Cell-cell fusion

Cell-cell fusion is a highly regulated and dramatic cellular event that is required for development and homeostasis. Fusion may also play a role in the development of cancer and in tissue repair by stem cells. While virus-cell fusion and the fusion of intracellular membranes have been the subject of intense investigation during the past decade, cell-cell fusion remains poorly understood. Given the importance of this cell-biological phenomenon, a number of investigators have begun analyses of the molecular mechanisms that mediate the specialized fusion events of a variety of cell types and species. We discuss recent genetic and biochemical studies that are beginning to yield exciting insights into the fusion mechanisms of Saccharomyces cerevisiae mating pairs, Caenorhabditis elegans epithelial cells and gametes, Drosophila melanogaster and mammalian myoblasts, and mammalian macrophages.     

Epigenetic natural variation in Arabidopsis thaliana

Cytosine methylation of repetitive sequences is widespread in plant genomes, occurring in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We used the methylation-dependent restriction enzyme McrBC to profile methylated DNA using tiling microarrays of Arabidopsis Chromosome 4 in two distinct ecotypes, Columbia and Landsberg erecta. We also used comparative genome hybridization to profile copy number polymorphisms. Repeated sequences and transposable elements (TEs), especially long terminal repeat retrotransposons, are densely methylated, but one third of genes also have low but detectable methylation in their transcribed regions. While TEs are almost always methylated, genic methylation is highly polymorphic, with half of all methylated genes being methylated in only one of the two ecotypes. A survey of loci in 96 Arabidopsis accessions revealed a similar degree of methylation polymorphism. Within-gene methylation is heritable, but is lost at a high frequency in segregating F₂ families. Promoter methylation is rare, and gene expression is not generally affected by differences in DNA methylation. Small interfering RNA are preferentially associated with methylated TEs, but not with methylated genes, indicating that most genic methylation is not guided by small interfering RNA. This may account for the instability of gene methylation, if occasional failure of maintenance methylation cannot be restored by other means.     

Endosymbiont phylogenesis in the dryophthoridae weevils: evidence for bacterial replacement

Intracellular symbiosis is widespread in the insect world where it plays an important role in evolution and adaptation. The weevil family Dryophthoridae (Curculionoidea) is of particular interest in intracellular symbiosis evolution with regard to the great economical and ecological features of these invasive insects, and the potential for comparative studies across a wide range of host plants and environments. Here, we have analyzed the intracellular symbiotic bacteria of 19 Dryophthoridae species collected worldwide, representing a wide range of plant species and tissues. All except one (Sitophilus linearis) harbor symbiotic bacteria within specialized cells (the bacteriocytes) assembled as an organ, the bacteriome. Phylogenetic analysis of the 16S rDNA gene sequence of the Dryophthoridae endosymbionts revealed three endosymbiotic clades belonging to gamma3-Proteobacteria and characterized by different GC contents and evolutionary rate. The genus name Candidatus Nardonella was proposed for the ancestral clade infesting Dryophthoridae 100 MYA and represented by five of nine bacterial genera studied. For this clade showing low GC content (40.5% GC) and high evolutionary rate (0.128 substitutions/site per 100 Myr), a single infection and subsequent cospeciation of the host and the endosymbionts was observed. In the two other insect lineage endosymbionts, with relatively high GC content (53.4% and 53.8% GC), competition with ancestral pathogenic bacteria might have occurred, leading to endosymbiont replacement in present-day last insects.     

The basolateral sorting signals of the thyrotropin and luteinizing hormone receptors: an unusual family of signals sharing an unusual distal intracellular localization, but unrelated in their structures

The mechanisms of the basolateral targeting of G protein-coupled receptors remain largely unknown. Mutagenesis experiments have allowed us to identify the basolateral sorting signals of the TSH and LH receptors expressed in Madin-Darby canine kidney cells and thyroid follicular FRT cells. Unexpectedly these signals (amino acids 731-746 and 672-689, respectively) share an unusual localization in the distal part of the intracellular domain of the receptors at a marked distance from the membrane. When grafted onto the p75-neurotropin receptor, these signals redirect this normally apically expressed protein to the basolateral cell surface. They are independent of the endocytosis signal. The basolateral sorting signals of TSH, LH, and FSH receptors do not exhibit primary sequence homology with each other or with any other known signal. Furthermore, circular dichroism studies show that the three signals exhibit distinct secondary structures. The TSH receptor has a stable helical structure, the LH receptor has both helix and beta-sheet structures, and the FSH receptor sorting signal has a main random coil structure. This means that even in closely-related receptors different secondary structures can be found for basolateral signals unrelated to internalization signals. This observation contrasts with what is known about basolateral signals related to internalization signals for which a common beta-turn structure has been described. Deletion of the basolateral sorting signals results in apical targeting of the receptors, suggesting the existence of apical sorting information. However, a soluble form of the TSH receptor, which harbors all N- and putative O-linked oligosaccharides, is secreted in a nonpolarized fashion. This implies that apical sorting information must be located elsewhere, either in the transmembrane or in the intracellular domains of the receptor.     

