B- to Z-DNA transition probed by oligonucleotides containing methylphosphonates.

The simulation of the B--Z-DNA transition by using space-filling models of the dimer d(C-G) shows the possibility of hydrogen-bond formation between the N-2 amino group of the partially rotated guanine and one of the 5'-phosphate oxygens of deoxyguanylic acid. To probe the importance of this postulated interaction, analogs of the hexamer d(C-G)3 were synthesized. These analogs contained a methylphosphonate linkage, of distinct stereochemistry, which replaced the first 5'-phosphate linkage of deoxyguanosine. The CD spectra in high salt concentration showed that the hexamer containing a methylphosphonate linkage with the RP stereochemistry formed Z-DNA to the same extent as d(C-G)3, whereas the hexamer containing a methylphosphonate linkage with the SP stereochemistry did not form Z-DNA. These results are consistent with a mechanism in which an interaction between the N-2 amino group of guanine and the prochiral SP oxygen of deoxyguanosine 5'-phosphate kinetically controls the formation of Z-DNA. A water bridge between the N-2 amino group of guanine and the 3'-phosphate oxygen of deoxyguanylic acid has been implicated in the stabilization of Z-DNA. To probe the importance of this water bridge, two additional analogs of the hexamer d(C-G)3 were synthesized. These analogs contained a methylphosphonate linkage, of distinct stereochemistry, that replaced the first deoxyguanosine 3'-phosphate. The CD spectra showed that the hexamer containing a methylphosphonate linkage of the RP stereochemistry underwent the transition to Z-DNA to the same extent as d(C-G)3, whereas the hexamer containing a methylphosphonate linkage of the SP stereochemistry underwent the transition to Z-DNA to a 35% lesser extent. Thus the water bridge involving the prochiral SP oxygen provides modest stabilization energy for Z-DNA. These studies, therefore, suggest that the B--Z-DNA transition is regulated both thermodynamically and kinetically through hydrogen-bond interactions involving phosphate oxygens and the N-2 amino group of guanine.     

HuR binding to cytoplasmic mRNA is perturbed by heat shock

AU-rich elements (AREs) located in the 3' untranslated region target the mRNAs encoding many protooncoproteins, cytokines, and lymphokines for rapid degradation. HuR, a ubiquitously expressed member of the embryonic lethal abnormal vision (ELAV) family of RNA-binding proteins, binds ARE sequences and selectively stabilizes ARE-containing reporter mRNAs when overexpressed in transiently transfected cells. HuR appears predominantly nucleoplasmic but has been shown to shuttle between the nucleus and cytoplasm via a novel shuttling sequence HNS. We report generation of a mouse monoclonal antibody 3A2 that both immunoblots and immunoprecipitates HuR protein; it recognizes an epitope located in the first of HuR's three RNA recognition motifs. This antibody was used to probe HuR interactions with mRNA before and after heat shock, a condition that has been reported to stabilize ARE-containing mRNAs. At 37 degrees C, approximately one-third of the cytoplasmic HuR appears polysome associated, and in vivo UV crosslinking reveals that HuR interactions with poly(A)(+) RNA are predominantly cytoplasmic rather than nuclear. This comprises evidence that HuR directly interacts with mRNA in vivo. After heat shock, 12-15% of HuR accumulates in discrete foci in the cytoplasm, but surprisingly the majority of HuR crosslinks instead to nuclear poly(A)(+) RNA, whose levels are dramatically increased in the stressed cells. This behavior of HuR differs from that of another ARE-binding protein, hnRNP D, which has been implicated as an effector of mRNA decay rather than mRNA stabilization and of the general pre-RNA-binding protein hnRNP A1. We interpret these differences to mean that the temporal association of HuR with ARE-containing mRNAs is different from that of these other two proteins.     

Left-handed Z-DNA and in vivo supercoil density in the Escherichia coli chromosome.

