Quantifying the temperature dependence of glycine-betaine RNA duplex destabilization.

Glycine-betaine (GB) stabilizes folded protein structure because of its unfavorable thermodynamic interactions with amide oxygen and aliphatic carbon surface area exposed during protein unfolding. However, GB can attenuate nucleic acid secondary structure stability, although its mechanism of destabilization is not currently understood. Here we quantify GB interactions with the surface area exposed during thermal denaturation of nine RNA dodecamer duplexes with guanine-cytosine (GC) contents of 17-100%. Hyperchromicity values indicate increasing GB molality attenuates stacking. GB destabilizes higher-GC-content RNA duplexes to a greater extent than it does low-GC-content duplexes due to greater accumulation at the surface area exposed during unfolding. The accumulation is very sensitive to temperature and displays characteristic entropy-enthalpy compensation. Since the entropic contribution to the m-value (used to quantify GB interaction with the RNA solvent-accessible surface area exposed during denaturation) is more dependent on temperature than is the enthalpic contribution, higher-GC-content duplexes with their larger transition temperatures are destabilized to a greater extent than low-GC-content duplexes. The concentration of GB at the RNA surface area exposed during unfolding relative to bulk was quantified using the solute-partitioning model. Temperature correction predicts a GB concentration at 25 °C to be nearly independent of GC content, indicating that GB destabilizes all sequences equally at this temperature.

GC-rich heterochromatin in silver stained nucleolar organizer regions (NORs) fluoresces with Chromomycin A3 (CMA3) staining in three species of teleostean fishes (Pisces).

In a bid to ascertain the molecular architecture of the silver positive regions (NORs) in chromosomes of three species of fish, namely, Hemibagrus menoda (Hamilton), Sperata seenghala (Sykes) (Fam: Bagridae) and Mastacembelus armatus (Lacep6de) (Fam: Mastacembelidae), an additional staining methodology using a fluorochrome dye (Chromomycin A3) was deployed along with the AgNO3 technique. The nucleolar organizing regions (NORs) were located terminally at the shorter arms (Tp) of one pair of submetacentric chromosomes (No.3) in H. menoda (2n=58), at the longer arms (Tq) of one pair of submetacentric chromosomes (No.5) in S. seenghala (2n=50) and at the shorter arm (Tp) of one pair of homologous submetacentric chromosomes (No.6) in M. armatus (2n=48). Staining with Chromomycin A3 produced bright fluorescing zones in GC-rich heterochromatin of Ag-positive NORs. The results indicate a more general trend of existence of an overlapping region between NOR and GC-rich fluorescing zones, the active sites of rRNA genes (rDNA) in this primitive group of vertebrates although exceptions to this situation has been reported in a couple of extant fish species earlier. More data utilizing such combined methodologies are warranted to understand the structural organization of fish chromosomes more precisely.

Phenobarbital induces progressive patterns of GC-rich and gene-specific altered DNA methylation in the liver of tumor-prone B6C3F1 mice.

Altered DNA methylation contributes to tumorigenesis by affecting gene expression in a heritable fashion. Phenobarbital (PB) is a nongenotoxic rodent carcinogen which induces global hypomethylation and regions of hypermethylation in mouse liver. Liver tumor-sensitive (B6C3F1) and -resistant (C57BL/6) male mice were administered 0.05% (wt/wt) PB in drinking water for 2 or 4 weeks, and a 2-week recovery was included following each dosing period. DNA was isolated from liver (target) and kidney (nontarget) tissues. The methylation status of GC-rich regions of DNA was assessed via methylation-sensitive restriction digestion, arbitrarily primedpolymerase chain reaction, and capillary electrophoretic separation of products. PB-induced regions of altered methylation (RAMs) which carry forward from an early to a later time point are more likely to be mechanistically relevant as compared to those that do not. Twelve of 69 RAMs (17%) present in B6C3F1 liver at 2 weeks were also seen at 4 weeks, while only 1 of the 123 RAMs (< 1%) present in C57BL/6 liver was seen at 4 weeks. In the B6C3F1 mice, 57 unique (as compared to the C57BL/6) regions of altered hepatic methylation (RAMs), predominantly hypomethylation, were observed at 2 weeks, increasing to 86 at 4 weeks. Changes in methylation were largely reversible. Altered methylation in liver was highly dissimilar to that of kidney. Following 4 weeks PB, bisulfite sequencing revealed hypomethylation of Ha-ras in B6C3F1, but not C57BL/6, which correlated with increased gene expression. These data indicate that (1) progressive, nonrandom changes in methylation provide an epigenetic mechanism underlying the ability of PB to cause mouse liver tumorigenesis and (2) susceptibility to tumorigenesis is related inversely to the capacity to maintain normal patterns of methylation.

