Dissimilar molecular and morphological patterns in an introgressed peripheral population of a sand dune species (Armeria pungens, Plumbaginaceae).

Introgression is a poorly understood evolutionary outcome of hybridisation because it may remain largely undetected whenever it involves the transfer of small parts of the genome from one species to another. Aiming to understand the early stages of this process, a putative case from the southernmost border of the Armeria pungens range from its congener A. macrophylla is revisited following the discovery of a subpopulation that does not show phenotypic signs of introgression and resembles typical A. pungens. We analysed morphometrics, nuclear ribosomal DNA ITS and plastid DNA (trnL-trnF) sequences, genome size, 45S and 5S rDNA loci-FISH data and nrDNA IGS sequences. Within the study site, most individuals match morphologies of either of the two hybridising species, particularly the new subpopulation, with intermediate phenotypes being scarce. This pattern does not fully fit molecular evidence revealing two ITS ribotypes co-occurring intragenomically in most plants from the study site and one single plastid haplotype. Genome size and structural features of the IGS sequences both indicate that A. pungens from the study site is genetically more similar to its sympatric congener than to the remainder of its conspecifics. Introgression of A. macrophylla into A. pungens and plastid capture explain all the evidence analysed. However, important features to understand the origin and fate of the introgressed population, such as the degree and direction of introgression, which are important for understanding early stages of hybridisation in plants with low reproductive barriers, should be addressed with new data.

Encapsulation of VEGF165 into magnetic PLGA nanocapsules for potential local delivery and bioactivity in human brain endothelial cells.

Angiogenesis is an important repairing mechanism in response to ischemia. The administration of pro-angiogenic proteins is an attractive therapeutic strategy to enhance angiogenesis after an ischemic event. Their labile structures and short circulation times in vivo are the main obstacles that reduce the bioactivity and dosage of such proteins at the target site. We report on poly(d,l-lactic-co-glycolic acid) (PLGA) nanocapsules (diameter < 200 nm) containing bioactive vascular endothelial growth factor-165 (VEGF165) in the inner core and superparamagnetic iron oxide nanoparticles (SPIONs) embedded in the polymeric shell. The system showed good encapsulation efficiencies for both VEGF165 and SPIONs and a sustained protein release over 14 days. In vitro studies confirmed protein bioactivity in the form of significantly increased proliferation in human microvascular brain endothelial cell cultures once the protein was released. Through magnetic resonance imaging (MRI) measurements we demonstrated excellent T2 contrast image properties with r2 values as high as 213 mM-1 s-1. In addition, magnetic VEGF165-loaded PLGA nanocapsules could be displaced and accumulated under an external magnetic field for guiding and retention purposes. We therefore suggest that using VEGF165-loaded magnetic PLGA nanocapsules may become a new targeted protein-delivery strategy in the development of future pro-angiogenic treatments, as for instance those directed to neurorepair after an ischemic event.

Role for the silencing protein Dot1 in meiotic checkpoint control.

During the meiotic cell cycle, a surveillance mechanism called the "pachytene checkpoint" ensures proper chromosome segregation by preventing meiotic progression when recombination and chromosome synapsis are defective. The silencing protein Dot1 (also known as Pch1) is required for checkpoint-mediated pachytene arrest of the zip1 and dmc1 mutants of Saccharomyces cerevisiae. In the absence of DOT1, the zip1 and dmc1 mutants inappropriately progress through meiosis, generating inviable meiotic products. Other components of the pachytene checkpoint include the nucleolar protein Pch2 and the heterochromatin component Sir2. In dot1, disruption of the checkpoint correlates with the loss of concentration of Pch2 and Sir2 in the nucleolus. In addition to its checkpoint function, Dot1 blocks the repair of meiotic double-strand breaks by a Rad54-dependent pathway of recombination between sister chromatids. In vegetative cells, mutation of DOT1 results in delocalization of Sir3 from telomeres, accounting for the impaired telomeric silencing in dot1.

Pch2 links chromatin silencing to meiotic checkpoint control.

The PCH2 gene of Saccharomyces cerevisiae is required for the meiotic checkpoint that prevents chromosome segregation when recombination and chromosome synapsis are defective. Mutation of PCH2 relieves the checkpoint-induced pachytene arrest of the zip1, zip2, and dmc1 mutants, resulting in chromosome missegregation and low spore viability. Most of the Pch2 protein localizes to the nucleolus, where it represses meiotic interhomolog recombination in the ribosomal DNA, apparently by excluding the meiosis-specific Hop1 protein. Nucleolar localization of Pch2 depends on the silencing factor Sir2, and mutation of SIR2 also bypasses the zip1 pachytene arrest. Under certain circumstances, Sir3-dependent localization of Pch2 to telomeres also provides checkpoint function. These unexpected findings link the nucleolus, chromatin silencing, and the pachytene checkpoint.

SWM1, a developmentally regulated gene, is required for spore wall assembly in Saccharomyces cerevisiae.

