Absence of cytosine methylation at C-C-G-G and G-C-G-C sites in the rDNA coding regions and intervening sequences of Drosophila and the rDNA of other insects.

Cytosine residues in C-G dinucleotides are frequently methylated in eukaryote DNA. In DNA of the dinoflagellate C. cohnii, the sequence C-MeC-G-G apparently renders Hpa II (C-C-G-G) incapable of digesting whole cell DNA in general, and rDNA in particular. Msp I, which also recognizes C-C-G-G but cleaves irrespective of methylation, degrades C. cohnii DNA and produces rDNA segments of 10.2 to 1.4 kb. We have applied this Hpa II/Msp I test to unfractionated DNA, and to rDNA and the rDNA intervening sequence of Drosophila virilis embryos and adults. There is no evidence of C-MeC-G-G sequences in either developmental stage of this species. Absence of G-MeC-G-C from coding and intervening sequences of rDNA was shown in comparisons of Hha I (G-C-G-C) cleavage patterns of unfractionated DNA and cloned (unmodified) segments of rDNA. Comparisons of Hpa II and Msp I cleavage products of DNA from the house fly, the flesh fly and a bumblebee also revealed no internal cytosine methylation in the sequence C-C-G-G. Because amounts of McC in C-G dinucleotides vary greatly among species, from apparent nonexistence to substantial proportions, no inference may yet be drawn about the role of such base modifications in DNA.     

Influence of Colloidal Iron on the Respiration of a Species of the Genus Acinetobacter

Washed suspensions of Acinetobacter sp. isolated from water caused the precipitation of iron from a suspension of colloidal ferric iron at pH 6.0 and 7.6. Iron-encrusted cells of the bacterium formed large aggregates. The amount of iron removed from the colloidal preparation in the form of aggregates was from 21 to 52% at pH 7.6 and 49% an pH 6.0 by the bacterial cells. Endogenous respiration rates of the iron-encrusted cells were from 32 to 72% lower than the rates for unencrusted cells. Respiration rates, measured polarographically in the presence of glucose, were also greatly reduced by the coating of iron on the cells.     

Uncoupler and anaerobic resistant transport of phosphate in Escherichia coli

Active transport of inorganic phosphate into whole cells of a strain (AB3311) derived from $\text{Escherichia coli}$ K12 was found to be partially resistant to 50 μM carbonyl cyanide $\text{m}$-chlorophenyl hydrazone (CCCP), a powerful uncoupler of oxidative phosphorylation. The presence of 10 mM dithiothreitol (DTT) before the addition of CCCP completely prevented the inhibition of phosphate uptake caused by the uncoupler. The addition of DTT to the CCCP-inhibited system restored phosphate uptake to the control rate even when added 5 min after the phosphate transport assay was started. This uncoupler resistant transport is insensitive to anaerobiosis, or the addition of 10 mM KCN which reduces oxygen consumption to less than 1% that of aerobic controls. Additional studies of transport in a mutant (CBT302) deficient in membranebound Ca²⁺-, Mg²⁺-ATPase activity also demonstrated the retention of appreciable inorganic phosphate uptake under anaerobic conditions.     

Genome-wide scan reveals important additive and non-additive genetic effects associated with resistance to Haemonchus contortus in Florida Native sheep

Florida Native sheep is among the sheep breeds best adapted to humid and hot climatic conditions such as those of Florida, USA, and have shown a superior ability to regulate nematode burdens. This is one of the oldest sheep breeds in North America and is an endangered species. To ensure genetic diversity and long-term survival of the breed, protection of the current genetic stock is critical and conservation efforts are required to promote its breeding and production. The objective of the present study was to investigate the importance of additive and non-additive genetic effects on resistance to natural Haemonchus contortus infections in Florida Native sheep using a whole genome scan. A total of 200 sheep were evaluated in the present study. Phenotypic records included faecal egg count (FEC, eggs/gram), FAMACHA® score, packed cell volume (PCV, %), body condition score and average daily gain (ADG, kg). Sheep were genotyped using the GGP Ovine 50K SNP chip and 45.2 k single nucleotide polymorphism (SNP) markers spanning the entire genome were available for quality control procedures. Mixed models were used to analyse the response variables and included the identity by state matrix to control for population structure. Bonferroni correction was used to control for multiple testing and a second arbitrary threshold (0.1 × 10^?3) was used. Fifteen SNPs with additive and non-additive genetic effects and located in Ovis aries chromosome OAR1, 2, 3, 6, 8, 10, 11, 12, 13 and 21 were associated with FEC, FAMACHA® score, PCV and ADG. These SNPs could be potential genetic markers for resistance to natural H. contortus exposure in Florida Native sheep.     