A system for studying Z-DNA formation in the Escherichia coli chromosome was developed. Prior investigations in recombinant plasmids showed that alternating (Pur-Pyr) sequences can adopt a left-handed Z-DNA conformation both in vitro and in vivo. We constructed mobile, transposon-based cassettes carrying cloned (Pur-Pyr) sequences containing an EcoRI site in the center. These cassettes were subsequently inserted into different locations in the E. coli chromosome in a random fashion. A number of stable insertions were characterized by Southern analysis and pulsed-field gel electrophoresis mapping. A cloned temperature-sensitive MEcoRI methylase was expressed in trans as the probe to study Z-DNA formation in vivo. In this system, the control EcoRI sites were quickly methylated when cells were placed at the permissive temperature. Strong inhibition of the methylation was observed, however, only for the EcoRI sites embedded in a 56-bp run of (C-G). In contrast, the shorter sequence of 32 bp did not show this behavior. Prior in vitro determinations revealed that the longer tract required less energy to stabilize the Z-helix than the shorter block. We conclude that the observed inhibition of methylation is due to Z-DNA formation in the E. coli chromosome. In vitro, these sequences undergo the B- to Z-DNA transition at a supercoil density of -0.026 for the 56-bp insert and -0.032 for the 32-bp block. Since only the longer (C-G) tract but not the shorter run adopted the left-handed conformation in the chromosome, we propose that these densities establish the boundaries in the different chromosomal loci investigated; these boundaries are in good agreement with the extremes found in plasmids.     

Right-handed and left-handed DNA: studies of B- and Z-DNA by using proton nuclear Overhauser effect and P NMR.

We have differentiated between syn and anti glycosidic torsion angles in nucleic acid duplexes by measuring the transient nuclear Overhauser effect (NOE) between the sugar H-1' protons and the purine H-8 and pyrimidine H-6 base protons. The transient NOE measurements demonstrate a syn glycosidic torsion angle at guanosine and an anti glycosidic torsion angle at cytidine in poly(dG-dC) in 4 M NaCl and in poly(dG-m5dC) in 1.5 M NaCl solution. These features have been observed previously in the left-handed Z-DNA conformation of (dC-dG)3 in the crystalline state. By contrast, transient NOE studies demonstrate that both guanosine and cytidine residues adopt the anti conformation about the glycosidic bond for the right-handed poly(dG-dC) and poly(dG-m5dC) conformation in a low-salt solution. We have used P NMR to monitor the equilibrium between B- and Z-DNA forms of poly(dG-dC) in LiCl solutions; at high temperatures, the equilibrium shifts from B- to Z-DNA.     

Oxidation of apolipoprotein B-100 in circulating LDL is related to LDL residence time. In vivo insights from stable-isotope studies

5-Hydroxy-2-aminovaleric acid (HAVA) has been suggested to be a specific marker of oxidation of apolipoprotein (apo) B-100 proline (Pro) and arginine (Arg) side-chain residues in low density lipoprotein (LDL) in vitro. Here we describe the application of sensitive mass spectrometric techniques to the characterization of Pro/Arg-modified apoB-100 in LDL(1) (S(f) 7 to 12) and LDL(2) (S(f) 0 to 7) in vivo. We studied 7 subjects with familial defective apoB-100 (FDB) and 8 normolipidemic controls. In FDB subjects, the presence of a mutant apoB-100 (FDB(3500Q)) in LDL markedly reduced its affinity for the LDL receptor, leading to increased residence times (RTs) of LDL(1) (65+/-21 versus 32+/-12 hours, P<0.005) and LDL(2) (230+/-40 versus 53+/-7 hours, P:<0.001) when compared with controls, as determined by stable-isotope turnover studies. LDL(1) HAVA content was not different between the groups (FDB, 0.004+/-0. 001 mol/mol apoB-100 versus controls, 0.003+/-0.001 mol/mol apoB-100, P=0.200). LDL(2) HAVA content was higher in FDB subjects (0. 374+/-0.088 versus 0.013+/-0.002 mol/mol apoB-100, P<0.001). In both groups, LDL(2) HAVA was positively associated with LDL(2) RT (FDB, r=0.893, P:=0.003; controls, r=0.976, P=0.000) and negatively correlated with LDL(2) alpha-tocopherol content (FDB, r=-0.929, P=0. 003; controls, r=-0.903, P=0.002). No significant correlations could be found between LDL(1) HAVA, LDL(1) RT, and alpha-tocopherol, respectively. The low LDL(1) HAVA content observed in both FDB and control groups was thought to be due to the relatively lower RT as well as the higher alpha-tocopherol content of these lipoproteins. In contrast, LDL(2) seemed to be strongly prone to direct oxidation of apoB-100 in vivo. The longer these particles linger in the circulation, the more apoB-100 Pro/Arg residues become modified.     

