Locally adapted traits maintained in the face of high gene flow.

Gene flow between phenotypically divergent populations can disrupt local adaptation or, alternatively, may stimulate adaptive evolution by increasing genetic variation. We capitalised on historical Trinidadian guppy transplant experiments to test the phenotypic effects of increased gene flow caused by replicated introductions of adaptively divergent guppies, which were translocated from high- to low-predation environments. We sampled two native populations prior to the onset of gene flow, six historic introduction sites, introduction sources and multiple downstream points in each basin. Extensive gene flow from introductions occurred in all streams, yet adaptive phenotypic divergence across a gradient in predation level was maintained. Descendants of guppies from a high-predation source site showed high phenotypic similarity with native low-predation guppies in as few as ~12 generations after gene flow, likely through a combination of adaptive evolution and phenotypic plasticity. Our results demonstrate that locally adapted phenotypes can be maintained despite extensive gene flow from divergent populations.

KNAT2: evidence for a link between knotted-like genes and carpel development.

The KNAT2 (for KNOTTED-like from Arabidopsis thaliana 2) homeobox gene is expressed in the vegetative apical meristem. It is also active during flower development, suggesting a function in the structuring of flowers. To investigate its role, we used a DEXAMETHASONE (DEX)-inducible system to generate transgenic plants that overexpressed a fusion of KNAT2 with the hormone binding domain of the glucocorticoid receptor. DEX-induced plants were similar to plants overexpressing the closely related KNAT1 gene, indicating overlapping functions, although we observed differences as well. In particular, KNAT2-GR activation induced ectopic carpel features. First, KNAT2 induced the homeotic conversion of nucellus into carpel-like structures. Second, KNAT2 induced stigmatic papillae on rosette leaves in the ap2-5 background. Third, ectopic expression of the carpel identity gene AGAMOUS (AG) was observed in carpels and ovules. Interestingly, the homeotic conversion was not dependent on AG activity, because it was maintained in the ag-1 ap2-5 double mutant. Therefore, our data indicate that KNAT2 also must activate other carpel regulators. Together, these results suggest that KNAT2 plays a role in carpel development.

PIN-FORMED 1 regulates cell fate at the periphery of the shoot apical meristem.

The process of organ positioning has been addressed, using the pin-formed 1 (pin1) mutant as a tool. PIN1 is a transmembrane protein involved in auxin transport in Arabidopsis. Loss of function severely affects organ initiation, and pin1 mutants are characterised by an inflorescence meristem that does not initiate any flowers, resulting in the formation of a naked inflorescence stem. This phenotype, combined with the proposed role of PIN1 in hormone transport, makes the mutant an ideal tool to study organ formation and phyllotaxis, and here we present a detailed analysis of the molecular modifications at the shoot apex caused by the mutation. We show that meristem structure and function are not severely affected in the mutant. Major alterations, however, are observed at the periphery of the pin1 meristem, where organ initiation should occur. Although two very early markers of organ initiation, LEAFY and AINTEGUMENTA, are expressed at the periphery of the mutant meristem, the cells are not recruited into distinct primordia. Instead a ring-like domain expressing those primordium specific genes is observed around the meristem. This ring-like domain also expresses a boundary marker, CUP-SHAPED COTYLEDON 2, involved in organ separation, showing that the zone at the meristem periphery has a hybrid identity. This implies that PIN1 is not only involved in organ outgrowth, but that it is also necessary for organ separation and positioning. A model is presented in which PIN1 and the local distribution of auxin control phyllotaxis.

Microsphere-adenoviral complexes target and transduce the glomerulus in vivo.

