Gene expression analysis in a murine model of allergic asthma reveals overlapping disease and therapy dependent pathways in the lung.

Accumulating evidence in animal models and human asthma support a central role for IL-13 signaling in disease pathogenesis. In order to identify asthma and therapy associated genes, global transcriptional changes were monitored in mouse lung following antigen challenge (ovalbumin (OVA)), either alone or in the presence of a soluble IL-13 antagonist. Changes in whole lung gene expression after instillation of mIL-13 were also measured both in wild type and STAT6 deficient mice. A striking overlap in the gene expression profiles induced by either OVA challenge or mIL-13 was observed, further strengthening the relationship of IL-13 signaling to asthma. Consistent with results from functional studies, a subset of the OVA-induced gene expression was significantly inhibited by a soluble IL-13 antagonist while IL-13-modulated gene expression was completely attenuated in the absence of STAT6-mediated signaling. Results from these experiments greatly expand our understanding of asthma and provide novel molecular targets for therapy and potential biomarkers of IL-13 antagonism.

Oligonucleotide arrays for expression monitoring.

This unit provides protocols for the amplification and labeling of mRNA (and the necessary controls) for hybridization to oligonucleotide arrays. It also describes methods for processing and normalizing the raw gene expression data in preparation for clustering and further analysis.

Preparation of mRNA for expression monitoring.

The ability to construct comprehensive gene expression profiles comprising hundreds to thousands of genes whose RNA levels are monitored simultaneously represents an exciting new capability in molecular biology. This is accomplished by hybridizing mRNA, which has been quantitatively amplified and labeled with biotin, to DNA chips that display thousands of nucleotides complementary to the mRNAs of interest. In this unit, rationale for starting with poly(A(+)) versus total RNA is discussed, and strategies for choosing oligonucleotides for chip design is presented. Protocols on RNA amplification and labeling, and purifying and quantifying the cDNA and in vitro transcription products are included.

Preparation of mRNA for expression monitoring.

The ability to construct comprehensive gene expression profiles comprising hundreds to thousands of genes whose RNA levels are monitored simultaneously represents an exciting new capability in molecular biology. This is accomplished by hybridizing mRNA, which has been quantitatively amplified and labeled with biotin, to DNA chips that display thousands of nucleotides complementary to the mRNAs of interest. In this unit, rationale for starting with poly(A(+)) vs. total RNA is discussed, and strategies for choosing oligonucleotides for chip design is presented. Protocols on RNA amplification and labeling, and purifying and quantifying the cDNA and in vitro transcription products are included.

Expression monitoring by hybridization to high-density oligonucleotide arrays.

The human genome encodes approximately 100,000 different genes, and at least partial sequence information for nearly all will be available soon. Sequence information alone, however, is insufficient for a full understanding of gene function, expression, regulation, and splice-site variation. Because cellular processes are governed by the repertoire of expressed genes, and the levels and timing of expression, it is important to have experimental tools for the direct monitoring of large numbers of mRNAs in parallel. We have developed an approach that is based on hybridization to small, high-density arrays containing tens of thousands of synthetic oligonucleotides. The arrays are designed based on sequence information alone and are synthesized in situ using a combination of photolithography and oligonucleotide chemistry. RNAs present at a frequency of 1:300,000 are unambiguously detected, and detection is quantitative over more than three orders of magnitude. This approach provides a way to use directly the growing body of sequence information for highly parallel experimental investigations. Because of the combinatorial nature of the chemistry and the ability to synthesize small arrays containing hundreds of thousands of specifically chosen oligonucleotides, the method is readily scalable to the simultaneous monitoring of tens of thousands of genes.

Histidine patch thioredoxins. Mutant forms of thioredoxin with metal chelating affinity that provide for convenient purifications of thioredoxin fusion proteins.

A cluster of surface amino acid residues on Escherichia coli thioredoxin were systematically mutated in order to provide the molecule with an ability to chelate metal ions. The combined effect of two histidine mutants, E30H and Q62H, gave thioredoxin the capacity to bind to nickel ions immobilized on iminodiacetic acid- and nitrilotriacetic acid-Sepharose resins. Even though these two histidines were more than 30 residues apart in thioredoxin's primary sequence, they were found to satisfy the geometric constraints for metal ion coordination as a result of the thioredoxin tertiary fold. A third histidine mutation, S1H, provided additional metal ion chelation affinity, but the native histidine at position 6 of thioredoxin was found not to participate in binding. All of the histidine mutants exhibited decreased thermal stability as compared with wild-type thioredoxin; however, the introduction of an additional mutation, D26A, increased their melting temperatures beyond that of wild-type thioredoxin. The metal chelating abilities of these histidine mutants of thioredoxin were successfully utilized for convenient purifications of human interleukin-8 and -11 expressed in E. coli as soluble thioredoxin fusion proteins.

Effect of different levels of aspartokinase on the lysine production by Corynebacterium lactofermentum.

