Vectors and Methods for Enhanced Cell Longevity and Protein Expression

It is the object of the current invention to provide methods and compositions relating to the expression of vankyrin proteins in cell lines to increase their viability, longevity and capacity for protein production. The inventors have discovered that the expression of P-ank-1 and I2-ank-3 proteins in cell culture has increased the cells\u27 longevity and capacity for endogenous and/or heterologous target protein production. Specifically, the present invention relates to the enhanced expression of endogenous and/or heterologous target proteins/polypeptides in recombinant cells that are also expressing P-ank-1 and/or I2-ank-3 protein compared to expression host cells that are not expressing P-ank-1 and/or I2-ank-3 protein

Cell Lines Having Enhanced Cell Longevity and Protein Expression

It is the object of the current invention to provide methods and compositions relating to the expression of vankyrin proteins in cell lines to increase their viability, longevity and capacity for protein production. The inventors have discovered that the expression of P-ank-1 and I2-ank-3 proteins in cell culture has increased the cells\u27 longevity and capacity for endogenous and/or heterologous target protein production. Specifically, the present invention relates to the enhanced expression of endogenous and/or heterologous target proteins/polypeptides in recombinant cells that are also expressing P-ank-1 and/or I2-ank-3 protein compared to expression host cells that are not expressing P-ank-1 and/or I2-ank-3 protein

Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification

Transcript analysis and comparative evaluation of shaker and slowmo gene homologues from the European corn borer, Ostrinia nubilalis

The movement and dispersal of larval Lepidoptera impact their survival and distribution within the natural landscape. Homologues of the Drosophila behaviour-linked genes shaker (shkr) and slowmo (slmo) were identified from Ostrinia nubilalis (Lepidoptera: Crambidae). Onshkr was isolated as a 1610-nucleotide (nt) constitutively expressed transcript encoding a membrane-localized 469-amino-acid (aa) protein with a conserved tetramerization domain and the six-domain architecture necessary for the molecule to fold into an active K(+) channel. Three expressed splice variants of 682, 970 and 1604 nt were identified for the Onslmo gene, and encode predicted 141 and 228 aa proteins with a conserved protein of relevant evolutionary and lymphoid interest (PRELI) domain that may function in mitochondrial protein sorting and perinuclear protein localization. Onshkr and Onslmo protein sequences aligned within monophyletic lepidopteran groups

A single major QTL controls expression of larval Cry1F resistance trait in Ostrinia nubilalis (Lepidoptera: Crambidae) and is independent of midgut receptor genes

The European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae), is an introduced crop pest in North America that causes major damage to corn and reduces yield of food, feed, and biofuel materials. The Cry1F toxin from Bacillus thuringiensis (Bt) expressed in transgenic hybrid corn is highly toxic to O. nubilalis larvae and effective in minimizing feeding damage. A laboratory colony of O. nubilalis was selected for high levels of Cry1F resistance (\u3e12,000-fold compared to susceptible larvae) and is capable of survival on transgenic hybrid corn. Genetic linkage maps with segregating AFLP markers show that the Cry1F resistance trait is controlled by a single quantitative trait locus (QTL) on linkage group 12. The map position of single nucleotide polymorphism (SNP) markers indicated that midgut Bt toxin-receptor genes, alkaline phosphatase, aminopeptidase N, and cadherin, are not linked with the Cry1F QTL. Evidence suggests that genes within this genome interval may give rise to a novel Bt toxin resistance trait for Lepidoptera that appears independent of known receptor-based mechanisms of resistance

Transcriptome Sequencing, and Rapid Development and Application of SNP Markers for the Legume Pod Borer Maruca vitrata (Lepidoptera: Crambidae)

