Insect oviposition behavior affects the evolution of adaptation to <Emphasis Type="Italic">Bt</Emphasis> crops: consequences for refuge policies

The major lepidopteran insect pests of cotton and maize harbor intra-specific variation for behavior determining the selection of host plants for oviposition. Yet, the consequences of behavioral adaptation for fitness have neither been modeled nor monitored for Bt cotton and maize crops, the most widely grown transgenic herbivore-resistant plants. Here, we present a general two-locus heuristic model to examine potential outcomes of natural selection when pest populations initially have low frequencies of alleles for both physiological and behavioral adaptation to Bt crops. We demonstrate that certain ecological conditions allow for the evolution of behavioral choices favoring alternative oviposition hosts that limit the increase in resistance alleles, even when they are phenotypically dominant. These results have implications for current refuge policies, which should be adapted to promote the evolution of certain behavioral choices for alternative oviposition hosts in addition to dilution of physiological resistance alleles. Collection of data on oviposition host preference as a component of monitoring schemes will provide important insights into mechanisms underlying the durability of Bt-transgenic host-plant resistance.     

Historical and Contemporary DNA Indicate Fisher Decline and Isolation Occurred Prior to the European Settlement of California

Establishing if species contractions were the result of natural phenomena or human induced landscape changes is essential for managing natural populations. Fishers (Martes pennanti) in California occur in two geographically and genetically isolated populations in the northwestern mountains and southern Sierra Nevada. Their isolation is hypothesized to have resulted from a decline in abundance and distribution associated with European settlement in the 1800s. However, there is little evidence to establish that fisher occupied the area between the two extant populations at that time. We analyzed 10 microsatellite loci from 275 contemporary and 21 historical fisher samples (1880–1920) to evaluate the demographic history of fisher in California. We did not find any evidence of a recent (post-European) bottleneck in the northwestern population. In the southern Sierra Nevada, genetic subdivision within the population strongly influenced bottleneck tests. After accounting for genetic subdivision, we found a bottleneck signal only in the northern and central portions of the southern Sierra Nevada, indicating that the southernmost tip of these mountains may have acted as a refugium for fisher during the anthropogenic changes of the late 19^th and early 20^th centuries. Using a coalescent-based Bayesian analysis, we detected a 90% decline in effective population size and dated the time of decline to over a thousand years ago. We hypothesize that fisher distribution in California contracted to the two current population areas pre-European settlement, and that portions of the southern Sierra Nevada subsequently experienced another more recent bottleneck post-European settlement.     

Generation and molecular characterization of new temperature-sensitive plasmids intended for genetic engineering of Pasteurellaceae

Temperature-sensitive (TS) plasmids were generated through chemical mutagenesis of a derivative of the streptomycin resistance parent plasmid pD70, isolated from Mannheimia hemolytica serotype 1. Three TS plasmids which failed to replicate at or above 42 degrees C in M. hemolytica but which were fully functional below 31 degrees C were selected for further analysis. Two of the TS plasmids were shown by sequencing to possess unique single-base-pair mutations. The third TS plasmid contained a unique base pair substitution and a second mutation that had been previously identified. These mutations were clustered within a 200-bp region of the presumed plasmid origin of replication. Site-directed single-nucleotide substitutions were introduced into the wild-type pD70 origin of replication to confirm that mutations identified by sequencing had conferred thermoregulated replication. Deletion analysis on the wild-type pD70 plasmid replicon revealed that approximately 720 bp are necessary for plasmid maintenance. Replication of the TS plasmids was thermoregulated in Pasteurella multocida and Haemophilus somnus as well. To consistently transform H. somnus with TS plasmid, in vitro DNA methylation with commercially available HhaI methyltransferase was necessary to protect against the organism's restriction enzyme HsoI (recognition sequence 5'-GCGC-3') characterized herein.     

Neurotransmitter release regulated by a MALS-liprin-alpha presynaptic complex

Synapses are highly specialized intercellular junctions organized by adhesive and scaffolding molecules that align presynaptic vesicular release with postsynaptic neurotransmitter receptors. The MALS/Veli-CASK-Mint-1 complex of PDZ proteins occurs on both sides of the synapse and has the potential to link transsynaptic adhesion molecules to the cytoskeleton. In this study, we purified the MALS protein complex from brain and found liprin-alpha as a major component. Liprin proteins organize the presynaptic active zone and regulate neurotransmitter release. Fittingly, mutant mice lacking all three MALS isoforms died perinatally with difficulty breathing and impaired excitatory synaptic transmission. Excitatory postsynaptic currents were dramatically reduced in autaptic cultures from MALS triple knockout mice due to a presynaptic deficit in vesicle cycling. These findings are consistent with a model whereby the MALS-CASK-liprin-alpha complex recruits components of the synaptic release machinery to adhesive proteins of the active zone.     

