Energetics of interaction of oligopeptide (lac 53-57) with DNA base sequences and origin of sequence-specific recognition

Computer model building with a dynamic energy minimization procedure is used here to study the interaction of a pentapeptide sequence from the lac repressor headpiece (lac 53-57) with different base sequences of DNA. The peptide fragment for this purpose was considered in the classical beta-antiparallel as well as the beta-associated conformation. The model of its interaction with DNA was optimised for various binding positions and base sequences. Partitioning of energy is analysed for different dielectric constant values and the main contributing factors to sequence-specific binding are discussed.     

Computer simulation of zinc finger motifs from cellular nucleic acid binding protein and their interaction with consensus DNA sequences

We report here a computer simulation of the three-dimensional structures of seven zinc finger motifs from cellular nucleic acid binding protein involved in negative feedback inhibition of cholesterol biosynthesis. The structures are optimised using steric constraints imposed by tetrahedral coordination of the zinc ion with Cys and His residues, by molecular mechanics technique. We have also optimised the structure of a finger-I with GpT sequence. The model for the interaction of seven fingered protein with single-stranded d(GTGCGGTG) from sterol regulatory element (SRE) is given on the basis of these results. We also propose a scheme for recognition of a multifingered regulatory protein with small single-stranded DNA fragments.     

Stereochemical aspects of interaction of DNA binding domain of human progesterone receptor with d(AGGTCATGCT)2.

Structures of (i) 66 amino-acid fragment (residues 567-633) from DNA binding domain of human progesterone receptor (hPR), (ii) a ten base pair DNA sequence d(AGGTCATGCT)2 from hormone responsive element (HRE) and (iii) a complex of these two are optimised by computer modelling and molecular mechanics technique using extensive steric constraints from secondary structure predictions, comparison with the structures of known metalloproteins, geometric constraints imposed by tetrahedral coordination with the zinc ion and comparison with structures of DNA binding domains of human glucocorticoid and estrogen receptors (hGR and hER). Structure of the complex was obtained using genetic modification data on steroid receptors and general consensus about protein-DNA interaction. DNA is in distorted B conformation. Sequence dependent as well as protein-induced conformation changes are noticed. There is change in propeller twist, buckle and angle between glycosyl bonds. However, H-bonding network is preserved. The complex is stabilized with eighteen hydrogen-bonds, mainly between peptide side-chains and backbone phosphate. There are five specific H-bonds between basic amino acid side chains, Lys 22, Lys 26 and Arg 27, and DNA bases, A1, G3, G16 and A17. Gly 19, Ser 20 and Val 23 are in close proximity of DNA.     

A new approach for the detection of DNA sequences in amplified nucleic acids by a surface plasmon resonance biosensor

In this paper, a simple and useful approach for DNA sensing based on surface plasmon resonance (SPR) transduction is reported. A new DNA sample pre-treatment has been optimised to allow fast and simple detection of hybridisation reaction between a target sequence in solution and a probe immobilised on the sensing surface. This pre-treatment consisted in a denaturation procedure of double stranded DNA containing the target sequence and was based on an high temperature treatment (95 degrees C, 5 min) followed by a 1 min incubation with small oligonucleotides. The oligonucleotides are designed to prevent the re-hybridising of the denatured strands, while enabling the target sequence to bind the immobilised probe. The important parameters of the procedure, i.e. incubation time, length and concentration of the oligonucleotides, have been studied in detail. The optimised DNA denaturation procedure has been successfully applied to the detection of amplified DNA with a commercially available SPR biosensor (Biacore X). DNA samples extracted from plant and human blood were tested after amplification by polymerase chain reaction (PCR).     

Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.     

A single-step competitive binding assay for mapping of single DNA molecules

Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification of pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence.     

In vitro mitogenic and steroidogenic effects of ACTH analogues on an adrenal tumor cell line (Y-1)

Native porcine adrenocorticotropin (ACTH_1–39) as well as synthetic adrenocorticotropin (ACTH_1–24) increase cAMP and steroid production and inhibit DNA synthesis in an adrenal cell line. The COOH terminal sequence of both peptides as well as β-endorphin have no effects, while the NH₂ terminal sequence of ACTH as well as α-MSH which have very low stimulatory effect on cAMP production, have a mitogenic effect. These results suggest that ACTH might have in vitro some mitogenic action on adrenal cell, but this effect is blunted by cAMP accumulation during hormonal stimulation. The results can also explain the in vivo and in vitro contradictory effects of the hormone on adrenal cell replication.     