The role of binding domains for dsRNA and Z-DNA in the in vivo editing of minimal substrates by ADAR1

RNA editing changes the read-out of genetic information, increasing the number of different protein products that can be made from a single gene. One form involves the deamination of adenosine to form inosine, which is subsequently translated as guanosine. The reaction requires a double-stranded RNA (dsRNA) substrate and is catalyzed by the adenosine deaminase that act on dsRNA (ADAR) family of enzymes. These enzymes possess dsRNA-binding domains (DRBM) and a catalytic domain. ADAR1 so far has been found only in vertebrates and is characterized by two Z-DNA-binding motifs, the biological function of which remains unknown. Here the role of the various functional domains of ADAR1 in determining the editing efficiency and specificity of ADAR1 is examined in cell-based assays. A variety of dsRNA substrates was tested. It was found that a 15-bp dsRNA stem with a single base mismatch was sufficient for editing. The particular adenosine modified could be varied by changing the position of the mismatch. Editing efficiency could be increased by placing multiple pyrimidines 5' to the edited adenosine. With longer substrates, editing efficiency also increased and was partly due to the use of DRBMs. Additional editing sites were also observed that clustered on the complementary strand 11-15 bp from the first. An unexpected finding was that the DRBMs are not necessary for the editing of the shorter 15-bp substrates. However, mutation of the Z-DNA-binding domains of ADAR1 decreased the efficiency with which such a substrate was edited.     

In Z-DNA the sequence G-C-G-C is neither methylated by Hha I methyltransferase nor cleaved by Hha I restriction endonuclease.

Plasmids carrying 24- or 32-base-pair inserts of alternating (dG-dC) residues were used to analyze the level of methylation of the G-C-G-C sites by Hha I DNA methyltransferase and their cleavage by Hha I endonuclease in the B-DNA or Z-DNA conformation. In supercoiled plasmids in which the inserts formed Z-DNA, the extent of methylation at the insert G-C-G-C sites was dramatically lower than the level of methylation at the G-C-G-C sites located outside the insert in the same plasmid. Similarly, cleavage by Hha I endonuclease was sharply lowered when the insert was in the Z-DNA form. In the relaxed plasmid, all its G-C-G-C sites were methylated to the same extent and the unmethylated sites were readily cleaved. After treatment with the methylase, the supercoiled plasmid was linearized and then digested with Hha I restriction endonuclease. This exposed unmethylated G-C-G-C sites from the insert that had been protected against cleavage in the Z conformation. A chemical reaction was used to study the distribution of the unmethylated cytosine residues. No accumulation of unmethylated cytosine residues was found anywhere along the entire 32-base-pair insert, which is consistent with a cooperative B-Z transition.     

The crystal structure of the second Z-DNA binding domain of human DAI (ZBP1) in complex with Z-DNA reveals an unusual binding mode to Z-DNA

Mammalian DAI (DNA-dependent activator of IFN-regulatory factors), an activator of the innate immune response, senses cytosolic DNA by using 2 N-terminal Z-DNA binding domains (ZBDs) and a third putative DNA binding domain located next to the second ZBD. Compared with other previously known ZBDs, the second ZBD of human DAI (hZβ_DAI) shows significant variation in the sequence of the residues that are essential for DNA binding. In this article, the crystal structure of the hZβ_DAI/Z-DNA complex reveals that hZβ_DAI has a similar fold to that of other ZBDs, but adopts an unusual binding mode for recognition of Z-DNA. A residue in the first β-strand rather than residues in the β-loop contributes to DNA binding, and part of the (α3) recognition helix adopts a 3₁₀ helix conformation. The role of each residue that makes contact with DNA was confirmed by mutational analysis. The 2 ZBDs of DAI can together bind to DNA and both are necessary for full B-to-Z conversion. It is possible that binding 2 DAIs to 1 dsDNA brings about dimerization of DAI that might facilitate DNA-mediated innate immune activation.     

Fate of lipid vesicles in vivo: a gamma-ray perturbed angular correlation study.

The structural integrity of unilamellar vesicles has been studied in vitro and in vivo by use of gamma-ray perturbed angular correlation techniques. These studies utilize 111In3+ weakly bound to the chelator nitrilotriacetic acid as a probe to monitor the percentage of intact vesicles. When complexes of 111In3+ and nitrilotriacetic acid is encapsulated in vesicles, 111In3+ exhibits a fast tumbling rate. Upon alteration of the membrane, 111In3+ is released from the liposomes and becomes bound to macromolecules, consequently exhibiting a decrease in 111In3+ tumbling rate. The in vitro experiments show that the present technique is capable of determining quantitatively the percentage of material released from the vesicles upon the addition of serum or Triton X-100 to vesicles. The percentage of vesicles remaining intact in vivo can also be monitored continuously by the present technique. In mice, the half-life of dipalmitoyl phosphatidylcholine-cholesterol vesicles after intraperitoneal injection was estimated to be 10-13 hr. The present study suggest that the vesicles remain intact in various tissues for extended periods, thereby allowing a slow release of the encapsulated material at those sites.     

