Directed evolution of ionizing radiation resistance in Escherichia coli.

We have generated extreme ionizing radiation resistance in a relatively sensitive bacterial species, Escherichia coli, by directed evolution. Four populations of Escherichia coli K-12 were derived independently from strain MG1655, with each specifically adapted to survive exposure to high doses of ionizing radiation. D(37) values for strains isolated from two of the populations approached that exhibited by Deinococcus radiodurans. Complete genomic sequencing was carried out on nine purified strains derived from these populations. Clear mutational patterns were observed that both pointed to key underlying mechanisms and guided further characterization of the strains. In these evolved populations, passive genomic protection is not in evidence. Instead, enhanced recombinational DNA repair makes a prominent but probably not exclusive contribution to genome reconstitution. Multiple genes, multiple alleles of some genes, multiple mechanisms, and multiple evolutionary pathways all play a role in the evolutionary acquisition of extreme radiation resistance. Several mutations in the recA gene and a deletion of the e14 prophage both demonstrably contribute to and partially explain the new phenotype. Mutations in additional components of the bacterial recombinational repair system and the replication restart primosome are also prominent, as are mutations in genes involved in cell division, protein turnover, and glutamate transport. At least some evolutionary pathways to extreme radiation resistance are constrained by the temporally ordered appearance of specific alleles.

SNP-specific array-based allele-specific expression analysis.

We have developed an optimized array-based approach for customizable allele-specific gene expression (ASE) analysis. The central features of the approach are the ability to select SNPs at will for detection, and the absence of need to PCR amplify the target. A surprisingly long probe length (39-49 nt) was needed for allelic discrimination. Reconstitution experiments demonstrate linearity of ASE over a broad range. Using this approach, we have discovered at least two novel imprinted genes, NLRP2, which encodes a member of the inflammasome, and OSBPL1A, which encodes a presumed oxysterol-binding protein, were both preferentially expressed from the maternal allele. In contrast, ERAP2, which encodes an aminopeptidase, did not show preferential parent-of-origin expression, but rather, cis-acting nonimprinted differential allelic control. The approach is scalable to the whole genome and can be used for discovery of functional epigenetic modifications in patient samples.

Genome-wide in situ exon capture for selective resequencing.

Increasingly powerful sequencing technologies are ushering in an era of personal genome sequences and raising the possibility of using such information to guide medical decisions. Genome resequencing also promises to accelerate the identification of disease-associated mutations. Roughly 98% of the human genome is composed of repeats and intergenic or non-protein-coding sequences. Thus, it is crucial to focus resequencing on high-value genomic regions. Protein-coding exons represent one such type of high-value target. We have developed a method of using flexible, high-density microarrays to capture any desired fraction of the human genome, in this case corresponding to more than 200,000 protein-coding exons. Depending on the precise protocol, up to 55-85% of the captured fragments are associated with targeted regions and up to 98% of intended exons can be recovered. This methodology provides an adaptable route toward rapid and efficient resequencing of any sizeable, non-repeat portion of the human genome.

Biochemical limitations to high-level expression of humanized monoclonal antibodies in transgenic maize seed endosperm.

Transgenic plants are potentially valuable systems for the large scale manufacture of therapeutic proteins. To improve this technology, determining the importance of transgene transcript levels on protein accumulation in sink tissues during their development is crucial. In transgenic maize (Zea mays L.) plants expressing humanized monoclonal antibodies (mAbs) in their seed endosperm, steady-state kappa light chain (LC) and gamma heavy chain (HC) mRNA levels were quantified during development and compared to the levels of fully-assembled mAb protein present at seed maturity. RNA blots and non-reducing SDS-PAGE western immunoblots revealed that steady-state LC and HC mRNA and protein levels were undetectable at 10 days after pollination (DAP) but increased quickly thereafter in three transgenic events expressing different mAb molecules. Similar to gamma-zein mRNA, LC and HC messages were highly abundant between 15 and 25 DAP. Quantitative RNA blots and western immunoblots showed that steady-state LC transcript levels during development correlated extremely closely with protein levels in mature seed (r(2)=0.99). For HC, this correlation was not as strong (r(2)=0.85). Consistent with this finding, concomitantly increasing the zygosity levels of the LC and HC transgenes enhanced mAb concentration in mature seed, in contrast to increasing the copy number of the transgene insert, which did not correlate with high seed mAb levels. The results indicate that high-level expression of fully-assembled mAb protein in maize endosperm was favored by high LC and HC mRNA levels and was largely limited by HC protein concentration.