The two neutrophil members of the formylpeptide receptor family activate the NADPH-oxidase through signals that differ in sensitivity to a gelsolin derived phosphoinositide-binding peptide

Background  

Upon serial passaging of mouse skeletal muscle cells, a small number of cells will spontaneously develop the ability to proliferate indefinitely while retaining the ability to differentiate into multinucleate myotubes. Possible gene changes that could underlie myogenic cell immortalization and their possible effects on myogenesis had not been examined.     

Development of Efficient Plant Regeneration and Transformation System for Impatiens Using <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>and Multiple Bud Cultures as Explants

Background  

Impatiens (Impatiens walleriana) is a top selling floriculture crop. The potential for genetic transformation of Impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. We have established a regeneration and transformation system for Impatiens that provides new alternatives to genetic improvement of this crop.     

Ectopic expression of MdSPDS1 in sweet orange (Citrus sinensis Osbeck) reduces canker susceptibility: involvement of H2O2 production and transcriptional alteration

Background  

Enormous work has shown that polyamines are involved in a variety of physiological processes, but information is scarce on the potential of modifying disease response through genetic transformation of a polyamine biosynthetic gene.     

One-step immortalization of primary human airway epithelial cells capable of oncogenic transformation

Background

The ability to transform normal human cells into cancer cells with the introduction of defined genetic alterations is a valuable method for understanding the mechanisms of oncogenesis. Easy establishment of immortalized but non-transformed human cells from various tissues would facilitate these genetic analyses.

Results

We report here a simple, one-step immortalization method that involves retroviral vector mediated co-expression of the human telomerase protein and a shRNA targeting the CDKN2A gene locus. We demonstrate that this method could successfully immortalize human small airway epithelial cells while maintaining their chromosomal stability. We further showed that these cells retain p53 activity and can be transformed by the KRAS oncogene.

Conclusions

Our method simplifies the immortalization process and is broadly applicable for establishing immortalized epithelial cell lines from primary human tissues for cancer research.

     

Genetic analysis of the capsule polysaccharide (K antigen) and exopolysaccharide genes in pandemic <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> O3:K6

Background  

Pandemic Vibrio parahaemolyticus has undergone rapid changes in both K- and O-antigens, making detection of outbreaks more difficult. In order to understand these rapid changes, the genetic regions encoding these antigens must be examined. In Vibrio cholerae and Vibrio vulnificus, both O-antigen and capsular polysaccharides are encoded in a single region on the large chromosome; a similar arrangement in pandemic V. parahaemolyticus would help explain the rapid serotype changes. However, previous reports on "capsule" genes are controversial. Therefore, we set out to clarify and characterize these regions in pandemic V. parahaemolyticus O3:K6 by gene deletion using a chitin based transformation strategy.     

Genetic transformation of Vitis vinifera via organogenesis

Background  

Efficient transformation and regeneration methods are a priority for successful application of genetic engineering to vegetative propagated plants such as grape. The current methods for the production of transgenic grape plants are based on Agrobacterium-mediated transformation followed by regeneration from embryogenic callus. However, grape embryogenic calli are laborious to establish and the phenotype of the regenerated plants can be altered.     

Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes

Background  

Infection with high-risk type human papilloma viruses (HPVs) is associated with cervical carcinomas and with a subset of head and neck squamous cell carcinomas. Viral E6 and E7 oncogenes cooperate to achieve cell immortalization by a mechanism that is not yet fully understood. Here, human keratinocytes were immortalized by long-term expression of HPV type 16 E6 or E7 oncoproteins, or both. Proteomic profiling was used to compare expression levels for 741 discrete protein features.     

Island selection on mammalian life-histories: genetic differentiation in offspring size

Background  

Since Darwin's pioneering work, evolutionary changes in isolated island populations of vertebrates have continued to provide the strongest evidence for the theory of natural selection. Besides macro-evolutionary changes, micro-evolutionary changes and the relative importance of natural selection vs. genetic drift are under intense investigation. Our study focuses on the genetic differentiation in morphological and life-history traits in insular populations of a small mammal the bank vole Myodes glareolus.     