Insecticidal activity of recombinant baculovirus co-expressing Bacillus thuringiensis crystal protein and Kunitz-type toxin isolated from the venom of bumblebee Bombus ignitus

To improve the insecticidal activity of Autographa californica nucleopolyhedrovirus (AcMNPV), using co-expression of Bacillus thuringiensis crystal protein and a Kunitz-type toxin isolated from bumblebee Bombus ignitus venom, a recombinant AcMNPV, ApPolh5-3006BiKTI, expressing Bi-KTI under the control of early promoter from Cotesia plutellae bracovirus (CpBV) was constructed. In this recombinant virus, B. thuringiensis cry1-5 crystal protein gene was introduced into the genome by the fusion of polyhedrin-cry1-5 under the control of polyhedrin gene promoter. RT-PCR analysis indicated that both Bi-KTI and polyhedrin-cry1-5 fusion protein were successfully expressed from the infected cells. In addition, SDS-PAGE revealed that polyhedrin-cry1-5 fusion protein expressed by recombinant viruses was occluded into the polyhedra. ApPolh5-3006BiKTI showed an improved insecticidal activity against larvae of Plutella xylostella and Spodoptera exigua. At low dosage rates, it was more effective against S. exigua than on P. xylostella, but more rapid insecticidal activity was shown in P. xylostella. These results strongly suggest that co-expression of Bt toxin and Kunitz-type toxins could be successfully applied to improve the insecticidal activity of baculoviruses.     

Differential effects of ret and TRKB on axonal branching and survival of parasympathetic neurons

Interactions between neurons and their targets of innervation influence many aspects of neural development. To examine how synaptic activity interacts with neurotrophic signaling, we determined the effects of blocking neuromuscular transmission on survival and axonal outgrowth of ciliary neurons from the embryonic chicken ciliary ganglion. Ciliary neurons undergo a period of cell loss due to programmed cell death between embryonic Days (E) 8 and 14 and they innervate the striated muscle of the iris. The nicotinic antagonist d‐tubocurarine (dTC) induces an increase in branching measured by counting neurofilament‐positive voxels (NF‐VU) in the iris between E14‐17 while reducing ciliary neuron survival. Blocking ganglionic transmission with dihyro‐β‐erythroidin and α‐methyllycacontine does not mimic dTC. At E8, many trophic factors stimulate neurite outgrowth and branching of neurons placed in cell culture; however, at E13, only GDNF stimulates branching selectively in cultured ciliary neurons. The GDNF‐induced branching at E13 could be inhibited by BDNF. Blocking ret signaling in vivo with a dominant negative (dn)ret decreases survival of ciliary and choroid neurons at E14 and prevents dTC induced increases in NF‐VU in the iris at E17. Blocking TRKB signaling with dn TRKB increases NF‐VU in the iris at E17 and decreases neuronal survival at E17, but not at E14. Thus, RET promotes survival during programmed cell death in the ciliary ganglion and contributes to promoting branching when synaptic transmission is blocked while TRKB inhibits branching and promotes maintenance of neuronal survival. These studies highlight the multifunctional nature of trophic molecule function during neuronal development. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

The Use of Combining Ability Analysis to Identify Elite Parents for Artemisia annua F1 Hybrid Production

Artemisia annua is an important medicinal crop used for the production of the anti-malarial compound artemisinin. In order to assist in the production of affordable high quality artemisinin we have carried out an A. annua breeding programme aimed at improving artemisinin concentration and biomass. Here we report on a combining ability analysis of a diallel cross to identify robust parental lines for hybrid breeding. The parental lines were selected based on a range of phenotypic traits to encourage heterosis. The general combining ability (GCA) values for the diallel parental lines correlated to the positive alleles of quantitative trait loci (QTL) in the same parents indicating the presence of beneficial alleles that contribute to parental performance. Hybrids generated from crossing specific parental lines with good GCA were identified as having an increase in both artemisinin concentration and biomass when grown either in glasshouse or experimental field trials and compared to controls. This study demonstrates that combining ability as determined by a diallel cross can be used to identify elite parents for the production of improved A. annua hybrids. Furthermore, the selection of material for breeding using this approach was found to be consistent with our QTL-based molecular breeding approach.     

Bumblebee Venom Serine Protease Increases Fungal Insecticidal Virulence by Inducing Insect Melanization

Insect-killing (entomopathogenic) fungi have high potential for controlling agriculturally harmful pests. However, their pathogenicity is slow, and this is one reason for their poor acceptance as a fungal insecticide. The expression of bumblebee, Bombus ignitus, venom serine protease (VSP) by Beauveria bassiana (ERL1170) induced melanization of yellow spotted longicorn beetles (Psacothea hilaris) as an over-reactive immune response, and caused substantially earlier mortality in beet armyworm (Spodopetra exigua) larvae when compared to the wild type. No fungal outgrowth or sporulation was observed on the melanized insects, thus suggesting a self-restriction of the dispersal of the genetically modified fungus in the environment. The research is the first use of a multi-functional bumblebee VSP to significantly increase the speed of fungal pathogenicity, while minimizing the dispersal of the fungal transformant in the environment.