BACKGROUND: Developing new treatments for glomerulonephritis makes the glomerulus a logical target for gene therapy. Microspheres may lodge in the glomerulus, and replication-deficient recombinant adenoviruses are potent mediators of gene transfer. We postulated that adenoviral-microsphere complexes could result in DNA transfer (transduction) into glomerular cells in vivo. METHODS: Two adenoviruses, each one containing a luciferase or beta-galactosidase (beta-gal) transgene expression cassette, were complexed to polystyrene microspheres. To assess in vivo glomerular transduction with this tool, male Sprague-Dawley rats underwent aortic injections with adenovirus linked to 11 or 16 microm diameter microspheres. RESULTS: After 48 hours, adenoviral-microsphere complexes resulted in transduction of up to 19% of glomeruli per kidney section. Endothelial and mesangial cells were transduced with this approach, and transprotein expression persisted for 21 days. Transduction efficiency was greater in the 16 microm group. For all rats, there was a strong correlation between kidney luciferase levels and the number of beta-gal-positive glomeruli (r = 0.87), indicating that transgene expression was primarily glomerular in location. This was supported by reverse transcriptase in situ polymerase chain reaction, which demonstrated glomerular localization of the beta-gal transgene. CONCLUSIONS: The aortic injection of adenoviral-microsphere complexes transduces the glomerulus in vivo and may be a useful tool in developing approaches to gene therapy of glomerular disease.

Successful DNA transfer in cultured human mesangial cells using replication deficient recombinant adenovirus.

BACKGROUND: Efficient transfer of DNA into human mesangial cells is an essential first step in the development of gene therapies for mesangial cell-mediated glomerulopathies. In the present studies, we assessed the ability of replication deficient recombinant adenovirus to transfer DNA (transduce) into primary cultures of human mesangial cells. METHODS: Primary cultures of human mesangial cells were transduced with an adenoviral vector (rAv beta-gal) containing a CMVllacZ promoter-reporter expression cassette coding for beta-galactosidase (beta-gal). We assessed soluble and histologic beta-gal activity, morphology, and phenotypic expression of mesangial cell transductants, durability of transduced mesangial cells by measuring transgene expression following trypsinization or after prolonged periods in culture and metabolic stability following transduction (as assessed by fibronectin biosynthesis). RESULTS: We showed that rAv beta-gal efficiently transduced mesangial cells in a dose-dependent fashion at a multiplicity of infectious units (MOI) ranging from 1 to 400 plaque forming units/cell (pfu/cell). One hundred percent of mesangial cells were transduced at an MOI of 100 pfu/cell. By electron microscopic evaluation, viral particles of approximately 85-90 nm were demonstrated in the cytoplasm of transduced cells. Following transduction, legal levels rose rapidly and were 10-fold greater than baseline levels after 2 hours. Beta-gal levels continued to rise for 7 days following transduction. Transduction with rAv beta-gal was well tolerated; mesangial cell transductants maintained normal morphology and phenotype, tolerated 3 cycles of trypsinization and maintained normal constitutive production of fibronectin. CONCLUSIONS: Gene transfer with adenovirus is an effective, well tolerated approach for introducing DNA into primary cultures of human mesangial cells.

MGOUN1 and MGOUN2: two genes required for primordium initiation at the shoot apical and floral meristems in Arabidopsis thaliana.

We report two new recessive mutations in Arabidopsis, mgoun1 and mgoun2 which cause a reduction in the number of leaves and floral organs, larger meristems and fasciation of the inflorescence stem. Although meristem structure is affected in the mutants, we provide evidence that its overall organisation is normal, as shown by the expression patterns of two meristem markers. Microscopical analyses suggest that both mutations affect organ primordia production. mgo1 strongly inhibits leaf production in a weak allele of shoot meristemless, stm-2. In addition, mgo1 and 2 severely reduce the ability of the fasciata1 and 2 mutants to initiate organs, although meristem formation per se was not inhibited. The strong allele, stm-5, is epistatic to mgo1, showing that the presence of meristematic cells is essential for MGO1 function. These results suggest a role for the MGO genes in primordia initiation although a more general role in meristem function can not be excluded. We describe a form of fasciation which is radically different from that described for clavata, which is thought to have an increased size of the meristem centre. Instead of one enlarged central meristem mgo1 and 2 show a continuous fragmentation of the shoot apex into multiple meristems, which leads to the formation of many extra branches. The phenotype of mgo1 clv3 and mgo2 clv3 double mutants suggest that the MGO and CLV genes are involved in different events. In conclusion, our results reveal two new components of the regulatory network controlling meristem function and primordia formation. A model for MGO genes is discussed.

Angiotensin II induction of fibronectin biosynthesis in cultured human mesangial cells: association with CREB transcription factor activation.