Whole ovary immunohistochemistry for monitoring cell proliferation and ovulatory wound repair in the mouse

Background  

Ovarian surface epithelial cells are thought to be a precursor cell type for ovarian carcinoma. It has been proposed that an increased rate of ovarian surface epithelial cell proliferation during ovulatory wound repair contributes to the accumulation of genetic changes and cell transformation. The proliferation of ovarian surface epithelial cells during ovulatory wound repair has been studied primarily using immunohistochemical staining of paraffin-embedded ovary sections. However, such analyses require complex reconstruction from serially-cut ovary sections for the visualization and quantification of the cells on the ovarian surface. In order to directly visualize the proliferation and organization of the ovarian surface epithelial cells, we developed a technique for immunohistochemical staining of whole mouse ovaries. Using this method, we analyzed cell proliferation and morphologic changes in mouse ovarian surface epithelial cells during follicle growth and ovulatory wound repair.     

Transformation of SV40-immortalized human uroepithelial cells by 3-methylcholanthrene increases IFN- and Large T Antigen-induced transcripts

Background  

Simian Virus 40 (SV40) immortalization followed by treatment of cells with 3-methylcholanthrene (3-MC) has been used to elicit tumors in athymic mice. 3-MC carcinogenesis has been thoroughly studied, however gene-level interactions between 3-MC and SV40 that could have produced the observed tumors have not been explored. The commercially-available human uroepithelial cell lines were either SV40-immortalized (HUC) or SV40-immortalized and then 3-MC-transformed (HUC-TC).     

Epstein-barr virus immortalization of human B-cells leads to stabilization of hypoxia-induced factor 1 alpha, congruent with the warburg effect

Background

Epstein-Barr virus (EBV) encodes six nuclear transformation-associated proteins that induce extensive changes in cellular gene expression and signaling and induce B-cell transformation. The role of HIF1A in EBV-induced B-cell immortalization has not been previously studied.

Methods and Findings

Using Western blotting and Q-PCR, we found that HIF1A protein is stabilized in EBV-transformed lymphoblastoid cells. Western blotting, GST pulldown assays, and immunoprecipitation showed that EBV-encoded nuclear antigens EBNA-5 and EBNA-3 bind to prolylhydroxylases 1 and 2, respectively, thus inhibiting HIF1A hydroxylation and degradation. Immunostaining and Q-PCR showed that the stabilized HIF1A translocates to the nucleus, forms a heterodimer with ARNT, and transactivates several genes involved in aerobic glycolysis. Using biochemical assays and Q-PCR, we also found that lymphoblastoid cells produce high levels of lactate, lactate dehydrogenase and pyruvate.

Conclusions

Our data suggest that activation of the aerobic glycolytic pathway, corresponding to the Warburg effect, occurs in EBV-transformed lymphoblastoid cells, in contrast to mitogen-activated B-cells.     

Construction of non-polar mutants in <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> using FLP recombinase technology

Background  

Nontypeable Haemophilus influenzae (NTHi) is a gram-negative bacterium that causes otitis media in children as well as other infections of the upper and lower respiratory tract in children and adults. We are employing genetic strategies to identify and characterize virulence determinants in NTHi. NTHi is naturally competent for transformation and thus construction of most mutants by common methodologies is relatively straightforward. However, new methodology was required in order to construct unmarked non-polar mutations in poorly expressed genes whose products are required for transformation. We have adapted the lambda red/FLP-recombinase-mediated strategy used in E. coli for use in NTHi.     

Investigations of fine-scale phylogeography in Tigriopus californicus reveal historical patterns of population divergence

Background  

The intertidal copepod Tigriopus californicus is a model for studying the process of genetic divergence in allopatry and for probing the nature of genetic changes that lead to reproductive isolation. Although previous studies have revealed a pattern of remarkably high levels of genetic divergence between the populations of this species at several spatial scales, it is not clear what types of historical processes are responsible. Particularly lacking are data that can yield insights into population history from the finest scales of geographic resolution.     