Meiosis in Saccharomyces cerevisiae is followed by encapsulation of haploid nuclei within multilayered spore walls. Formation of this spore-specific wall requires the coordinated activity of enzymes involved in the biosynthesis of its components. Completion of late events in the sporulation program, leading to spore wall formation, requires the SWM1 gene. SWM1 is expressed at low levels during vegetative growth but its transcription is strongly induced under sporulating conditions, with kinetics similar to those of middle sporulation-specific genes. Homozygous swm1Delta diploids proceed normally through both meiotic divisions but fail to produce mature asci. Consistent with this finding, swm1Delta mutant asci display enhanced sensitivity to enzymatic digestion and heat shock. Deletion of SWM1 specifically affects the expression of mid-late and late sporulation-specific genes. All of the phenotypes observed are similar to those found for the deletion of SPS1 or SMK1, two putative components of a sporulation-specific MAP kinase cascade. However, epistasis analyses indicate that Swm1p does not form part of the Sps1p-Smk1p-MAP kinase pathway. We propose that Swm1p, a nuclear protein, would participate in a different signal transduction pathway that is also required for the coordination of the biochemical and morphological events occurring during the last phase of the sporulation program.

SSG1, a gene encoding a sporulation-specific 1,3-beta-glucanase in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the meiotic process is accompanied by a large increase in 1,3-beta-glucan-degradative activity. The molecular cloning of the gene (SSG1) encoding a sporulation-specific exo-1,3-beta-glucanase was achieved by screening a genomic library with a DNA probe obtained by polymerase chain reaction amplification using synthetic oligonucleotides designed according to the nucleotide sequence predicted from the amino-terminal region of the purified protein. DNA sequencing indicates that the SSG1 gene specifies a 445-amino-acid polypeptide (calculated molecular mass, 51.8 kDa) showing extensive similarity to the extracellular exo-1,3-beta-glucanases encoded by the EXG1 gene (C. R. Vazquez de Aldana, J. Correa, P. San Segundo, A. Bueno, A. R. Nebreda, E. Mendez, and F. del Rey, Gene 97:173-182, 1991). The N-terminal domain of the putative precursor is a very hydrophobic segment with structural features resembling those of signal peptides of secreted proteins. Northern (RNA) analysis reveals a unique SSG1-specific transcript, 1.7 kb long, which can be detected only in sporulating diploids (MATa/MAT alpha) but does not appear in vegetatively growing cells or in nonsporulating diploids (MAT alpha/MAT alpha) when incubated under nitrogen starvation conditions. The meiotic time course of SSG1 induction indicates that the gene is transcribed only in the late stages of the process, beginning at the time of meiosis I and reaching a maximum during spore formation. Homozygous ssg1/ssg1 mutant diploids are able to complete sporulation, although with a significant delay in the appearance of mature asci.

Genetic mapping of 1,3-beta-glucanase-encoding genes in Saccharomyces cerevisiae.

The map position of three 1,3-beta-glucanase-encoding genes in S. cerevisiae has been determined following conventional meiotic and mitotic mapping combined with recombinant DNA techniques. EXG1, EXG2 and SSG1 were localized to chromosomes XII, IV and XV, respectively, by hybridizing the cloned genes to Southern blots of chromosomes separated by pulsed-field gel electrophoresis, in conjunction with the rad52-1-dependent chromosome-loss mapping technique. Meiotic tetrad analyses further localized the EXG1 gene 6.1 centimorgans centromere-proximal to CDC25 on the right arm of chromosome XII. EXG2 was positioned between LYS4 and GCN2 on the right arm of chromosome IV, at distances of 6.2 centimorgans from LYS4 and 4.9 centimorgans from GCN2. Finally, the SSG1 locus mapped on the right arm of chromosome XV, about 8.2 centimorgans to the centromere-proximal side of HIS3.

Nucleotide sequence of the exo-1,3-beta-glucanase-encoding gene, EXG1, of the yeast Saccharomyces cerevisiae.

The nucleotide (nt) sequence of the Saccharomyces cerevisiae gene (EXG1) encoding extracellular exo-1,3-beta-glucanases (EXG) I and II was determined. An open reading frame of 1344 bp codes for a 448-amino acid (aa) polypeptide, with a calculated Mr of 51,307, which contains two potential N-glycosylation sites. The EXG1 DNA hybridizes to a 1.7-kb transcript whose 5' end maps to a position 98 bp upstream from the site of initiation of protein synthesis. Comparison of the N-terminal aa sequence deduced from the nt sequence with that of the purified EXGII revealed the existence of an extra 40-aa peptide in the precursor protein containing a Lys-Arg peptidase-processing site at the junction with the mature, extracellular form. The N-terminal region of the putative precursor is a very hydrophobic segment with structural features resembling those of signal peptides of secreted proteins. The Mr of the mature EXG polypeptide deduced from the nt sequence is 46,385. The 5'- and 3'-flanking regions of the EXG1 gene have structural features in common with other yeast genes.