We assessed the effect of angiotensin II on fibronectin biosynthesis a nd transcription factor activation in adult human mesangial cells in culture. We found that 10(-5) mol/L angiotensin II tended to increase fibronectin mRNA expression within 1 hour (1.2-fold +/- 0.3-fold of that in controls), with a significant increase after 4 hours (0.3-fold +/- 0.1-fold of that in controls, p < 0.05) and 24 hours (1.9-fold +/- 0.3-fold of that in controls, p < 0.02). In conjunction with increased fibronectin mRNA levels, angiotensin II exposure resulted in a significant elevation in immunoreactive fibronectin concentrations and the incorporation of (35S)-labeled methionine into fibronectin after 2 hours (224% +/- 23% of controls, p < 0.05). Angiotensin II also induced mesangial cell activation of the cyclic adenosine monophosphate response element binding protein (CREB) transcription factor, a DNA binding protein known to recognize specific regulatory elements present on the fibronectin gene promoter. Using the electrophoretic mobility shift assay, we showed that angiotensin II increased mesangial cell expression of the activated form of CREB after 4 hours (1.2-fold +/- 0.04-fold of that in controls, p < 0.05). To determine the importance of the CREB regulatory elements in mediating angiotensin II induction of fibronectin gene transcription, JEG-3 cells were transfected with plasmids containing fibronectin promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs with (FN510) or without (FN122) the CREB regulatory motifs. Angiotensin II resulted in a significant increase in CAT activity in FN510 transfectants (1.6-fold +/- 0.2-fold of that in controls, p < 0.05), but there was no effect of angiotensin II on FN122 transfected cells. These data demonstrate that angiotensin II stimulates fibronectin biosynthesis in adult human mesangial cells and suggest that the process may be regulated at the transcriptional level.

Procuste1 mutants identify two distinct genetic pathways controlling hypocotyl cell elongation, respectively in dark- and light-grown Arabidopsis seedlings.

Plant morphogenesis is dependent on a tight control of cell division and expansion. Cell elongation during post-embryonic hypocotyl growth is under the control of a light-regulated developmental switch. Light is generally believed to exert its effects on hypocotyl elongation through a phytochrome-and blue-light receptor-mediated inhibitory action on a so far unknown cell elongation mechanism. We describe here a new class of allelic mutants in Arabidopsis, at the locus PROCUSTE1 (prc1-1 to -4), which have a hypocotyl elongation defect specifically associated with the dark-grown development program. Normal hypocotyl elongation is restored in plants grown in white, blue or red light. In agreement with this, the constitutive photomorphogenic mutation cop1-6, which induces a de-etiolated phenotype in the dark, is epistatic to prc1-2 for the hypocotyl phenotype. Epistasis analyses in red and blue light respectively, indicate that phytochrome B but not the blue light receptor HY4, is required for the switch from PRC1-dependent to PRC1-independent elongation. The conditional hypocotyl growth defect is associated with a deformation of the hypocotyl surface due to an uncontrolled swelling of epidermal, cortical or endodermal cells, suggesting a defect in the structure of the expanding cell wall. A similar phenotype was observed in elongating roots, which was however, independent of the light conditions. The aerial part of mature mutant plants grown in the light was indistinguishable from the wild type. prc1 mutants provide a means of distinguishing, for the first time, two genetic pathways regulating hypocotyl cell elongation respectively in dark- and light-grown seedlings, whereby light not only inhibits hypocotyl growth, but also activates a PRC1-independent cell elongation program.

The higher plant Arabidopsis thaliana encodes a functional CDC48 homologue which is highly expressed in dividing and expanding cells.