Sphingosine 1-phosphate prevents platelet-activating factor-induced increase in hydraulic conductivity in rat mesenteric venules: pertussis toxin sensitive

Sphingosine 1-phosphate (S1P) is a biologically active lipid. In vitro, S1P tightens the endothelial barrier, as assessed by a rapid increase in electrical resistance and a decrease in solute permeability. We hypothesized that this activity of S1P would also occur in vivo. Hydraulic conductivity (Lp), an assessment of endothelial barrier function, was measured in individually perfused venules in rat mesenteries. S1P (1 microM) decreased basal Lp by 63% when basal Lp was between 3.6 and 4.1 x 10(-7) cm x s(-1) x cmH2O(-1) but showed no effect when basal Lp was below 2 x 10(-7) cm x s(-1) x cmH2O(-1). Under either condition, S1P blocked the sixfold increase in Lp induced by platelet-activating factor (PAF, 10 nM). Perfusion of venules with pertussis toxin (0.1 microg/ml), a specific inhibitor of the inhibitory G protein, Gi, for 3 h did not affect basal Lp or the increased Lp induced by PAF. Pertussis toxin, however, significantly attenuated the inhibitory action of S1P on the PAF-induced increase in Lp, indicating the involvement of the Gi protein. Measurement of endothelial cytoplasmic Ca2+ concentration ([Ca2+]i) in venules loaded with fura-2 AM showed that S1P alone transiently increased basal endothelial [Ca2+]i (from 89 nM to 193 nM) but had no effect on the magnitude and time course of the PAF-induced increase in endothelial [Ca2+]i. These results indicate that S1P functions in vivo to prevent the PAF-induced increase in microvessel permeability. The inhibitory action of S1P involves the pertussis toxin-sensitive Gi protein and is not mediated by prevention of the PAF-induced increase in endothelial [Ca2+]i.     

Yeast N(alpha)-terminal acetyltransferases are associated with ribosomes

N-terminal acetylation is one of the most common modifications, occurring on the vast majority of eukaryotic proteins. Saccharomyces cerevisiae contains three major NATs, designated NatA, NatB, and NatC, with each having catalytic subunits Ard1p, Nat3p, and Mak3p, respectively. Gautschi et al. (Gautschi et al. [2003] Mol Cell Biol 23: 7403) previously demonstrated with peptide crosslinking experiments that NatA is bound to ribosomes. In our studies, biochemical fractionation in linear sucrose density gradients revealed that all of the NATs are associated with mono- and polyribosome fractions. However only a minor portion of Nat3p colocalized with the polyribosomes. Disruption of the polyribosomes did not cause dissociation of the NATs from ribosomal subparticles. The NAT auxiliary subunits, Nat1p and Mdm20p, apparently are required for efficient binding of the corresponding catalytic subunits to the ribosomes. Deletions of the genes corresponding to auxiliary subunits significantly diminish the protein levels of the catalytic subunits, especially Nat3p, while deletions of the catalytic subunits produced less effect on the stability of Nat1p and Mdm20p. Also two ribosomal proteins, Rpl25p and Rpl35p, were identified in a TAP-affinity purified NatA sample. Moreover, Ard1p copurifies with Rpl35p-TAP. We suggest that these two ribosomal proteins, which are in close proximity to the ribosomal exit tunnel, may play a role in NatA attachment to the ribosome.     

Isolation and characterization of lectins and lectin-alliinase complexes from bulbs of garlic (Allium sativum) and ramsons (Allium ursinum)

A procedure developed to separate the homodimeric and heterodimeric mannose-binding lectins from bulbs of garlic (Allium sativum L.) and ramsons (Allium ursinum L.) also enabled the isolation of stable lectin-alliinase complexes. Characterization of the individual lectins indicated that, in spite of their different molecular structure, the homomeric and heteromeric lectins resemble each other reasonably well with respect to their agglutination properties and carbohydrate-binding specificity. However, a detailed analysis of the lectin-alliinase complexes from garlic and ramsons bulbs demonstrated that only the heterodimeric lectins are capable of binding to the glycan chains of the alliinase molecules (EC 4.4.1.4). Moreover, it appears that only a subpopulation of the alliinase molecules is involved in the formation of lectin-alliinase complexes and that the complexed alliinase contains more glycan chains than the free enzyme. Finally, some arguments are given that the lectin-alliinase complexes do not occur in vivo but are formed in vitro after homogenization of the tissue. This revised version was published online in November 2006 with corrections to the Cover Date.