High fidelity extrachromosomal recombination and gene targeting in plants

The precision of extrachromosomal homologous recombination and gene targeting in plant cells was investigated. Recombination was directed to introns of selectable marker genes where potential changes could persist without affecting the function and therefore the selectability of the genes. Approximately 9 kb of crossover regions was rescued and sequenced. Changes were detected at a frequency below one point mutation per 1000 bp, indicating that extrachromosomal recombination and gene targeting both appear to occur with high fidelity.     

A minichromosome-like structure in wheat embryos

A crude nuclear fraction of resting wheat embryos was used as the source of putative plant minichromosomes: unique DNA sequences the size of genes and flanked by telomere-type repeats. Preliminary separation of low-molecular-weight DNA species from chromosomal DNA (Hirt's method), velocity sedimentation, and isopycnic centrifugation were followed by PCR amplification of minichromosome-like sequences. The most abundant PCR product was cloned and sequenced. In addition to telomeric repeats (defined by a PCR primer), which were the expected sequences, the linear DNA molecule (637 pb) contained an ARS-like element, RAP1-binding site, and two relatively long ORFs. The whole sequence seems to represent a naturally occurring plant minichromosome.     

Different haplotypes for cystic fibrosis-linked DNA polymorphisms in Polish and Dutch populations

Summary We analyzed DNA from 34 Polish and 63 Dutch cystic fibrosis (CF) patients and their families using the polymorphic markers XV2c and KM19, which are in linkage disequilibrium with the CF mutation. Strong linkage disequilibrium was found in the Dutch population sample, but the haplotypes of the Polish chromosomes showed a significantly less extreme disequilibrium. Our data and previous studies indicate that the highest degree of homogeneity of the CF defect and hence the best possible use of the XV2c/KM19/CF linkage disequilibrium for CF carrier detection/exclusion is in populations of northern European origin.     

Crystal structure of MboIIA methyltransferase

DNA methyltransferases (MTases) are sequence-specific enzymes which transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the amino group of either cytosine or adenine within a recognized DNA sequence. Methylation of a base in a specific DNA sequence protects DNA from nucleolytic cleavage by restriction enzymes recognizing the same DNA sequence. We have determined at 1.74 A resolution the crystal structure of a beta-class DNA MTase MboIIA (M.MboIIA) from the bacterium Moraxella bovis, the smallest DNA MTase determined to date. M.MboIIA methylates the 3' adenine of the pentanucleotide sequence 5'-GAAGA-3'. The protein crystallizes with two molecules in the asymmetric unit which we propose to resemble the dimer when M.MboIIA is not bound to DNA. The overall structure of the enzyme closely resembles that of M.RsrI. However, the cofactor-binding pocket in M.MboIIA forms a closed structure which is in contrast to the open-form structures of other known MTases.     

Nonadditive Effects of Genes in Human Metabolomics

Genome-wide association studies (GWAS) are widely applied to analyze the genetic effects on phenotypes. With the availability of high-throughput technologies for metabolite measurements, GWAS successfully identified loci that affect metabolite concentrations and underlying pathways. In most GWAS, the effect of each SNP on the phenotype is assumed to be additive. Other genetic models such as recessive, dominant, or overdominant were considered only by very few studies. In contrast to this, there are theories that emphasize the relevance of nonadditive effects as a consequence of physiologic mechanisms. This might be especially important for metabolites because these intermediate phenotypes are closer to the underlying pathways than other traits or diseases. In this study we analyzed systematically nonadditive effects on a large panel of serum metabolites and all possible ratios (22,801 total) in a population-based study [Cooperative Health Research in the Region of Augsburg (KORA) F4, N = 1,785]. We applied four different 1-degree-of-freedom (1-df) tests corresponding to an additive, dominant, recessive, and overdominant trait model as well as a genotypic model with two degree-of-freedom (2-df) that allows a more general consideration of genetic effects. Twenty-three loci were found to be genome-wide significantly associated (Bonferroni corrected P ≤ 2.19 × 10⁻¹²) with at least one metabolite or ratio. For five of them, we show the evidence of nonadditive effects. We replicated 17 loci, including 3 loci with nonadditive effects, in an independent study (TwinsUK, N = 846). In conclusion, we found that most genetic effects on metabolite concentrations and ratios were indeed additive, which verifies the practice of using the additive model for analyzing SNP effects on metabolites.     

Theoretical Investigation of the Molecular Structure of the πκ DNA Base Pair

Abstract

The structure of the nonclassical πκ base pair (7–methyl-oxoformycin … 2,4-diaminopyrimidine) was studied at the ab initio Hartree-Fock (HF) and MP2 levels using the 6–31G* and 6–31G** basis sets. The πκ base pair is bound by three parallel hydrogen bonds with the donor-acceptor-donor recognition pattern. Recently, these bases were proposed as an extension of the genetic alphabet from four to six letters (Piccirilli et al. Nature 343, 33(1990)). By the HF/6- 31G* method with full geometry optimization we calculated the 12 degree propeller twist for the minimum energy structure of this complex. The linearity of hydrogen bonds is preserved in the twisted structure by virtue of the pyramidal arrangement of the κ-base amino groups. The rings of both the π and κ molecules remain nearly planar. This nonplanar structure of the πκ base pair is only 0.1 kcal/mol more stable than the planar (Cs) conformation. The HF/6- 31G* level gas-phase interaction energy of πκ (—13.5 kcal/mol) calculated by us turned out to be nearly the same as the interaction energy obtained previously for the adenine-thymine base pair (—13.4 kcal/mol) at the same computational level. The inclusion of p-polarization functions on hydrogens, electron correlation effects (MP2/6–31G** level), and the correction for the basis set superposition error (BSSE) increase this energy to -14.0 kcal/mol.     

Cumulation of mycotoxins in maize cobs infected with<Emphasis Type="Italic">Fusarium gramihearum</Emphasis>

Maize cobs withFusarium ear rot were collected at 1986 season and five infected byFusarium graminearum were analyzed for presence of triohothecenes and zearalenone. Collected material was subsampled forFusarium damaged kernels and corresponding axial stems and healthy looking kernels. All investigated cobs contained deoxynivalenol (DON) (range 18.0–131.5 mg/kg) and zearalenone (ZEA) (range 0.38–2.17 mg/kg), in four cobs 15-acetyl-deoxynivalenol (15-AcDON) (range 5.2–6.2 mg/kg) was present and two cobs besides three all metabolites contained 3-acetyl-deoxynivalenol (3-AcD0N) (range 0.5–0.8 mg/kg).The average of individual toxins amount in axial stems: in mg/kg was equal to: DON — 110.36, ZEA — 4.57, 15-AcD0N — 16.66, and 3-AcD0N — 1.32.Fusarium damaged kernels contained in average the following amount (mg/kg) of: DON 77.00, ZEA 0.98, 15-AcD0N 3.78 and 3-AcD0N 0.06. Healthy looking kernels contained DON 1.96 mg/kg and ZEA 0.07 mg/kg only. Cooccurrence of 3-AcDON and 15-AcDON in two samples was an interesting finding. The amount of DON in total cob was highly correlated (r = 0.94) with percentage ofFusarium damaged kernels in given ear.