Efficient conversion of 11-deoxycortisol to cortisol (hydrocortisone) by recombinant fission yeast Schizosaccharomyces pombe

Genetically engineered microorganisms are being increasingly used for the industrial production of complicated chemical compounds such as steroids; however, there have been few reports on the use of the fission yeast Schizosaccharomyces pombe for this purpose. We previously have demonstrated that this yeast is a unique host for recombinant expression of human CYP11B2 (aldosterone synthase), and here we report the functional production of human CYP11B1 (steroid 11beta-hydroxylase) in S. pombe using our new integration vector pCAD1. In the human adrenal, the mitochondrial cytochrome P450 enzyme CYP11B1 catalyses the conversion of 11-deoxycortisol to cortisol, a key reaction in cortisol biosynthesis that in addition is of fundamental interest for the technical synthesis of glucocorticoids. We observed that the endogenous mitochondrial electron transport system detected previously by us is capable of supplying this enzyme with the reducing equivalents necessary for steroid hydroxylation activity. Under optimised cultivation conditions the transformed yeasts show in vivo the inducible ability to efficiently and reliably convert deoxycortisol to cortisol at an average rate of 201 microM d(-1) over a period of 72h, the highest value published to date for this biotransformation.     

Hormone-induced changes in the in vitro DNA-binding activity of the chicken progesterone receptor

Previous analyses have indicated that steroid hormone receptors undergo an allosteric change in structure upon binding by the steroid ligand. This structural change was envisioned as an intramolecular unmasking of the protein's DNA-binding domain, thus allowing the receptor to function in gene regulation. We report an analysis of the effect of hormone on the DNA-binding activity of the chicken progesterone receptor. Using an isocratic elution of DNA affinity columns we show that unliganded receptor (aporeceptor) can bind a 23-basepair progesterone response element with high affinity and a high degree of sequence preference. Hormone causes a 1.5-fold increase in affinity for the PRE sequence and a 2-fold decrease in affinity for non-specific DNA. Kinetic analysis of the off-rate of receptor-DNA complexes is consistent with this minor effect of hormone. In addition, gel retardation analysis of receptor-progesterone response element complexes further substantiates that hormone is not required for sequence-specific DNA binding. These results indicate that hormone is not necessary for the progesterone receptor to fold into a conformation that recognizes specific gene regulatory sequences.     

Maxiprep genomic DNA extractions for molecular epidemiology studies and biorepositories

Molecular epidemiology and genomic characterisation studies require the screening of large numbers of individuals to achieve statistical significance. Although many of the novel DNA extraction methods offer convenient, high-throughput capabilities, their use for the processing of larger sample volumes becomes very expensive. We are currently compiling the Mexican Genomic DNA Collection in order to address specific health priorities through molecular techniques. Our approach employs a low-cost laundry detergent based DNA extraction technique that maximizes DNA yield and quality. We have optimised four different modalities (maxiprep, midiprep, miniprep and microprep) for two different sources (leukocyte concentrates and whole blood). Our optimised protocol produces 4.5 mg of DNA from 15 ml of blood-bank discarded leukocyte concentrates with spectrophotometric quality, genomic integrity and PCR suitability that rivals that of phenol–chloroform extracted samples. We present evidence of many PCR applications that we have carried out on samples extracted with this technique including Killer-cell Immunoglobulin-like Receptor genotyping, Short Tandem Repeat profiling as well as nucleic acid screening for hepatitis B and human immunodeficiency type-1 viruses. This paper highlights many of the advantages that this DNA extraction technique provides over existing methodologies, whether it is used to establish large genomic DNA collections (as was our main intention) or as a routine DNA extraction method for PCR applications.     

Detection of luciferase gene sequence in nonluminescent Vibrio cholerae by colony hybridization and polymerase chain reaction.

Bioluminescence is a trait observed among approximately 10% of Vibrio cholerae isolates. We have demonstrated that not only do some strains of V. cholerae produce low levels of light, undetectable by the human eye, but the luciferase gene sequence is present in strains of V. cholerae which emit no detectable light, evidenced by hybridization with a luciferase DNA probe. Comparisons of the amino acid sequences of luciferase enzymes of marine species have shown that these proteins have diverged to the point where they have only short regions of amino acid identity. The polymerase chain reaction method of DNA amplification with oligonucleotide primers based on these regions was used to isolate a region of the luxA gene from both luminescent and nonluminescent V. cholerae strains. The nucleotide sequence of this region was determined and reveals that nonluminescent V. cholerae have 99.7% nucleotide sequence similarity in this region with the luminescent biovar V. cholerae bv. albensis as well as significant similarity to other species of bioluminescent bacteria, a finding that is in accord with the hypothesis that these species have a common luminescent ancestor, most probably from the marine environment.     