Diethanolamine and phenobarbital produce an altered pattern of methylation in GC-rich regions of DNA in B6C3F1 mouse hepatocytes similar to that resulting from choline deficiency.

DNA methylation is an epigenetic mechanism regulating transcription, which when disrupted, can alter gene expression and contribute to carcinogenesis. Diethanolamine (DEA), a non-genotoxic alkanolamine, produces liver tumors in mice. Studies suggest DEA inhibits choline uptake and causes biochemical changes consistent with choline deficiency (CD). Rodents fed methyl-deficient diets exhibit altered methylation of hepatic DNA and an increase in liver tumors, e.g., CD causes liver tumors in B6C3F1 mice. We hypothesize that DEA-induced CD leads to altered methylation patterns which facilitates tumorigenesis. B6C3F1 hepatocytes in primary culture were grown in the presence of either 4.5 mM DEA, 3 mM Phenobarbital (PB), or CD media for 48 h. These concentrations induced comparable increases in DNA synthesis. PB, a nongenotoxic rodent liver carcinogen known to alter methylation in mouse liver, was included as a positive control. Global, average, DNA methylation status was not affected. The methylation status of GC-rich regions of DNA, which are often associated with promoter regions, were assessed via methylation-sensitive restriction digestion and arbitrarily primed PCR with capillary electrophoretic separation and detection of PCR products. DEA, PB, and CD treatments resulted in 54, 63, and 54 regions of altered methylation (RAMs), respectively, and the majority were hypomethylations. A high proportion of RAMs (72%) were identical when DEA was compared to CD. Similarly, 70% were identical between PB and CD. Altered patterns of methylation in GC-rich regions induced by DEA and PB resemble that of CD and indicate that altered DNA methylation is an epigenetic mechanism involved in the facilitation of mouse liver tumorigenesis.

Thalidomide down-regulates transcript levels of GC-rich promoter genes in multiple myeloma.

Thalidomide (Thd), a potent teratogen, was shown to have therapeutic potential in cancer, primarily in multiple myeloma (MM), yet its mechanism of action has not been elucidated. It was recently suggested that its teratogenicity is derived from interference in expression of genes regulated by GC-rich promoters by blocking the binding of SP1 transcription factor to its motif. We explored the validation of the proposed model by focusing on potential molecular targets associated with MM pathogenesis. Cell lines RPMI 8226, U266, and ARH-77 were exposed for 24 h to racemic Thd and analyzed for apoptosis, membranal expression of CD29 and CD63, transcript level of hTERT, CD63, and IGFI-R (characterized by GC-rich motifs) and telomerase activity. Analysis of an hTERT core promoter reporter gene expression [enhanced green fluorescent protein (EGFP)] in transiently transfected RPMI 8226 incubated with racemic and steric (+/-)-enantiomers of Thd was performed. A consistent reduction ( approximately 10-40%) in transcript levels of all three assayed genes in all three cell lines was demonstrated in the presence of racemic Thd. Significant reduction of EGFP was demonstrated in cells transfected with hTERT reporter gene and treated with racemic and (S)-Thd. Our results show that Thd's antimyeloma activity can be ascribed to the same mechanism responsible for its teratogenic effect and that the inhibition of GC-rich promoter genes is mostly attributed to the S-racemate. Indeed, this selectivity delineates GC-rich promoter genes as a unique group eligible for specific drug targeting.

GC clusters and the stability of mitochondrial genomes of Saccharomyces cerevisiae and related yeats.

The occurrence of GC clusters in Saccharomyces spp. and related yeasts was examined to clarify their association with the stability of intact mitochondrial genome. Abundance of nonspecific or specific GC clusters in these species decreases with phylogenetic distance from S. cerevisiae. Their number but not the number of replication origins correlates with the ability to form respiration-deficient mutants induced by ethidium bromide. This effect is not associated with the nuclear background since the cybrids having identical nuclei and mitochondria from different species gave similar results. In contrast to grand genomes, the presence of GC clusters in rho- mutants does not play any role in ethidium bromide induced mtDNA loss. The most plausible explanation for mitotically lost petite mtDNA seems to be dilution during the distribution.

