rT₃ induced by TSH stimulation. Thus, this phenomenon seems not to depend on intrathyroidal iodothyromine deiodinating processes.     

Characterization of Three Novel Human Cadherin Genes (CDH7, CDH19, and CDH20) Clustered on Chromosome 18q22–q23 and with High Homology to Chicken Cadherin-7

Full-length coding sequences of two novel human cadherin cDNAs were obtained by sequence analysis of several EST clones and 5′ and 3′ rapid amplification of cDNA ends (RACE) products. Exons for a third cDNA sequence were identified in a public-domain human genomic sequence, and the coding sequence was completed by 3′ RACE. One of the sequences (CDH7L1, HGMW-approved gene symbol CDH7) is so similar to chicken cadherin-7 gene that we consider it to be the human orthologue. In contrast, the published partial sequence of human cadherin-7 is identical to our second cadherin sequence (CDH7L2), for which we propose CDH19 as the new name. The third sequence (CDH7L3, HGMW-approved gene symbol CDH20) is almost identical to the mouse “cadherin-7” cDNA. According to phylogenetic analysis, this mouse cadherin-7 and its here presented human homologue are most likely the orthologues of Xenopus F-cadherin. These novel human genes, CDH7, CDH19, and CDH20, are localized on chromosome 18q22–q23, distal of both the gene CDH2 (18q11) encoding N-cadherin and the locus of the six desmosomal cadherin genes (18q12). Based on genetic linkage maps, this genomic region is close to the region to which Paget's disease was linked. Interestingly, the expression patterns of these three closely related cadherins are strikingly different.     

Massive parallel analysis of the binding specificity of histone-like protein HU to single- and double-stranded DNA with generic oligodeoxyribonucleotide microchips

A generic hexadeoxyribonucleotide microchip has been applied to test the DNA-binding properties of HU histone-like bacterial protein, which is known to have a low sequence specificity. All 4096 hexamers flanked within 8mers by degenerate bases at both the 3′- and 5′-ends were immobilized within the 100 × 100 × 20 mm polyacrylamide gel pads of the microchip. Single-stranded immobilized oligonucleotides were converted in some experiments to the double-stranded form by hybridization with a specified mixture of 8mers. The DNA interaction with HU was characterized by three type of measurements: (i) binding of FITC-labeled HU to microchip oligonucleotides; (ii) melting curves of complexes of labeled HU with single-stranded microchip oligonucleotides; (iii) the effect of HU binding on melting curves of microchip double-stranded DNA labeled with another fluorescent dye, Texas Red. Large numbers of measurements of these parameters were carried out in parallel for all or many generic microchip elements in real time with a multi-wavelength fluorescence microscope. Statistical analysis of these data suggests some preference for HU binding to G/C-rich single-stranded oligonucleotides. HU complexes with double-stranded microchip 8mers can be divided into two groups in which HU binding either increased the melting temperature (T_m) of duplexes or decreased it. The stabilized duplexes showed some preference for presence of the sequence motifs AAG, AGA and AAGA. In the second type of complex, enriched with A/T base pairs, the destabilization effect was higher for longer stretches of A/T duplexes. Binding of HU to labeled duplexes in the second type of complex caused some decrease in fluorescence. This decrease also correlates with the higher A/T content and lower T_m. The results demonstrate that generic microchips could be an efficient approach in analysis of sequence specificity of proteins.     

Polyadenylated H3 histone transcripts and H3 histone variants in alfalfa

Histone H3 mRNAs were found in polyA(+) fractions of total RNA prepared from alfalfa plants, calli and somatic embryos. The sequence analysis of cDNAs revealed the presence of a polyA tail on independent alfalfa H3 mRNAs. A highly conserved sequence motif AAUGAAA identified about 20bp upstream from the 3' ends of the alfalfa H3 cDNAs was suggested to be one of the possible regulatory elements in the 3' end formation and polyadenylation. Three out of the four analysed H3 cDNAs have more than 97% homology with a genomic clone and encode the same protein. While the fourth represents a minor species with only 78.8% homology to the coding region of the genomic clone and encodes a H3 histone with four amino acid replacements. On the basis of compilation analysis we suggest a consensus sequence for plant H3 histones which differs from that of animal's by four amino acid changes.