Toll-like receptors (TLRs) constitute an essential family of pattern recognition molecules that, through the direct recognition of conserved microbial components, initiate inflammatory responses after infection. Phylogenetic evidence suggests that vertebrate TLRs are under strong purifying selection for the maintenance of function. Our lab is focused on a related group of vertebrate TLRs that comprise the TLR2 subfamily. The most closely related members, TLRs 1 and 6, appear to have arisen from a recent gene duplication event and have acquired differential microbial recognition specificity. We have characterized two single nucleotide polymorphisms (SNPs), P315L, and I602S, in human TLR1 that effect receptor function through different mechanisms. The 315L variant is associated with deficient recognition of microbial products whereas the 602S variant is associated with aberrant trafficking of the receptor to the cell surface. It is surprising to note that the 602S allele is associated with a decreased incidence of leprosy suggesting that Mycobacterium leprae subverts the TLR system as a mechanism of immune evasion. TLR1 I602S exhibits strikingly different allele frequencies among different races suggesting that if strong purifying selection took place, it was restricted by either additional genetic or environmental factors that were geographically constrained.
High expression of transgenes is desired for molecular farming. However, transgenes are often subjected to gene silencing pathways in plant cells. Gene silencing may be triggered by the production of aberrant (hairpin) RNA molecule from a complex integration locus in plant genome or by the overexpression of transgene. Excessively transcribed RNA are subject to gene silencing even if they are produced from a single-copy locus. Therefore, we sought to determine how highly a transgene can be expressed before its transcript is subjected to gene silencing. In a previous study, we demonstrated that precise single-copy locus generated by Cre/lox-mediated site-specific integration (SSI) in rice is stably expressed at predictable levels through subsequent generations, and that its expression invariably doubled in homozygous progenies. To further explore the stability of the SSI locus and determine the expression-threshold of rice genomic sites, we generated transformation vectors containing 1–3 copies of 35S-GUS and 35S-GFP transgenes. These vectors were used to develop SSI locus containing 1–3 copies of each transgene in two different varieties of rice, Nipponbare and Taipei 309. SSI lines will be analyzed by PCR and Southern blotting to ascertain the presence of precise integration structures consisting of 1–3 copies of each trans-gene. The precise SSI lines will be subjected to quantitative GUS and GFP assay to determine if gene expression indeed increased with the increase in gene dosage. Molecular and expression data of SSI lines will be presented.
Tobacco plants (Nicotiana tabacum L.) were transformed with a construct based on pCAMBIA 2301 containing a “hairpin” inverted repeat of 598 nucleotides derived from the Potato Virus Y (PVY) replicase (NIb gene) of the N strain (Robaglia et al., J. Gen. Virol. 70, 935, 1989). Such constructs confer virus resistance by a post translational gene silencing mechanism. Homozygous (T3) plants were challenged with a range of PVY strains and resistance was measured by symptom expression, ELISA titer, and back inoculation of controls with extracts from resistant plants. The nucleotide homology of PVY strains to the transgene was: WP (99.5%) PVY-NTN (96.3), PVY-H (95.6%), PVY-O (88.9%), strain 52 (88.3%), and local field isolates from tomato (86.8%), and pepper (86.3%). A transgenic tobacco line was immune to the five PVY strains with which the transgene had the greatest homology (WP, NTN, H, O, 52). In
