T-DNA integration: a mode of illegitimate recombination in plants.

Transferred DNA (T-DNA) insertions of Agrobacterium gene fusion vectors and corresponding insertional target sites were isolated from transgenic and wild type Arabidopsis thaliana plants. Nucleotide sequence comparison of wild type and T-DNA-tagged genomic loci showed that T-DNA integration resulted in target site deletions of 29-73 bp. In those cases where integrated T-DNA segments turned out to be smaller than canonical ones, the break-points of target deletions and T-DNA insertions overlapped and consisted of 5-7 identical nucleotides. Formation of precise junctions at the right T-DNA border, and DNA sequence homology between the left termini of T-DNA segments and break-points of target deletions were observed in those cases where full-length canonical T-DNA inserts were very precisely replacing plant target DNA sequences. Aberrant junctions were observed in those transformants where termini of T-DNA segments showed no homology to break-points of target sequence deletions. Homology between short segments within target sites and T-DNA, as well as conversion and duplication of DNA sequences at junctions, suggests that T-DNA integration results from illegitimate recombination. The data suggest that while the left T-DNA terminus and both target termini participate in partial pairing and DNA repair, the right T-DNA terminus plays an essential role in the recognition of the target and in the formation of a primary synapsis during integration.     

Synergistic activity of a Bacillus thuringiensis delta-endotoxin and a bacterial endochitinase against Spodoptera littoralis larvae.

In an attempt to increase the insecticidal effect of the delta-endotoxin crystal protein CryIC on the relatively Cry-insensitive larvae of Spodoptera littoralis, a combination of CryIC and endochitinase was used. CryIC comprising the first 756 amino acids from Bacillus thuringiensis K26-21 and endochitinase ChiAII encoded by Serratia marcescens were separately produced in Escherichia coli carrying the genes in overexpression vectors. The endochitinase on its own, even at very low concentrations (0.1 microgram/ml), perforated the larval midgut peritrophic membrane. When applied together with low concentrations of CryIC, a synergistic toxic effect was obtained. In the absence of chitinase, about 20 micrograms of CryIC per ml was required to obtain maximal reduction in larval weight, while only 3.0 micrograms of CryIC per ml caused a similar toxic effect in the presence of endochitinase. Thus, a combination of the Cry protein and an endochitinase could result in effective insect control in transgenic systems in which the Cry protein is not expressed in a crystalline form.     

Corticotroph,somatotroph and mammotroph cell kinetics in the postnatal infant female rat

The aim of this work was to detect if hypothalamic-pituitary maturation was accompanied by significant proliferation changes in differentiated pituitary cell pools. For this purpose, pituitary corticotroph (Ct), mammotroph (Mt) and somatotroph (St) proliferation activities were scanned in intact female rats during the postnatal (P) period (1–35 postnatal days). The techniques of tritiated thymidine labelling, immunostaining and autoradiography were combined to visualize DNA synthesis of hormone containing cells. Immunoreactive cell densities were measured using image analysis, and double labelled cells were counted. Corticotroph proliferation activity increased significantly on day P12, followed by an increase in the Ct proportion on days P13–14. This is the first observation of a spontaneous change of corticotroph proliferation at the end of the stress nonresponsive period. The mammotroph density and proliferation rate increased gradually during postnatal maturation, until the Mt pool overran other cell types of the female hypophysis on day 35. The somatotroph pool was the most numerous until day P20; the proliferation rate remained constant while St proportions increased reaching a plateau between days P13 and 20, then decreased to the adult level. Each cell type examined showed a characteristic, individual density and proliferation pattern.     

Cloning of mtDNA fragments homologous to mitochondrial S2 plasmid-like DNA in maize

Summary Mitochondria from S-type cytoplasmic male-sterile maize contain two small DNA species, S1 and S2, which are absent from other fertile and male-sterile cytoplasms. These species have been cloned in plasmid pBR322 by the homopolymer extension method. Probes made with these recombinant plasmids have been used to establish the homology between high molecular weight mitochondrial DNAs of fertile and male-sterile cytoplasms, and small mitochondrial plasmid-like molecules. Hybridization and mapping data show that S2 DNA copies are homologuous with sequences of the normal mitochondrial genome. A comparison of physical maps of different isolated mtDNA fragments indicates a heterogeneous arrangement of S2 sequences in the mtDNA population of normal fertile maize cytoplasm. The origin of this heterogeneity is discussed.     

Evaluating the accuracy of SHAPE-directed RNA secondary structure predictions

Recent advances in RNA structure determination include using data from high-throughput probing experiments to improve thermodynamic prediction accuracy. We evaluate the extent and nature of improvements in data-directed predictions for a diverse set of 16S/18S ribosomal sequences using a stochastic model of experimental SHAPE data. The average accuracy for 1000 data-directed predictions always improves over the original minimum free energy (MFE) structure. However, the amount of improvement varies with the sequence, exhibiting a correlation with MFE accuracy. Further analysis of this correlation shows that accurate MFE base pairs are typically preserved in a data-directed prediction, whereas inaccurate ones are not. Thus, the positive predictive value of common base pairs is consistently higher than the directed prediction accuracy. Finally, we confirm sequence dependencies in the directability of thermodynamic predictions and investigate the potential for greater accuracy improvements in the worst performing test sequence.