The legume pod borer, Maruca vitrata (Lepidoptera: Crambidae), is an insect pest species of crops grown by subsistence farmers in tropical regions of Africa. We present the de novo assembly of 3729 contigs from 454- and Sanger-derived sequencing reads for midgut, salivary, and whole adult tissues of this non-model species. Functional annotation predicted that 1320 M. vitrata protein coding genes are present, of which 631 have orthologs within the Bombyx mori gene model. A homology-based analysis assigned M. vitrata genes into a group of paralogs, but these were subsequently partitioned into putative orthologs following phylogenetic analyses. Following sequence quality filtering, a total of 1542 putative single nucleotide polymorphisms (SNPs) were predicted within M. vitrata contig assemblies. Seventy one of 1078 designed molecular genetic markers were used to screen M. vitrata samples from five collection sites in West Africa. Population substructure may be present with significant implications in the insect resistance management recommendations pertaining to the release of biological control agents or transgenic cowpea that express Bacillus thuringiensis crystal toxins. Mutation data derived from transcriptome sequencing is an expeditious and economical source for genetic markers that allow evaluation of ecological differentiation

Polydnavirus genomes reflect their dual roles as mutualists and pathogens

AbstractSymbionts often exhibit significant reductions in genome complexity while pathogens often exhibit increased complexity through acquisition and diversification of virulence determinants. A few organisms have evolved complex life cycles in which they interact as symbionts with one host and pathogens with another. How the predicted and opposing influences of symbiosis and pathogenesis affect genome evolution in such instances, however, is unclear. The Polydnaviridae is a family of double-stranded (ds) DNA viruses associated with parasitoid wasps that parasitize other insects. Polydnaviruses (PDVs) only replicate in wasps but infect and cause severe disease in parasitized hosts. This disease is essential for survival of the parasitoid's offspring. Thus, a true mutualism exists between PDVs and wasps as viral transmission depends on parasitoid survival and parasitoid survival depends on viral infection of the wasp's host. To investigate how life cycle and ancestry affect PDVs, we compared the genomes of Campoletis sonorensis ichnovirus (CsIV) and Microplitis demolitor bracovirus (MdBV). CsIV and MdBV have no direct common ancestor, yet their encapsidated genomes share several features including segmentation, diversification of virulence genes into families, and the absence of genes required for replication. In contrast, CsIV and MdBV share few genes expressed in parasitized hosts. We conclude that the similar organizational features of PDV genomes reflect their shared life cycle but that PDVs associated with ichneumonid and braconid wasps have likely evolved different strategies to cause disease in the wasp's host and promote parasitoid survival

Stat3 controls cell death during mammary gland involution by regulating uptake of milk fat globules and lysosomal membrane permeabilization.

We have previously demonstrated that Stat3 regulates lysosomal-mediated programmed cell death (LM-PCD) during mouse mammary gland involution in vivo. However, the mechanism that controls the release of lysosomal cathepsins to initiate cell death in this context has not been elucidated. We show here that Stat3 regulates the formation of large lysosomal vacuoles that contain triglyceride. Furthermore, we demonstrate that milk fat globules (MFGs) are toxic to epithelial cells and that, when applied to purified lysosomes, the MFG hydrolysate oleic acid potently induces lysosomal leakiness. Additionally, uptake of secreted MFGs coated in butyrophilin 1A1 is diminished in Stat3-ablated mammary glands and loss of the phagocytosis bridging molecule MFG-E8 results in reduced leakage of cathepsins in vivo. We propose that Stat3 regulates LM-PCD in mouse mammary gland by switching cellular function from secretion to uptake of MFGs. Thereafter, perturbation of lysosomal vesicle membranes by high levels of free fatty acids results in controlled leakage of cathepsins culminating in cell death.This work was supported by a grant from the Medical Research Council programme grant no. MR/J001023/1 (T.J.S. and B. L-L.) and a Cancer Research UK Cambridge Cancer Centre PhD studentship (H.K.R.).This is the accepted manuscript. The final version is available from Nature Publishing at http://www.nature.com/ncb/journal/vaop/ncurrent/full/ncb3043.html