In vivo stability of ester- and ether-linked phospholipid-containing liposomes as measured by perturbed angular correlation spectroscopy.

To evaluate liposome formulations for use as intracellular sustained-release drug depots, we have compared the uptake and degradation in rat liver and spleen of liposomes of various compositions, containing as their bulk phospholipid an ether-linked phospholipid or one of several ester-linked phospholipids, by perturbed angular correlation spectroscopy. Multilamellar and small unilamellar vesicles (MLVs and SUVs), composed of egg phosphatidylcholine, sphingomyelin, distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC) or its analog dihexadecylglycerophosphorylcholine (DHPC), and cholesterol plus phosphatidylserine, and containing 111In complexed to nitrilotriacetic acid, were injected intravenously in rats. Recovery of 111In-labeled liposomes in blood, liver, and spleen was assessed at specific time points after injection and the percentage of liposomes still intact in liver and spleen was determined by measurement of the time-integrated angular perturbation factor [G22(infinity)] of the 111In label. We found that MLVs but not SUVs, having DHPC as their bulk phospholipid, showed an increased resistance against lysosomal degradation as compared to other phospholipid-containing liposomes. The use of diacyl phospholipids with a high gel/liquid-crystalline phase-transition temperature, such as DPPC and DSPC, also retarded degradation of MLV, but not of SUV in the dose range tested, while the rate of uptake of these liposomes by the liver was lower.     

Unusual conformational effect exerted by Z-DNA upon its neighboring sequences.

Supercoiled plasmid DNA harboring an insert of (dG-dC)16, a sequence known to form Z-DNA upon negative supercoiling, was reacted with chloroacetaldehyde. Chloroacetaldehyde, like bromoacetaldehyde, was found to be a specific probe for detecting unpaired DNA bases in supercoiled plasmid DNA. Under torsional stress (at bacterial superhelical density), chloroacetaldehyde reacted at multiple discrete regions within the neighboring sequences of the (dG-dC)16 insert. When the plasmid population was considered as a whole, the distribution of the chemically reactive bases exhibited a pattern of inversion symmetry with the center of inversion in the middle of the (dG-dC)16 insert. However, when a single supercoiled plasmid molecule was considered, chloroacetaldehyde reacted with only one of the neighboring sequences, either 5' or 3' of the (dG-dC)16 insert, but not with both. The possibility that the supercoiled plasmid DNA is in equilibrium with these two structural forms is discussed.     

Isolation of Drosophila proteins that bind selectively to left-handed Z-DNA.

An affinity column for isolating Z-DNA binding proteins was made by attaching brominated poly(dG-dC) to Sephadex. Proteins from Drosophila nuclei were prepared and those that could bind to Escherichia coli B-DNA were removed from the solution. The remaining proteins were passed over the Z-DNA affinity column and then eluted with NaCl. Using both direct and competitive filter binding assays, we found that the eluted proteins bind to brominated poly(dG-dC) (Z-DNA) and poly(dG-m5dC) but not to poly(dG-dC) (B-DNA), native or denatured E. coli or calf thymus DNA, or brominated oligonucleotides. The proteins also bind to negatively supercoiled plasmids carrying Z-DNA sequences but not to relaxed or linearized plasmids in which the Z-DNA conformation is no longer present. Gel analysis reveals a mixture of several large proteins up to approximately 150,000 daltons.     

Z-DNA immunoreactivity in fixed metaphase chromosomes of primates.

Antibodies against Z-DNA bind to fixed metaphase chromosomes of man and Cebus albifrons (Platyrrhini, Primate). By indirect immunofluorescence and indirect immunoperoxidase techniques, a heavy staining is detected in some segments of chromosomes of C. albifrons. These segments correspond to R-band-positive heterochromatin, which has a high G + C-base content. Euchromatin of human and Cebus chromosomes show a weak and heterogeneous staining that consistently reproduces an R- and T-banding pattern in both species. Because chromosome homologies previously were demonstrated between these distantly related species by chromosome banding, our results suggest that Z-DNA has been conserved during the course of primate evolution.     