We have identified an Arabidopsis thaliana CDC48 gene which, unlike the putative mammalian homologue vasolin-containing protein (VCP), functionally complements Saccharomyces cerevisiae cdc48 mutants. CDC48 is an essential gene in S. cerevisiae and genetic studies suggest a role in spindle pole body separation. Biochemical studies link VCP function to membrane trafficking and signal transduction. We have described the AtCDC48 expression pattern in a multicellular eukaryote; the zones of cell division, expansion and differentiation are physically separated in higher plants, thus allowing the analysis of in situ expression patterns with respect to the state of cell proliferation. AtCDC48 is highly expressed in the proliferating cells of the vegetative shoot, root, floral inflorescence and flowers, and in rapidly growing cells. AtCDC48 mRNA and the encoded protein are up-regulated in the developing microspores and ovules. AtCDC48 expression is down-regulated in most differentiated cell types. AtCDC48p was primarily localized to the nucleus and, during cytokinesis, to the phragmoplast, a site where membrane vesicles are targeted in the deposition of new cell wall materials. This study shows that the essential cell division function of CDC48 has been conserved by, at least, some multicellular eukaryotes and suggests that in higher plants, CDC48 functions in cell division and growth processes.

Cloning and expression of distinct nitrite reductases in tobacco leaves and roots.

Three tobacco nitrite reductase (NiR) cDNA clones were isolated using spinach NiR cDNA as a probe. Sequence analysis and Southern blot hybridization revealed four genes in tobacco. Two of these genes presumably derived from the ancestral species Nicotiana tomentosiformis, the other two from the ancestor N. sylvestris. Northern blot analysis showed that one gene from each ancestral genome was expressed predominantly in leaves, whilst RNA from the other was detected mostly in roots. The accumulation of both leaf and root NiR mRNAs was induced by nitrate and repressed by nitrate- or ammonium-derived metabolites. In addition, the expression of the root NiR gene was detectable in leaves of a tobacco nitrate reductase (NR)-deficient mutant. Thus, the regulation of expression of tobacco NiR genes is comparable to the regulation of expression of barley NR genes.

Inhibition of tobacco nitrite reductase activity by expression of antisense RNA.

A tobacco nitrite reductase (NiR) cDNA and its corresponding gene were isolated from cDNA and genomic libraries. An NiR antisense mRNA was expressed in transgenic tobacco under the control of a double 35S promoter. Transformants were obtained on a medium containing ammonium as the sole source of nitrogen. One plant growing normally on ammonium but displaying drastically reduced development and chlorotic leaves when grown on nitrate as the sole source of nitrogen was studied further. This plant accumulated nitrite fivefold over wild-type level and showed reduced amounts of ammonium (11% wild-type level), glutamine (19%), and total protein (8%). NiR mRNA and activity were below detectable levels. Under these conditions, nitrate reductase (NR) activity and mRNA were overexpressed, suggesting that N-metabolites resulting from nitrate reduction are responsible for the repression of the expression of the NR gene, independently from the presence or absence of a functional NR protein.

Coaction of light, nitrate and a plastidic factor in controlling nitrite-reductase gene expression in tobacco.

Nitrite reductase (NIR; EC 1.7.7.1) - a key enzyme of nitrate reduction - is known to be induced by nitrate and light. In the present study with tobacco (Nicotiana tabacum L.) seedlings the dependency of NIR gene expression on nitrate, light and a plastidic factor was investigated to establish the nature of the coaction between these controlling factors. A cDNA clone coding for tobacco plastidic NIR was available as a probe. The major results were as follows: (i) The light effect on the appearance of NIR occurred predominantly through phytochrome. However, a specific blue-light effect was also involved. (ii) There was no effect of light on NIR appearance in the absence of nitrate while light exerted a strong effect when nitrate was provided. (iii) Anion-exchange chromatography revealed only a single form of NIR. While experiments involving plastid photooxidation indicated that this NIR is plastidic, a small residual level could not be eliminated by photooxidation. (iv) Northern blot analysis of NIR-transcript levels indicated that a low transcript level existed in the absence of nitrate and light; however, this level appeared to be increased slightly by light (in the absence of nitrate) and by nitrate (in the absence of light). A high transcript level was detected only when light as well as nitrate were provided. A low level was found when the plastids were damaged by photooxidation. It is concluded that plastidic NIR gene expression in tobacco requires positive control by a plastidic factor. Moreover, a synergistic action of phytochrome and nitrate is required to bring about a high transcript level. As found previously with mustard and spinach seedlings, there is no quantitative relationship between the transcript level and the rate of enzyme synthesis.

