Agrobacterium tumefaciens-mediated transformation of Solanum gilo Raddi as influenced by explant type

An efficient system for Agrobacterium tumefaciens-mediated transformation of Solanum gilo was established. The marker genes for kanamycin resistance and ß-glucuronidase expression were introduced. A comparison between cotyledon and hypocotyl explants showed that while regeneration was better from hypocotyl explants, cotyledon explants gave better transformation efficiency (46% vs. 32%). Four levels of kanamycin selection (100, 150, 200 and 250 mg/l) were tested for effect on transformation efficiency with each type of explant. Lower levels of kanamycin worked better using cotyledon explants, while higher levels of kanamycin worked better for hypocotyl explants. All nine t₀ plants tested for expression of the kan ^r gene were positive. The progeny of three of these plants showed a pattern of classical Mendelian inheritance (3 to 1) for both the kan ^r and the ß-glucuronidase genes.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-Dichlorophenoxyacetic acid - NPTII neomycin phosphotransferase - GUS ß-glucuronidase     

Competition between bound and free peptides in an ELISA-based procedure that assays peptides derived from protein digests

Background  

We describe an ELISA-based method that can be used to identify and quantitate proteins in biological samples. In this method, peptides in solution, derived from proteolytic digests of the sample, compete with substrate-attached synthetic peptides for antibodies, also in solution, generated against the chosen peptides. The peptides used for the ELISA are chosen on the basis of their being (i) products of the proteolytic (e.g. tryptic) digestion of the protein to be identified and (ii) unique to the target protein, as far as one can know from the published sequences.     

A genetics‐based approach confirms immune associations with life history across multiple populations of an aquatic vertebrate (Gasterosteus aculeatus)

Understanding how wild immune variation covaries with other traits can reveal how costs and trade‐offs shape immune evolution in the wild. Divergent life history strategies may increase or alleviate immune costs, helping shape immune variation in a consistent, testable way. Contrasting hypotheses suggest that shorter life histories may alleviate costs by offsetting them against increased mortality, or increase the effect of costs if immune responses are traded off against development or reproduction. We investigated the evolutionary relationship between life history and immune responses within an island radiation of three‐spined stickleback, with discrete populations of varying life histories and parasitism. We sampled two short‐lived, two long‐lived and an anadromous population using qPCR to quantify current immune profile and RAD‐seq data to study the distribution of immune variants within our assay genes and across the genome. Short‐lived populations exhibited significantly increased expression of all assay genes, which was accompanied by a strong association with population‐level variation in local alleles and divergence in a gene that may be involved in complement pathways. In addition, divergence around the eda gene in anadromous fish is likely associated with increased inflammation. A wider analysis of 15 populations across the island revealed that immune genes across the genome show evidence of having diverged alongside life history strategies. Parasitism and reproductive investment were also important sources of variation for expression, highlighting the caution required when assaying immune responses in the wild. These results provide strong, gene‐based support for current hypotheses linking life history and immune variation across multiple populations of a vertebrate model.     

Ecological and evolutionary diversification in the Australo-Papuan scrubwrens (<Emphasis Type="Italic">Sericornis</Emphasis>) and mouse-warblers (<Emphasis Type="Italic">Crateroscelis</Emphasis>), with a revision of the subfamily Sericornithinae (Aves: Passeriformes: Acanthizidae)

