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PubMed1997

Spectroscopic and thermodynamic studies of DNA duplexes containing alpha-anomeric C, A, and G nucleotides and polarity reversals: coexistence of localized parallel and antiparallel DNA.

We present a thermodynamic, enzymatic, and spectroscopic study of three self-complementary DNA decamer duplexes, d[GCGAATT-3'-3'-(alphaC)-5'-5'-GC]2 (alphaC), d[GCG-3'-3'-(alphaA)-5'-5'-ATTCGC]2 (alphaA), and d[GC-3'-3'-(alphaG)-5'-5'-AATTCGC]2 (alphaG), which are identical in sequence but contain one alpha-anomeric nucleotide per strand in a parallel orientation via 3'-3' and 5'-5' phosphodiester bonds; the results are placed in the context of our recent studies on the other members of this series, namely alphaT, d[GCGAAT-3'-3'-(alphaT)-5'-5'-CGC]2, and the unmodified control [Aramini, J. M., et al. (1996) Biochemistry 35, 9355-9365]. On the basis of UV hyperchromicity and melting profiles as well as 1H and 31P nuclear magnetic resonance (NMR) spectroscopic data, we conclude that all five constructs form stable duplexes, with very comparable structural features that are consistent with an overall right-handed, antiparallel B-DNA motif and Watson-Crick base pairing throughout. However, each of the alpha-containing sequences exhibits unique thermodynamic and structural differences ascribed to the nature (and position) of the alpha-nucleotide. First, the thermostability of these duplexes decreases from the control to alphaC in the following series: control > alphaT approximately alphaA approximately alphaG > alphaC. Second, in each of the four alpha-duplexes, 1H and 31P chemical shift differences compared to those of the control duplex are largely confined to the region encompassing the alpha-nucleotide and unnatural phosphodiester linkages, as well as neighboring nucleotides. Surprisingly, for alphaC, these modifications result in a significant alteration to the backbone conformation at the phosphodiester group directly across from the 3'-3' linkage. Finally, spin-spin (J) coupling data, specifically Sigma1', indicate that the vast majority of the furanose rings in these duplexes display a high propensity for adopting the S pucker. However, in alphaC, alphaA, and alphaT (but not alphaG), the sugar ring conformation in the nucleotide immediately following the 5'-5' linkage is described by an approximately equal distribution between the N and S conformers.

Base Composition↗
PubMed1998

Mutation detection and typing of polymorphic loci through double-strand conformation analysis.

Variations, such as nucleotide substitutions, deletions and insertions, within genes can affect the function of the gene product and in some cases be deleterious. Screening for known allelic variation is important for determining disease and gene associations. Techniques which target specific mutations such as restriction enzyme polymorphism and oligonucleotide probe or PCR primer reactivity are useful for the detection of specific mutations, but these techniques are not generally effective for the identification of new mutations. Approaches for measuring changes in DNA conformation have been developed, based on the principle that DNA fragments which differ in nucleotide composition exhibit different mobilities after separation by polyacrylamide gel electrophoresis (PAGE). Here we describe a conformation-based mutation detection system, double-strand conformation analysis (DSCA), which provides a simple means to detect genetic variants and to type complex polymorphic loci. We demonstrate the application of DSCA to detect genetic polymorphisms such as a single-nucleotide difference within DNA fragments of up to 979 base pairs in length. We present the application of DSCA in detecting four different mutations in the cystic fibrosis gene (CFTR) and 131 different alleles encoded by HLA class I genes.

DNA↗
PubMed1993

The minimal self-sufficient element in a murine G+C-rich promoter is a large element with imperfect dyad symmetry.

The murine adenosine deaminase gene has a structurally archetypal TATAA-box-deficient G+C-rich promoter. The three Sp1 binding sites of the promoter are neither necessary nor sufficient for promoter function. Minimal basal promoter activity resides within a 48-bp element downstream of the Sp1 binding sites. This element shows an imperfect dyad symmetry around the promoter's major transcriptional initiation site and contains at least two nuclear protein binding sites. The distinctive sequence characteristics and nuclear protein binding locations of this element led us to propose a model for how such promoters may function.

Adenosine Deaminase↗
PubMed1996

Competition for a unique response element mediates retinoic acid inhibition of vitamin D3-stimulated transcription.