Spectrum of disease-causing mutations in protein secondary structures

Background  

Most genetic disorders are linked to missense mutations as even minor changes in the size or properties of an amino acid can alter or prevent the function of the protein. Further, the effect of a mutation is also dependent on the sequence and structure context of the alteration.     

Isolation and antisense suppression of <Emphasis Type="Italic">flavonoid 3', 5'-hydroxylase</Emphasis>modifies flower pigments and colour in cyclamen

Background  

Cyclamen is a popular and economically significant pot plant crop in several countries. Molecular breeding technologies provide opportunities to metabolically engineer the well-characterized flavonoid biosynthetic pathway for altered anthocyanin profile and hence the colour of the flower. Previously we reported on a genetic transformation system for cyclamen. Our aim in this study was to change pigment profiles and flower colours in cyclamen through the suppression of flavonoid 3', 5'-hydroxylase, an enzyme in the flavonoid pathway that plays a determining role in the colour of anthocyanin pigments.     

Genetic transformation of Indian bread (<Emphasis Type="Italic">T. aestivum</Emphasis>) and pasta (<Emphasis Type="Italic">T. durum</Emphasis>) wheat by particle bombardment of mature embryo-derived calli

Background  

Particle bombardment has been successfully employed for obtaining transgenics in cereals in general and wheat in particular. Most of these procedures employ immature embryos which are not available throughout the year. The present investigation utilizes mature seeds as the starting material and the calli raised from the hexaploid Triticum aestivum and tetraploid Triticum durum display a high regeneration response and were therefore used as the target tissue for genetic transformation by the biolistic approach.     

Inference of demographic history from genealogical trees using reversible jump Markov chain Monte Carlo

Background  

Coalescent theory is a general framework to model genetic variation in a population. Specifically, it allows inference about population parameters from sampled DNA sequences. However, most currently employed variants of coalescent theory only consider very simple demographic scenarios of population size changes, such as exponential growth.     

The complete chloroplast genome sequence of Citrus sinensis (L.) Osbeck var 'Ridge Pineapple': organization and phylogenetic relationships to other angiosperms

Background  

The production of Citrus, the largest fruit crop of international economic value, has recently been imperiled due to the introduction of the bacterial disease Citrus canker. No significant improvements have been made to combat this disease by plant breeding and nuclear transgenic approaches. Chloroplast genetic engineering has a number of advantages over nuclear transformation; it not only increases transgene expression but also facilitates transgene containment, which is one of the major impediments for development of transgenic trees. We have sequenced the Citrus chloroplast genome to facilitate genetic improvement of this crop and to assess phylogenetic relationships among major lineages of angiosperms.     

The mitochondrial genome structure of Xenoturbella bocki (phylum Xenoturbellida) is ancestral within the deuterostomes

Background  

Mitochondrial genome comparisons contribute in multiple ways when inferring animal relationships. As well as primary sequence data, rare genomic changes such as gene order, shared gene boundaries and genetic code changes, which are unlikely to have arisen through convergent evolution, are useful tools in resolving deep phylogenies. Xenoturbella bocki is a morphologically simple benthic marine worm recently found to belong among the deuterostomes. Here we present analyses comparing the Xenoturbella bocki mitochondrial gene order, genetic code and control region to those of other metazoan groups.     

Combined inactivation of the <Emphasis Type="Italic">Clostridium cellulolyticum</Emphasis> lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations

Background  

The model bacterium Clostridium cellulolyticum efficiently degrades crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H₂ and CO₂, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels production. Therefore genetic engineering will likely be required to improve the ethanol yield. Plasmid transformation, random mutagenesis and heterologous expression systems have previously been developed for C. cellulolyticum, but targeted mutagenesis has not been reported for this organism, hindering genetic engineering.