Detection of luciferase gene sequence in nonluminescent Vibrio cholerae by colony hybridization and polymerase chain reaction.

Bioluminescence is a trait observed among approximately 10% of Vibrio cholerae isolates. We have demonstrated that not only do some strains of V. cholerae produce low levels of light, undetectable by the human eye, but the luciferase gene sequence is present in strains of V. cholerae which emit no detectable light, evidenced by hybridization with a luciferase DNA probe. Comparisons of the amino acid sequences of luciferase enzymes of marine species have shown that these proteins have diverged to the point where they have only short regions of amino acid identity. The polymerase chain reaction method of DNA amplification with oligonucleotide primers based on these regions was used to isolate a region of the luxA gene from both luminescent and nonluminescent V. cholerae strains. The nucleotide sequence of this region was determined and reveals that nonluminescent V. cholerae have 99.7% nucleotide sequence similarity in this region with the luminescent biovar V. cholerae bv. albensis as well as significant similarity to other species of bioluminescent bacteria, a finding that is in accord with the hypothesis that these species have a common luminescent ancestor, most probably from the marine environment.     

Chimeric receptors as a tool for luminescent measurement of biological activities of steroid hormones

Some applications of chimeric cellular models are presented to study the biological activities of steroid hormones. We have used several chimeric constructs encoding the DNA binding domain of Gal4 yeast protein fused to the hormone binding domain of various steroid receptors (MR, PR, GR and ER). Interactions of these chimeric receptors with a 17-mer DNA sequence, specific for Gal-4, control expression of the firefly luciferase as a reporter gene. Stable transfected cell lines expressing the firefly luciferase under the control of different steroids were established and an efficient and easy sub-cloning was allowed with the help of an imaging system using a single-photon-counting camera. In the cell lines obtained, the bioluminescent response can be easily measured and thus used to measure specific biological activities of steroid agonists or antagonists. We observed that the responses are effector-concentration-dependent and their biological activities will be compared to those of native receptors.     

The vitamin D receptor: a primitive steroid receptor related to thyroid hormone receptor

We have previously reported the cloning and sequencing of both the chicken and human vitamin D3 receptor cDNAs. A comparison of their deduced amino acid sequence with that of the other classic steroid hormone receptors and the receptor for thyroid hormone indicates that there are two regions of conservation between these molecules. The first is a 70 amino acid, cysteine-rich sequence (C1), the second region (C2) is a 62 amino acid region located towards the carboxyl terminus of the proteins. In other systems the former has been identified as a region responsible for DNA binding activity, whereas the latter represents the NH2-terminal boundary of the hormone binding domain. We present here evidence utilizing eucaryotic expression of cDNA encoding the hVDR C1 domain, followed by a DNA cellulose chromatography assay, which confirms that the DNA binding activity resides in this region of the receptor for vitamin D3. Additionally, the vitamin D3 receptor contains a 60 amino acid portion at its carboxyl terminus (C3) which exhibits homology with the receptor for thyroid hormone. Conservation in this region of the molecule is found only between homologous or closely related receptors. This indicates a relationship between the vitamin D3 receptor and the receptor for thyroid hormone and may suggest that they evolved from a single primordial gene.     

Genetic heterogeneity of steroid sulfatase deficiency revealed with cDNA for human steroid sulfatase

Three cDNA clones with inserts of 1.2-1.6 kb that reacted both with antibodies and oligonucleotides specific for steroid sulfatase were isolated from a human placental library in lambda gt11. The 5'-end of one of the inserts, STS-3, was sequenced and colinearity with the amino acid sequence of 3 peptides of steroid sulfatase encompassing 64 amino acids was demonstrated. STS-3 hybridized with 2.5, 4.6 and 6.3 kb species in poly(A)+RNA and with 2.5, 4 and 9 kb fragments of EcoRI digested human DNA. The frequency of the EcoRI fragments in DNA from females was approximately twice that in DNA from males. DNA from two patients with steroid sulfatase deficiency and X-linked ichthyosis did not hybridize with STS-3. DNA from a third patient showed a normal hybridization pattern. It is concluded that steroid sulfatase deficiency is a genetically heterogenous disorder.