We have identified a novel steroid hormone response element in the avian beta3 integrin promoter. This sequence, comprising three hexameric direct repeat half-sites separated by nine and three nucleotides binds vitamin D receptor (VDR)-retinoid X receptor (RXR) and retinoic acid receptor (RAR)-RXR heterodimers. VDR-RXR binds direct repeats separated by three base pairs, and RAR-RXR recognizes half-sites separated by nine bases, whereas the central half-site interacts with both heterodimers. Retinoic acid and 1, 25-dihydroxyvitamin D3 activate both a genomic fragment including the transcriptional start site and an oligonucleotide containing the three repeats, linked to a heterologous promoter. Co-addition of the steroids produces neither synergy nor an additive effect; rather the result equals that for retinoic acid alone. Scatchard analysis demonstrates that RAR-RXR has greater affinity than VDR-RXR for the composite element. Based on these findings we propose a model in which there is specific, polarity-defined binding of VDR-RXR and RAR-RXR to three half-sites, which form two overlapping steroid response elements, with the central half-site common to both. Our results identify a novel mechanism by which one steroid hormone can modulate the activity of a second, by competing for a shared half-site in a composite response element.

Animals↗
PubMed2003

Translational selection and yeast proteome evolution.

The primary structures of peptides may be adapted for efficient synthesis as well as proper function. Here, the Saccharomyces cerevisiae genome sequence, DNA microarray expression data, tRNA gene numbers, and functional categorizations of proteins are employed to determine whether the amino acid composition of peptides reflects natural selection to optimize the speed and accuracy of translation. Strong relationships between synonymous codon usage bias and estimates of transcript abundance suggest that DNA array data serve as adequate predictors of translation rates. Amino acid usage also shows striking relationships with expression levels. Stronger correlations between tRNA concentrations and amino acid abundances among highly expressed proteins than among less abundant proteins support adaptation of both tRNA abundances and amino acid usage to enhance the speed and accuracy of protein synthesis. Natural selection for efficient synthesis appears to also favor shorter proteins as a function of their expression levels. Comparisons restricted to proteins within functional classes are employed to control for differences in amino acid composition and protein size that reflect differences in the functional requirements of proteins expressed at different levels.

Adaptation, Physiological↗
PubMed1992

Formation of novel hairpin structures by telomeric C-strand oligonucleotides.

Telomeres are specialized structures at the ends of chromosomes that are required for long term chromosome stability and replication of the chromosomal terminus. Telomeric DNA consists of simple repetitive sequences with one strand G-rich relative to the other, C-rich, strand. Evolutionary conservation of this feature of telomeric repeat sequences suggests that they have specific structural characteristics involved in telomere function. Absorbance thermal denaturation, chemical modification and non-denaturing gel electrophoretic analyses showed that telomeric C-strand oligonucleotides form stable non-Watson-Crick hairpin structures containing C.C+ base pairs. Formation of such hairpins may facilitate previously reported G-strand exclusive interactions.

Animals↗
PubMed1981

Antigenic and genomic analyses of influenza A(H1N1) viruses from different regions of the world, February 1978 to March 1980.

Antigenic analysis of influenza A(H1N1) virus isolates by hemagglutination inhibition reactions with postinfection ferret sera and mouse monoclonal antibodies showed that, although the predominant virus in 1978 to 1979 had an A/Brazil/11/78-like hemagglutinin, several new antigenic variants could be detected which differed from those identified in 1977 to 1978. Most of the variants showed very minor changes and did not achieve epidemiological significance. Genome analysis by ribonucleic acid-ribonucleic acid hybridization, and supported by oligonucleotide mapping, indicated that nonrecombinant H1N1 viruses cocirculated in the world with recombinant H1N1 viruses, identified in 1978 to 1979. The results of our antigenic analysis and genomic analysis, in conjunction with genomic analysis by other authors, showed that recombinants with A/USSR/90/77-like and A/Brazil/11/78-like hemagglutinin had different genome compositions, indicating that H3N2 virus recombined separately with these different antigenic variants. Prevalence of the nonrecombinant and recombinant H1N1 viruses varied between regions of the world, and influenza A(H1N1) virus with a genotype almost identical to that of nonrecombinant A/Brazil/11/78 virus reappeared as a cause of local outbreaks in the United States in 1980 despite the predominance of recombinant H1N1 strains in the preceding winter.