Molecular cloning and characterisation of the two homologous genes coding for nitrate reductase in tobacco.

The two structural genes encoding tobacco nitrate reductases (NR) were isolated from tobacco genomic libraries constructed in lambda EMBL phages. Two independent genomic clones of 12.6 and 13.5 kbp, respectively, cross-hybridizing with a partial tobacco NR cDNA probe, were further characterized. Southern blot experiments were performed with the NR cDNA probe on genomic DNA derived from Nicotiana tabacum and from the ancestors of tobacco, N. sylvestris and N. tomentosiformis. They showed that the larger clone, referred to as nia-1, was related to the N. tomentosiformis parent, and the smaller one, referred to as nia-2, to the N. sylvestris parent. Both homeologous genes were found to be expressed in tobacco. The sequence of the gene nia-2, from which the cDNA previously cloned is derived, was determined. It encodes a 904 amino acid protein. Three intervening sequences were found interspersed with the coding sequence of the enzyme. The precise location of the transcription initiation site on the structural gene was mapped by primer extension experiments. A TATA consensus sequence was detected 32 bp upstream from the transcription initiation site. The leader sequence of the transcript is 138 nucleotides long and a stable secondary structure involving the translation initiation site has been proposed. The amino acid sequence of tobacco NR deduced from the nucleotide sequence of the gene shows that heme and FAD binding domains occupy the entire C-terminal moiety of the polypeptide. The remaining N-terminal part of the protein should thus carry the catalytic site of nitrate reduction by the molybdenum cofactor.

Cloning of DNA fragments complementary to tobacco nitrate reductase mRNA and encoding epitopes common to the nitrate reductases from higher plants.

Messenger RNAs encoding the nitrate reductase apoenzyme from tobacco can be translated in a cell-free system. Poly(A)+ mRNA fractions from the 23-32 S area of a sucrose gradient were used to build a cDNA library in the expression vector gt11 with an efficiency of cloning of approximately 10(4) recombinants/ng mRNA. Recombinant clones were screened with a rabbit polyclonal antibody directed against the corn nitrate reductase, which cross reacts specifically with the nitrate reductases from dicotyledons. Among 240000 recombinant plaques, eight clones were isolated containing inserts of sizes ranging from 1.6 kb to 2.1 kb and sharing sequence homologies. Seven of these clones contained a common internal 1.6 kb EcoRI fragment. The identity of these clones was confirmed as follows. A fusion protein of 170 kDa inducible by IPTG and recognized by the rabbit nitrate reductase antibody was expressed by a lysogen derived from one of the recombinants. The antibodies binding the fused protein were eluted and shown to be inhibitory to the catalytic activity of tobacco nitrate reductase. Two monoclonal antibodies directed against nitrate reductase were also able to bind the hybrid protein. The 1.6 kb EcoRI fragment was sequenced by the method of Sanger. The open reading frame corresponding to a translational fusion with the -galactosidase coding sequence of the vector shared strong homology at the amino acid level with the heme-binding domain of proteins of the cytochrome b5 superfamily and with human erythrocyte cytochrome b5 reductase. When the 1.6 kb EcoRI fragment was used as a probe for Northern blot experiments a signal corresponding to a 3.5 kb RNA was detected in tobacco and in Nicotiana plumbaginifolia mRNA preparations but no cross-hybridization with corn mRNAs was detected. The probe hybridized with low copy number sequences in genomic blots of tobacco DNA.

Food neophobia in wild and laboratory mice (Mus musculus domesticus).

In a conditioned taste aversion procedure we were specifically interested in the topic of food neophobia. Wild and laboratory mice were individually presented with a novel drink (0.1 % saccharin solution). Compared with the daily water consumption, the intake of this was lower. This decrease was greater: (1) in wild than in tame populations ; (2) in random-bred (Swiss-albinos) than in inbred (C57 B1/6, BALB/c) strains ; (3) in F1-hybrids (either wild x tame or inbred x inbred) than in the parental strains. These results are discussed: (1) in terms of a selective pressure linked to man's fight against rodents, leading to increased neophobia in wild mice ; and (2) by stressing the heterosis an inbreeding depression effects, which suggest that food neophobia is a component of Darwinian fitness.