Mutagenic fingerprint of ozone in human cells.

Ozone is an important factor in urban pollution and represents a major concern for human health. The chemical reactivity of ozone toward biological targets and particularly its genotoxicity supports a possible link between exposure and cancer risk, but no molecular data exist on its mutagenic potential in human cells. Using a shuttle vector, we showed that ozone is indeed a potent mutagen and we characterized the mutation spectrum it produced in human cells. Almost all mutations are base substitutions, essentially located at G:Cs (75%), typical of reactive oxygen species (ROS), but occurring in a specific pattern, i.e. a similar extent of GC:TA (28%), GC:CG (23%) and GC:AT (23%). The targeted distribution of mutations and identification of hotspot sequences define the first molecular fingerprint of mutations induced by ozone in human cells. Possible applications derived from our results with respect to ozone genotoxicity should help determining quantifiable biomarkers of ozone exposure in human health, especially for carcinogenesis.

Fluorescence lifetime imaging of nuclear DNA: effect of fluorescence resonance energy transfer.

BACKGROUND: DNA fluorescence dyes have been used to study DNA dynamics, chromatin structure, and cell cycle analysis. However, most microscopic fluorescence studies of DNA use only steady-state measurements and do not take advantage of the additional information content of the time-resolved fluorescence. In this paper, we combine fluorescence imaging of DNA with time-resolved measurements to examine the proximity of donors and acceptors bound to chromatin. METHODS: We used frequency-domain fluorescence lifetime imaging microscopy to study the spatial distribution of DNA-bound donors and acceptors in fixed 3T3 nuclei. Over 50 cell nuclei were imaged in the presence of an AT-specific donor, Hoechst 33258 (Ho), and a GC-specific acceptor, 7-aminoactinomycin D (7-AAD). RESULTS: The intensity images of Ho alone showed a spatially irregular distribution due to the various concentrations of DNA or AT-rich DNA throughout the nuclei. The lifetime imaging of the Ho-stained nuclei was typically flat. Addition of 7-AAD decreased the fluorescence intensity and lifetime of the Ho-stained DNA. The spatially dependent phase and modulation values of Ho in the presence of 7-AAD showed that the Ho decay becomes nonexponential, as is expected for a resonance energy transfer (RET) with multiple acceptors located over a range of distances. In approximately 40 nuclei, the intensity and lifetime decrease was spatially homogeneous. In approximately 10 nuclei, addition of 7-AAD resulted in a spatially nonhomogeneous decrease in intensity and lifetime. The RET efficiency was higher in G(2)/M than in G(0/1) phase cells. CONCLUSIONS: Because RET efficiency depends on the average distance between Ho and 7-AAD, data suggest that the heterogeneity of lifetimes and spatial variation of the RET efficiency are caused by the presence of highly condensed regions of DNA in nuclei.

Mechanism of action in thalidomide teratogenesis.

In this commentary, we describe a model to explain the mechanism of the embryopathy of thalidomide. We propose that thalidomide affects the following pathway during development: insulin-like growth factor I (IGF-I) and fibroblast growth factor 2 (FGF-2) stimulation of the transcription of alphav and beta3 integrin subunit genes. The resulting alphavbeta3 integrin dimer stimulates angiogenesis in the developing limb bud, which promotes outgrowth of the bud. The promoters of the IGF-I and FGF-2 genes, the genes for their binding proteins and receptors, as well as the alphav and beta3 genes, lack typical TATA boxes, but instead contain multiple GC boxes (GGGCGG). Thalidomide, or a breakdown product of thalidomide, specifically binds to these GC promoter sites, decreasing transcription efficiency of the associated genes. A cumulative decrease interferes with normal angiogenesis, which results in truncation of the limb. Intercalation into G-rich promoter regions of DNA may explain why certain thalidomide analogs are not teratogenic while retaining their anti-tumor necrosis factor-alpha (TNF-alpha) activity, and suggests that we look elsewhere to explain the action of thalidomide on TNF-alpha. On the other hand, the anti-cancer action of thalidomide may be based on its antiangiogenic action, resulting from specific DNA intercalation. The tissue specificity of thalidomide and its effect against only certain neoplasias may be explained by the fact that various developing tissues and neoplasias depend on different angiogenesis or vasculogenesis pathways, only some of which are thalidomide-sensitive.