Animals↗
PubMed1991

A single base pair dominates over the novel identity of an Escherichia coli tyrosine tRNA in Saccharomyces cerevisiae.

The Escherichia coli su+3 tyrosine tRNA was shown recently to be a leucine-specific tRNA in Saccharomyces cerevisiae. This finding raises the possibility that some determinants for tRNA identity in E. coli may be different in S. cerevisiae. To investigate whether the fungal system is sensitive to the major determinant for alanine acceptance in E. coli, a single G3 . U70 base pair was introduced into the acceptor helix of the su+3 tyrosine tRNA. This substitution converts the identity of the E. coli suppressor in S. cerevisiae from leucine to alanine. Thus, as in E. coli, G3 . U70 is a strong determinant for alanine acceptance that can dominate over other features in a tRNA that might be recognized by alternative charging enzymes.

Amino Acid Sequence↗
PubMed1997

Parallel-stranded duplexes and quartet assemblies formed by oligonucleotides containing isoguanine.

Oligonucleotides with isoguanine-cytosine base pairs form duplexes with parallel chain orientation. This strong base pair dictates the strand polarity when additional dA-dT or N7Ad-dT pairs are present. The same is observed for the isocytosine-guanine pair. Quartet structures are built by the self-assembly of oligonucleotides containing short runs of isoguanine or 7-deazaisoguanine. New base pairs between isoguanine or guanine and 5-aza-7-deazaguanine are presented.

Base Composition↗
PubMed2002

Solution composition and thermal denaturation for the production of single-stranded PCR amplicons: piperidine-induced destabilization of the DNA duplex?

Strategies to produce single-stranded PCR amplicons for detection by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) were investigated using modified electrospray solutions and by thermally denaturing the duplex structures with a resistively heated electrospray ionization source. A synthetic 20-mer oligonucleotide annealed to its complementary strand was used as a model system for initial experiments. Electrospray solutions were altered by varying the relative proportion of aqueous phase in efforts to induce destabilization of the double helix. When the electrospray solution contains a 25% aqueous content, the 20-mer oligonucleotide is detected in its double-stranded form. Increasing the proportion of aqueous phase in the electrospray solution to 60% destabilized the double helix, resulting in the detection of only single-stranded species. This strategy was extended to an 82-bp polymerase chain reaction (PCR) product derived from the human tyrosine hydroxylase gene (HUMTH01). In efforts to destabilize the 82-bp PCR product, electrospray solutions reaching 70% aqueous content were necessary to promote the detection of only single-stranded amplicons. Implementation of the resistively heated transfer line and an electrospray solution in which the oligonucleotide is on the threshold of duplex stability allowed for double-stranded and single-stranded species to be generated from the same ESI solutions at both ambient and elevated transfer line temperatures, respectively, without disruption of the electrospray process. The volatile base piperidine, present at 20 mM concentrations in the electrospray solution, was found to play a critical role in the formation of single-stranded species at the higher aqueous percentages and a duplex destabilization mechanism has been proposed.

Cyclotrons↗
PubMed2003

Hypothalamic melanin-concentrating hormone is induced by cold exposure and participates in the control of energy expenditure in rats.

Short-term cold exposure of homeothermic animals leads to higher thermogenesis and food consumption accompanied by weight loss. An analysis of cDNA-macroarray was employed to identify candidate mRNA species that encode proteins involved in thermogenic adaptation to cold. A cDNA-macroarray analysis, confirmed by RT-PCR, immunoblot, and RIA, revealed that the hypothalamic expression of melanin-concentrating hormone (MCH) is enhanced by exposure of rats to cold environment. The blockade of hypothalamic MCH expression by antisense MCH oligonucleotide in cold-exposed rats promoted no changes in feeding behavior and body temperature. However, MCH blockade led to a significant drop in body weight, which was accompanied by decreased liver glycogen, increased relative body fat, increased absolute and relative interscapular brown adipose tissue mass, increased uncoupling protein 1 expression in brown adipose tissue, and increased consumption of lean body mass. Thus, increased hypothalamic MCH expression in rats exposed to cold may participate in the process that allows for efficient use of energy for heat production during thermogenic adaptation to cold.