Understanding how the complex geotectonic and climatic history of the Australo-Papuan region has promoted the ecological and evolutionary diversification of its avifauna remains a challenge. Outstanding questions relate to the nature and timing of biogeographical connections between Australia and the emerging island of New Guinea and the mechanisms by which distinctive altitudinal replacement sequences have evolved amongst congeneric species in montane New Guinea. Here, we combine analyses of phylogenetic and eco-morphological data to investigate ecological and evolutionary patterns of diversification in the largely mesic-adapted Australo-Papuan scrubwrens (Sericornis) and mouse-warblers (Crateroscelis). We find evidence of ecological convergence and present a revised taxonomic and systematic treatment of the subfamily integrating information from new (ND2) and existing molecular phylogenetic reconstructions. Biogeographical connections indicate at least three phases of faunal interchange between Australia and New Guinea commencing in the mid to late Miocene. We also find little support for the proposed time dependency of ecological sorting mechanisms linked to divergence in foraging niche amongst altitudinal replacements. Instead, physiological adaptations to hypoxia and increased thermal efficiency at higher altitudes may better account for observed patterns of diversification in montane New Guinea. Indirect support for this hypothesis is derived from molecular clock calibrations that indicate a pulse of diversification across the Miocene-Pliocene boundary coincident with a phase of rapid mountain uplift. Simple ecological and climatic models appear inadequate for explaining observed patterns and mechanisms of diversification in the New Guinean montane avifauna. Further insights will require multidisciplinary research integrating geotectonic, palaeoclimatic, genetic, ecological and physiological approaches.     

Heat shock and light activation of aChlamydomonas HSP70 gene are mediated by independent regulatory pathways

Induction ofHSP70 heat shock genes by light has been demonstrated inChlamydomonas. Our aim was to establish whether this induction by light is mediated by the heat stress sensing pathway or by an independent signal chain. Inhibitors of cytoplasmic protein synthesis revealed an initial difference. Cycloheximide and other inhibitors of protein synthesis preventedHSP70A induction upon illumination but not during heat stress. Analysis ofHSP70A induction in cells that had differentiated into gametes revealed a second difference. While heat shock resulted in elevatedHSP70A mRNA levels, light was no longer able to serve as an inducer in gametes. To identify the regulatory sequences that mediate the response of theHSP70A gene to either heat stress or light we introduced a series of progressive 5 truncations into its promoter sequence. Analyses of the levels of mRNA transcribed from these deletion constructs showed that in most of them the responses to heat shock and light were similar, suggesting that light induction is mediated by a light-activated heat shock factor. However, we show that theHSP70A promoter also containscis-acting sequences involved in light induction that do not participate in induction by heat stress. Together, these results provide evidence for a regulation ofHSP70A gene expression by light through a heat shock-independent signal pathway.     

Seasonal influence on reproduction in chimpanzees of gombe national park

Although wild chimpanzees are not seasonal breeders, there are seasonal effects on several aspects of chimpanzee reproduction. I examined the seasonal incidence of anogenital swelling in cyclic, pregnant, and acyclic female chimpanzees in Gombe National Park, May 1975–April 1992, and surveyed important reproductive events to determine whether there is a seasonal effect. I analyzed data by season (wet vs. dry) and seasonal quarter;early dry season = May–July;late dry = August–October;early wet = November–January;late wet = February–April. When data for the 17 years are combined, the percentage of females in each reproductive state remains consistent throughout the year. In a given month, 30–35% of subjects were in the cyclic category, 11–15% were pregnant, and 54–61% were acyclic. Cyclic females showed full swelling more often during the late dry season. Pregnant females exhibited anogenital swelling more often during the late dry and early wet seasons. Acyclic females also exhibited a seasonal effect with more anogenital swelling during the late dry season. There is no seasonal difference in frequency of live births (dry, 20;wet, 23). However, the timing of conception showed a seasonal effect (dry, 32;wet, 16). Consistent with earlier reports, the onset of postpartum cycles is highly seasonal;30 occurred during dry season, 9 during wet season. The occurrence of first full swellings for young females is also concentrated in the late dry season. It appears that the dry season is a time of great change for Gombe chimpanzee reproductive physiology. Previous studies indicated that seasonal changes in food availability play a role in increasing group size during the dry season and social contact between females can enhance cyclicity. Accordingly, I suggest that seasonal changes in diet may play a role, either directly (food content) or indirectly (social contact), to alter reproductive physiology.     