Expansion and divergence of the GH locus between spider monkey and chimpanzee

Growth hormone (GH) has been previously described as showing distinct evolutionary stories between primates and other mammals. A burst of changes and successive amplification events took place in the primate lineage giving rise to a multigene family in the three Anthropoidea lineages. Polymerase chain reaction (PCR) was used to obtain the genes and the intergenic regions comprising the GH loci of the spider monkey (Ateles geoffroyi), a New-World primate, and of the chimpanzee (Pan troglodytes), an ape. The intergenic sequences of both species were screened by hybridization to detect copies of the Alu family, which have been implicated in the formation of the human GH locus. The GH locus of the spider monkey contains at least six GH-related genes, four of them were cloned. Likewise, five short intergenic sequences of approximately 3 kb were amplified and cloned. On the other hand, in the chimpanzee four new placental lactogen (PL) genes as well as four intergenic regions were amplified. Consequently, in this ape, six genes (two GHs, previously obtained, and four PLs) are clustered, separated by intergenic sequences of different lengths (two short ones of about 5 kb, and at least two long ones between 9 and 13 kb). The presence of Alu sequences within the intergenic regions of both GH loci corroborates the current hypothesis that they acted as a driving force for the locus expansion. GH sequence comparisons reveal that several gene-conversion events might have occurred during the formation of this genome region, which has undergone independent evolution in the three Anthropoidea branches. To establish the GH's evolutionary history may prove to be a difficult task due to these gene-conversion events.     

Raw cow milk bacterial population shifts attributable to refrigeration

We monitored the dynamic changes in the bacterial population in milk associated with refrigeration. Direct analyses of DNA by using temporal temperature gel electrophoresis (TTGE) and denaturing gradient gel electrophoresis (DGGE) allowed us to make accurate species assignments for bacteria with low-GC-content (low-GC%) (<55%) and medium- or high-GC% (>55%) genomes, respectively. We examined raw milk samples before and after 24-h conservation at 4 degrees C. Bacterial identification was facilitated by comparison with an extensive bacterial reference database ( approximately 150 species) that we established with DNA fragments of pure bacterial strains. Cloning and sequencing of fragments missing from the database were used to achieve complete species identification. Considerable evolution of bacterial populations occurred during conservation at 4 degrees C. TTGE and DGGE are shown to be a powerful tool for identifying the main bacterial species of the raw milk samples and for monitoring changes in bacterial populations during conservation at 4 degrees C. The emergence of psychrotrophic bacteria such as Listeria spp. or Aeromonas hydrophila is demonstrated.     

Global gene expression profiles reveal an increase in mRNA levels of collagens, MMPs, and TIMPs in late radiation enteritis

Radiation enteritis, a common complication of radiation therapy for abdominal and pelvic cancers, is characterized by severe transmural fibrosis associated with mesenchymal cell activation, tissue disorganization, and deposition of fibrillar collagen. To investigate the mechanisms involved in this pathological accumulation of extracellular matrix, we studied gene expression of matrix components along with that of genes involved in matrix remodeling, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs). Hybrid selection on high-density cDNA array, real-time RT-PCR, gelatin zymography and immunohistochemistry were used to characterize the mRNA expression profile, activity, and tissue location of extracellular matrix-related genes in radiation enteritis compared with healthy ileum. cDNA array analysis revealed a strong induction of genes coding for collagens I, III, IV, VI, and VIII, SPARC, and tenascin-C, extracellular-matrix degrading enzymes (MMP-1, -2, -3, -14, -18+19), and metalloproteinase inhibitors (TIMP-1, -2, plasminogen activator inhibitor-1) in radiation enteritis. This increase was correlated with the degree of infiltration of the mucosa by inflammatory cells, and the presence of differentiated mesenchymal cells in the submucosa and muscularis propria. Despite the fact that expression of collagens, MMPs, and TIMPs simultaneously increase, quantification of net collagen deposition shows an overall accumulation of collagen. Our results indicate that late radiation enteritis tissues are subjected to active process of fibrogenesis as well as fibrolysis, with a balance toward fibrogenesis. This demonstrates that established fibrotic tissue is not scarred fixed tissue but is subjected to a dynamic remodeling process.