Cultural and biological evolutionary processes: gene-culture disequilibrium.

The dynamics of the interaction between genetic and cultural transmission are studied by using a simple two-phenotype diallelic haploid genetic system. The value of an individual's phenotype is determined by cultural transmission from its parent or by a randomly chosen member of the parental population. In the absence of phenotypic selection, polymorphic equilibria of the gene and trait frequencies are obtained. The correlation between genotype and phenotype within or between populations depends on a quantity formally similar to linkage disequilibrium and is determined by a relationship among transmission coefficients analogous to a coefficient of epistasis. With natural selection on the phenotype and no mutation, only degenerate transmission rules allow polymorphic equilibria to be attained, and, in general, the genotype allowing the strongest transmission of the favored phenotype is successful.     

Transforming growth factor alpha is trophic to pancreatic cancer in vivo.

Pancreatic cancer cell lines overexpress epidermal growth factor (EGF) receptors and also have the capacity to produce transforming growth factor alpha (TGF alpha), the alternate agonist of the EGF receptor. The purpose of this study was to determine if TGF alpha had a trophic effect on the growth of pancreatic cancer in vivo. Syrian golden hamsters were inoculated with 50,000 H2T hamster ductal pancreatic cancer cells. The hamsters were then randomised to three equal groups: the groups received either saline (control), EGF, or TGF alpha, each by intraperitoneal injection, three times a day. Treatment continued for seven weeks, and each week the hamsters were weighed and tumour areas were measured. The hamsters were then killed, and the tumours were excised, weighed, and extracted for assay of DNA content as a measure of cellularity. From week four onwards both EGF and TGF alpha significantly stimulated tumour growth. Although tumour weights were not significantly different, tumour DNA content had nearly doubled after exposure to both EGF and TGF alpha. It is concluded that like EGF, TGF alpha can stimulate pancreatic cancer growth in vivo, and this may in part explain the aggressive nature of these cancers in clinical practice.     

The short-lived MAT alpha 2 transcriptional regulator is ubiquitinated in vivo.

The substrates of ubiquitin-dependent proteolytic pathways include both damaged or otherwise abnormal proteins and undamaged proteins that are naturally short-lived. Few specific examples of the latter class have been identified, however. Previous work has shown that the cell type-specific MAT alpha 2 repressor of the yeast Saccharomyces cerevisiae is an extremely short-lived protein. We now demonstrate that alpha 2 is conjugated to ubiquitin in vivo. More than one lysine residue of alpha 2 can be joined to ubiquitin, and some of the ubiquitin moieties form a Lys48-linked multiubiquitin chain. Overexpression of degradation-impaired ubiquitin variants was used to show that at least a significant fraction of alpha 2 degradation is dependent on its ubiquitination.     

Left-handed Z-DNA in bands of acid-fixed polytene chromosomes.

Antibodies to DNA in the left-handed (Z) conformation bind to acid-fixed polytene chromosomes of both Chironomus thummi and Drosophila melanogaster, as shown by direct and indirect immunofluorescence. Comparison of the phase-contrast, immunofluorescence, and DNA staining patterns shows a predominant localization of the antibody to the regions of high contrast and DNA density, the bands. The immunofluorescence is completely abolished by competition with polynucleotides in the Z conformation but not by those in the B form. DNase but not RNase treatment eliminates the antibody staining. Actinomycin D inhibits binding, whereas mithramycin has no effect. The highly reproducible immunofluorescence patterns obtained with the anti-Z-DNA antibodies demonstrate variations in fluorescence intensity between particular bands, which can be quantitated by laser scanning and photon counting techniques. The telomeric regions and DNA strands associated with end-to-end chromosome linkage and ectopic pairing are exceptionally bright. At saturation, average values of 1 IgG molecule per 3,000 base pairs and 1 per 15,000 base pairs are found in the intensely and weakly staining regions, respectively. An alternative statement is that the left-handed Z-DNA conformation is present at a frequency of 0.02-0.1%. The measured differences reflect variations in the local density of Z-DNA sites and not in the affinity for the specific antibody, which appears to be relatively constant throughout the chromosomes (Kd approximately equal to 10 nM). These observations taken together with results of biophysical studies on the properties of Z-DNA in solution suggest that regions of DNA in the left-handed conformation could be involved in higher-order structural organization of chromosomes and possibly in modulation of their functional state.