Purification and characterization of the integrase from the Haemophilus influenzae bacteriophage HP1; identification of a four-stranded intermediate and the order of strand exchange

The integrase encoded by the temperate phage HP1 promotes the site-specific recombination between DNA sites on its genome (the attP site) and on the genome of the host Haemophilus influenzae (the attB site). The protein has been overproduced in Escherichia coli , and purified to apparent homogeneity. HP1 integrase promotes recombination of supercoiled attP -containing molecules with linear segments with attB sites. Reaction was enhanced by spermidine and by the bacterial DNA-bending protein integration host factor. The rate of recombination showed complex and related dependence upon the integrase concentration and the concentration of the supercoiled attP substrate. These relationships probably originate from the need to assemble a multi-protein complex on the attP DNA. The reaction promoted by HP1 integrase produced a four-stranded initial reaction product in which one pair of DNA strands had undergone transfer while the other pair remained intact. This four-stranded component was produced more rapidly than any product, and its steady-state level was proportional to the overall rate of reaction. This component had the kinetic and structural properties of an intermediate in the recombination reaction. The existence of this intermediate was used to determine that the two strand exchanges required for recombination of the duplex substrates proceed in a defined order.     

Biogeographical history of cuckoo‐shrikes (Aves: Passeriformes): transoceanic colonization of Africa from Australo‐Papua

Aim Cuckoo‐shrikes and allies (Campephagidae) form a radiation of birds widely distributed in the Indo‐Pacific and Africa. Recent studies on the group have been hampered by poor taxon sampling, causing inferences about systematics and biogeography to be rather speculative. With improved taxon sampling and analyses within an explicit spatiotemporal framework, we elucidate biogeographical patterns of dispersal and diversification within this diverse clade of passerine birds. Location Africa, Asia, Australo‐Papua, the Pacific, the Philippines and Wallacea. Methods We use model‐based phylogenetic methods (Mr Bayes and garli ) to construct a phylogenetic hypothesis of the core Campephagidae (Campephagidae with the exclusion of Pericrocotus). The phylogeny is used to assess the biogeographical history of the group with a newly developed Bayesian approach to dispersal–vicariance analysis (Bayes‐diva) . We also made use of a partitioned beast analysis, with several calibration points taken from island ages, passerine mitochondrial substitution rates and secondary calibration points for passerine birds, to assess the timing of diversification and dispersal. Results We present a robust molecular phylogeny that includes all genera and 84% of the species within the core Campephagidae. Furthermore, we estimate divergence dates and ancestral area relationships. We demonstrate that Campephagidae originated in Australo‐Papua with a single lineage (Pericrocotus) dispersing to Asia early. Later, there was further extensive transoceanic dispersal from Australo‐Papua to Africa involving lineages within the core Campephagidae radiation. Main conclusions The phylogenetic relationships, along with the results of the ancestral area analysis and the timing of dispersal events, support a transoceanic dispersal scenario from Australo‐Papua to Africa by the core Campephagidae. The sister group to core Campephagidae, Pericrocotus, dispersed to mainland Asia in the late Oligocene. Asia remained uncolonized by the core Campephagidae until the Pliocene. Transoceanic dispersal is by no means an unknown phenomenon, but our results represent a convincing case of colonization over a significant water gap of thousands of kilometres from Australo‐Papua to Africa.     

Predator experience overrides learned aversion to heterospecifics in stickleback species pairs

Predation risk can alter female mating decisions because the costs of mate searching and selecting attractive mates increase when predators are present. In response to predators, females have been found to plastically adjust mate preference within species, but little is known about how predators alter sexual isolation and hybridization among species. We tested the effects of predator exposure on sexual isolation between benthic and limnetic threespine sticklebacks (Gasterosteus spp.). Female discrimination against heterospecific mates was measured before and after females experienced a simulated attack by a trout predator or a control exposure to a harmless object. In the absence of predators, females showed increased aversion to heterospecifics over time. We found that predator exposure made females less discriminating and precluded this learned aversion to heterospecifics. Benthic and limnetic males differ in coloration, and predator exposure also affected sexual isolation by weakening female preferences for colourful males. Predator effects on sexual selection were also tested but predators had few effects on female choosiness among conspecific mates. Our results suggest that predation risk may disrupt the cognitive processes associated with mate choice and lead to fluctuations in the strength of sexual isolation between species.