Adaptation, Physiological↗
PubMed2006

Nature of Cx30-containing channels in the adult mouse mammary gland.

Oligonucleotide microarray analysis uniquely shows that several members of the connexin family of gap junction proteins are expressed by the epithelium during mouse mammary gland development. Connexin 26 (Cx26) is present throughout pregnancy and lactation, is then undetectable shortly after weaning, but reappears during involution. Additionally, Cx30 is abundant in late-pregnant and early lactating gland epithelium. From mid-pregnancy into early lactation, Cx26 and Cx30 co-localize in junctional plaques between epithelial cells, forming hemichannels of mixed connexin content. Microarray analysis also shows Cx32 is developmentally restricted to parturition, suggesting that specific modification of gap junction channel composition and/or intercellular communication pathways occurs at parturition. Specifically, heteromeric channels of all pairwise combinations are formed when these connexins are expressed within the same cells. Of these hemichannels, Cx26/Cx32 pores are increasingly sensitive to closure by taurine (an osmolyte implicated in milk protein synthesis) with increasing Cx26 content. In contrast, physiological taurine concentrations have no effect on Cx26/Cx30 and Cx30/Cx32 channel activity. Such changes in connexin expression and channel composition and their chemical modulation are discussed in relation to the various stages of mammary gland development in the adult mouse.

Animals↗
PubMed2006

Liver-specific inhibition of acyl-coenzyme a:cholesterol acyltransferase 2 with antisense oligonucleotides limits atherosclerosis development in apolipoprotein B100-only low-density lipoprotein receptor-/- mice.

OBJECTIVE: The purpose of this study was to determine the effects of liver-specific inhibition of acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) on the development of hypercholesterolemia and atherosclerosis in mice. METHODS AND RESULTS: Apolipoprotein B100-only low-density lipoprotein (LDL) receptor-/- mice were given saline, a nontargeting control antisense oligonucleotide (ASO), or ASOs targeting ACAT2 biweekly for a period spanning 16 weeks. Mice treated with ACAT2 targeting ASOs had liver-specific reduction in ACAT2 mRNA, yet intestinal ACAT2 and cholesterol absorption was left undisturbed. ASO-mediated knockdown of ACAT2 resulted in reduction of total plasma cholesterol, increased levels of plasma triglyceride, and a shift in LDL cholesteryl ester (CE) fatty acid composition from mainly saturated and monounsaturated to polyunsaturated fatty acid enrichment. Furthermore, the liver-specific depletion of ACAT2 resulted in protection against diet-induced hypercholesterolemia and aortic CE deposition. This is the first demonstration that specific pharmacological inhibition of ACAT2, without affecting ACAT1, is atheroprotective. CONCLUSIONS: Hepatic ACAT2 plays a critical role in driving the production of atherogenic lipoproteins, and therapeutic interventions, such as the ACAT2-specific ASOs used here, which reduce acyltransferase 2 (ACAT2) function in the liver without affecting ACAT1, may provide clinical benefit for cardiovascular disease prevention.

Animals↗
PubMed1994

13C-NMR of the deoxyribose sugars in four DNA oligonucleotide duplexes: assignment and structural features.

Natural-abundance 13C-NMR spectra have been obtained for four self-complementary DNA oligonucleotides: [d(TAGCGCTA)]2, [d(GGTATACC)]2, [d(CG)3]2, and [d(TCGCG)]2; this paper focuses on the deoxyribose resonances. Assignments were made by a combination of the two-dimensional proton-detected heteronuclear correlation experiment and comparison of 1D spectra, accounting for 31P coupling, base composition, and similarities in chemical shift versus temperature profiles (delta vs T). Large shielding and deshielding of the sugar resonances (between 2.0 and -1.9 ppm) are observed upon thermal dissociation of the duplex. The shapes of the delta vs T profiles correlate strongly with the purine/pyrimidine nature of the base attached at C1' in these duplexes that have a substantial fraction of residues within alternating purine-pyrimidine sequences. The correlation is primarily associated with changes in the equilibrium distribution of furanose pseudorotational states that may arise in part from the relief of interstrand purine-purine steric clashes.

DNA↗
PubMed1997

DNA sequence context and protein composition modulate HMG-domain protein recognition of cisplatin-modified DNA.

