Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.     

Does Forest Continuity Enhance the Resilience of Trees to Environmental Change?

There is ample evidence that continuously existing forests and afforestations on previously agricultural land differ with regard to ecosystem functions and services such as carbon sequestration, nutrient cycling and biodiversity. However, no studies have so far been conducted on possible long-term (>100 years) impacts on tree growth caused by differences in the ecological continuity of forest stands. In the present study we analysed the variation in tree-ring width of sessile oak (Quercus petraea (Matt.) Liebl.) trees (mean age 115–136 years) due to different land-use histories (continuously existing forests, afforestations both on arable land and on heathland). We also analysed the relation of growth patterns to soil nutrient stores and to climatic parameters (temperature, precipitation). Tree rings formed between 1896 and 2005 were widest in trees afforested on arable land. This can be attributed to higher nitrogen and phosphorous availability and indicates that former fertilisation may continue to affect the nutritional status of forest soils for more than one century after those activities have ceased. Moreover, these trees responded more strongly to environmental changes – as shown by a higher mean sensitivity of the tree-ring widths – than trees of continuously existing forests. However, the impact of climatic parameters on the variability in tree-ring width was generally small, but trees on former arable land showed the highest susceptibility to annually changing climatic conditions. We assume that incompletely developed humus horizons as well as differences in the edaphon are responsible for the more sensitive response of oak trees of recent forests (former arable land and former heathland) to variation in environmental conditions. We conclude that forests characterised by a long ecological continuity may be better adapted to global change than recent forest ecosystems.     

RNA mediated formation of a phosphorothioate diester bond

Previous results showed that multimeric, tandemly sequence-repeated forms of satellite tobacco ringspot virus RNA of the encapsidated polarity (STobRV (+)RNA) autolytically process at a specific phosphodiester bond, the junction. Substituting a phosphorothioate diester bond for the STobRV (+)RNA junction drastically slowed autolytic processing. Here we show that for the complementary STobRV (-)RNA, in contrast, replacing sets of phosphodiester bonds with phosphorothioate diester bonds, even at the junction, did not greatly slow autolytic processing or spontaneous ligation, the usual reactions of the unmodified RNA. In the ligation reaction STobRV (-)RNA directed the formation of an ApG phosphorothioate diester bond.     

Stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments

The stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments was investigated using phosphorothioate analogs of GTP as chiral probes. (Sp)-Guanosine 5'-O-(1-thiotriphosphate) (Sp-GTP alpha S) is a substrate, whereas (Rp)-GTP alpha S is a competitive inhibitor (K1 = 0.1 mM), but not a substrate. (Sp)-GTP alpha S is converted into (Rp)-guanosine 3':5'-monophosphorothioate, showing that the reaction proceeds with inversion of configuration at the alpha-phosphorus atom. Km and Vmax for (Sp)-GTP alpha S (at low [Ca2+], 20 nM) are 3.7 mM and 1.1 nmol/min/mg of rhodopsin, respectively, compared with 1.1 mM and 23.1 nmol/min/mg of rhodopsin for GTP. Vmax for the cyclization of (Sp)-GTP alpha S, as for GTP, increases 10-20-fold when the calcium level is lowered. This activity change is centered at approximately 90 nM and has a Hill coefficient of 4.8. The configuration of the metal-substrate complex was determined by measuring the effectiveness of the Sp and Rp isomers of GTP alpha S and guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) in the presence of Mg2+ or Mn2+. (Sp)-GTP alpha S is a substrate with either Mg2+ or Mn2+, whereas (Rp)-GTP beta S is a substrate with only Mn2+. These findings suggest that the substrate is a metal-beta, gamma-bidentate complex with delta screwsense. We also found that the cyclization reaction catalyzed by the membrane-bound guanylate cyclase from sea urchin sperm proceeds with inversion of configuration at the alpha-phosphorus atom. The stereochemical course of the reactions catalyzed by all prokaryotic and eukaryotic adenylate cyclases and guanylate cyclases studied thus far is the same.     

Study of a hammerhead ribozyme containing 2'-modified adenosine residues

The improved synthesis of 2'-fluoro-2'-deoxyadenosine (2'-FA) starting from adenosine is described. This compound was converted to the phosphoramidite and incorporated into a hammerhead ribozyme RNA with the use of automated RNA synthesis techniques. Ribozymes containing 2'-deoxy-adenosine (2'-dA) were prepared in a similar manner. A kinetic rate comparison of the unmodified ribozyme with two ribozymes that had every adenosine replaced with 2'FA or 2'-dA revealed a large decrease in catalytic efficiency (kcat/Km) for the modified ribozymes resulting from a drop in kcat. The kinetic analysis of a number of partially substituted 2'-FA or 2'-dA containing hammerheads revealed that the decrease in activity was not associated with any particular residue but was the result of the accumulation of modified nucleosides within the structure.     

The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA.

M13 RF IV DNA may be prepared in vitro to contain phosphorothioate-modified internucleotidic linkages in the (-)strand only. Certain restriction enzymes react with this modified DNA to hydrolyze the (+)strand exclusively when a phosphorothioate linkage occurs at the normal cleavage point in the (-)strand. The reaction of Pvu I with M13mp2 RF IV DNA containing dCMPS residues in the (-)strand is of this type, and is exploited to allow subsequent digestion with exonuclease III of a portion of the (+)strand opposite different mutagenic mismatched oligonucleotide primers. Two methods are described by which this approach has been used to produce mutations in M13mp2 phage DNA with high efficiency as a result of simple and rapid in vitro manipulations. Plaques containing mutant phage in a genetically-pure form are obtained at a frequency of 40-66%, allowing their characterisation directly by sequence analysis without prior screening and plaque purification. The wide applicability of this approach is discussed.     

Ribozyme-mediated RNA degradation in nuclei suspension.

Ribozymes containing 2'-fluoro- and 2'-amino-modified pyrimidine nucleosides in combination with terminal phosphorothioate linkages were targeted against HTLV-I tax RNA. In order to examine the activity of such chemically modified ribozymes in the nuclear environment, they were incubated with nuclei of a Tax-transformed mouse fibroblast cell line. Ribozyme cleavage of tax RNA was analyzed by the RNase protection assay. Comparison of the cleavage of tax RNA isolated nuclei with that of tax RNA present in nuclei suspension revealed a 30 times more efficient cleavage of the latter one. Pre-treatment with proteinase K and SDS abolished the enhancement of the ribozyme-mediated RNA cleavage. Catalytically inactive ribozymes did not yield any cleavage products. These results demonstrate an augmenting effect of nuclear proteins on the ribozyme-mediated RNA cleavage.     

In vitro selection of hammerhead ribozymes containing a bulged nucleotide in stem II.

Hammerhead ribozymes were transcribed from a dsDNA template containing four random nucleotides between stems II and III, which replace the naturally occurring GAA nucleotides. In vitro selection was used to select hammerhead ribozymes capable of in cis cleavage using denaturing polyacrylamide gels for the isolation of cleaving sequences. Self-cleaving ribozymes were cloned after the first and second rounds of selection, sequenced and characterised. Only sequences containing 5'-HGAA-3', where H is A, C or U, between stems II and III were active; G was clearly not tolerated at this position. Thus, only three sequences out of the starting pool of 256 (4(4)) were active. The Michaelis-Menten parameters were determined for the in trans cleaving versions of these ribozymes and indicate that selected ribozymes are less efficient than the native sequence. We propose that the selected ribozymes accommodate the extra nucleotide as a bulge in stem II.