Proteins containing the high mobility group (HMG) DNA-binding domain form specific complexes with cisplatin-modified DNA which shield the major intrastrand d(GpG) and d(ApG) cross-links from excision repair. The molecular basis for the specificity of binding was investigated for the two isolated domains of HMG1 with a series of 15-bp oligonucleotides, d(CCTCTCN1G*G*N2TCTTC). (GAAGAN3CCN4GAGAGG), where asterisks denote N7-modification of guanosine with cisplatin. Alteration of the nucleotides flanking the platinum lesion modulated HMG1domA recognition in this series by over 2 orders of magnitude and revealed an unprecedented preference for N2 = dA > T > dC. The flanking nucleotide preference for HMG1domB interaction with this oligonucleotide series was less pronounced and had only a 20-fold range of binding affinities. For the N1 = N2 = dA 15-bp probe, 100-fold stronger binding occurred with HMG1domA (Kd = 1.6 +/- 0.2 nM) compared to HMG1domB (Kd = 134 +/- 18 nM). The platinum-dependent recognition of the N1 = N2 = dA 15-bp probe saturates at 1 equiv of HMG1domA and is highly specific, as evidenced by the 1000-fold decrease in HMG1domA binding affinity for the corresponding unplatinated oligonucleotide. HMG domains were unable to bind specifically to cisplatin-modified DNA-RNA hybrids, revealing the need for a deoxyribose sugar backbone for specific complex formation with HMG-domain proteins. Protein-DNA contacts which may account for these observed binding preferences are proposed, and potential implications for the biological processing of cisplatin-DNA adducts are discussed.

Amino Acid Sequence↗
PubMed1995

Accurate modification of the trypanosome spliced leader cap structure in a homologous cell-free system.

During RNA maturation in trypanosomatid protozoa, trans-splicing transfers the spliced leader (SL) sequence and its cap from the SL RNA to the 5' end of all mRNAs. In Trypanosoma brucei and Crithidia fasciculata the SL RNA has an unusual cap structure with four methylated nucleotides following the 7-methylguanosine residue (cap 4). Since modification of the 5' end of the SL RNA is a pre-requisite for trans-splicing activity in T. brucei, we have begun to characterize the enzyme(s) involved in this process. Here we report the development of a T. brucei cell-free system for modification of the cap of the SL RNA. Analysis of the nucleotide composition of the in vitro generated cap structure by two-dimensional thin layer chromatography established that the in vitro reaction is accurate. Cap 4 formation requires the SL RNA to be in a ribonucleoprotein particle and can be inhibited by annealing a complementary 2'-O-methyl RNA oligonucleotide to nucleotides 7-18 of the SL RNA. Methylation of the 5' end of the SL RNA is also required for trans-splicing in T. cruzi and Leishmania amazonensis and cell-free extracts from C. fasciculata and L. amazonensis are capable of modifying the cap structure on the T. brucei SL ribonucleoprotein particle.

Adenosine Triphosphate↗
PubMed1999

The type and the localization of cAMP-dependent protein kinase regulate transmission of cAMP signals to the nucleus in cortical and cerebellar granule cells.

cAMP signals are received and transmitted by multiple isoforms of cAMP-dependent protein kinases, typically determined by their specific regulatory subunits. In the brain the major regulatory isoform RIIbeta and the RII-anchor protein, AKAP150 (rat) or 75 (bovine), are differentially expressed. Cortical neurons express RIIbeta and AKAP75; conversely, granule cerebellar cells express predominantly RIalpha and RIIalpha. Cortical neurons accumulate PKA catalytic subunit and phosphorylated cAMP responsive element binding protein very efficiently into nuclei upon cAMP induction, whereas granule cerebellar cells fail to do so. Down-regulation of RIIbeta synthesis by antisense oligonucleotides inhibited cAMP-induced nuclear signaling in cortical neurons. Expression in cerebellar granule cells of RIIbeta and AKAP75 genes by microinjection of specific expression vectors, markedly stimulated cAMP-induced transcription of the lacZ gene driven by a cAMP-responsive element promoter. These data indicate that the composition of PKA in cortical and granule cells underlies the differential ability of these cells to transmit cAMP signals to the nucleus.

A Kinase Anchor Proteins↗