A 2.9-kb SacI fragment containing the ask-asd operon, encoding aspartokinase and aspartatesemialdehyde dehydrogenase, was cloned from an aminoethylcysteine-resistant, lysine-producing Corynebacterium lactofermentum strain. Enzymatic analysis showed that the aspartokinase (ASK) activity was completely resistant to inhibition by mixtures of lysine and threonine. Comparison of the deduced amino acid sequence of the beta submit of the ask gene showed three amino acid residue changes with ask gene encoding wild-type, feedback-sensitive enzymes. Three C. lactofermentum strains, one being aspartokinase-negative, one carrying two ask genes on the chromosome and one having a sixfold higher specific ASK activity than the parental strain, were constructed by transconjugation and electroporation, and used to analyse the role of ASK in the lysine production by C. lactofermentum. The results indicate that, in this study, feed-back-resistant ASK is necessary for high-level lysine production, but dispensable for lysine and diaminopimelate synthesis required for cell growth.

Effect of inducible thrB expression on amino acid production in Corynebacterium lactofermentum ATCC 21799.

Amplification of the operon homdr-thrB encoding a feedback-insensitive homoserine dehydrogenase and a wild-type homoserine kinase in a Corynebacterium lactofermentum lysine-producing strain resulted in both homoserine and threonine accumulation, with some residual lysine production. A plasmid enabling separate transcriptional control of each gene was constructed to determine the effect of various enzyme activity ratios on metabolite accumulation. By increasing the activity of homoserine kinase relative to homoserine dehydrogenase activity, homoserine accumulation in the medium was essentially eliminated and the final threonine titer was increased by about 120%. Furthermore, a fortuitous result of the cloning strategy was an unexplained increase in homoserine dehydrogenase activity. This resulted in a further decrease in lysine production along with a concomitant increase in threonine accumulation.

Gene structure and expression of the Corynebacterium flavum N13 ask-asd operon.

Two promoters required for expression of the ask-asd genes, encoding aspartokinase (AK) and aspartate-semialdehyde dehydrogenase (ASD), in Corynebacterium flavum N13, askP1 and askP2, have been identified by deletion analysis and S1 nuclease mapping. Transcription from askP1 initiates 35 and 38 bp upstream of the ask structural gene. A second promoter, askP2, lies within the ask coding region, upstream of the translation start site of the AK beta subunit and can direct the expression of AK beta and ASD. Western immunoblot analysis and heterologous expression in Escherichia coli demonstrate that two separate polypeptides, a 44.8-kDa alpha subunit and an 18.5-kDa beta subunit, are expressed from the C. flavum N13 ask gene from distinct, in-frame translation initiation sites. A second AK mutation, G345D, which reduces the sensitivity of AK to concerted feedback inhibition by threonine plus lysine, was identified.

The phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: molecular cloning, nucleotide sequence, and expression.

The ppc gene of Corynebacterium glutamicum encoding phosphoenolpyruvate (PEP) carboxylase was isolated by complementation of a ppc mutant of Escherichia coli using a cosmid gene bank of chromosomal C. glutamicum DNA. By subsequent subcloning into the plasmid pUC8 and deletion analysis, the ppc gene could be located on a 3.3 kb SalI fragment. This fragment was able to complement the E. coli ppc mutant and conferred PEP carboxylase activity to the mutant. The complete nucleotide sequence of the ppc gene including 5' and 3' flanking regions has been determined and the primary structure of PEP carboxylase was deduced. The sequence predicts a 919 residue protein product (molecular weight of 103 154) which shows 34% similarity with the respective E. coli enzyme.

Organization and regulation of the Corynebacterium glutamicum hom-thrB and thrC loci.

The genes encoding the three terminal enzymes in the threonine biosynthetic pathway, homoserine dehydrogenase (hom), homoserine kinase (thrB) and threonine synthase (thrC) have been isolated from Corynebacterium glutamicum. The C. glutamicum hom and thrB genes were subcloned on a 3.6 kb SalI-generated chromosomal fragment. The C. glutamicum thrC gene was shown not to be linked to the hom-thrB locus. L-methionine represses the cloned homoserine dehydrogenase and homoserine kinase similar to that of the chromosomally encoded hom and thrB gene products. Northern hybridization analysis demonstrates that this repression is mediated at the level of transcription and that hom-thrB represents an operon in C. glutamicum.

Nucleotide sequence and fine structural analysis of the Corynebacterium glutamicum hom-thrB operon.

The complete nucleotide sequence of the Corynebacterium glutamicum hom-thrB operon has been determined and the structural genes and promoter region mapped. A polypeptide of Mr 46,136 is encoded by hom and a polypeptide of Mr 32,618 is encoded by thrB. Both predicted protein sequences show amino acid sequence homology to their counterparts in Escherichia coli and Bacillus subtilis. The promoter region has been mapped by S1-nuclease and deletion analysis. Located between -88, RNA start site and -219 (smallest deletion clone with complete activity) are sequence elements similar to those found in E. coli and B. subtilis promoters. Although there are no obvious attenuator-like structures in the 5'-untranslated region, there is a dyad-symmetry element, which may act as an operator.

Molecular cloning and nucleotide sequence of the Corynebacterium glutamicum pheA gene.

The pheA gene of Corynebacterium glutamicum encoding prephenate dehydratase was isolated from a gene bank constructed in C. glutamicum. The specific activity of prephenate dehydratase was increased six-fold in strains harboring the cloned gene. Genetic and structural evidence is presented which indicates that prephenate dehydratase and chorismate mutase were catalyzed by separate enzymes in this species. The C. glutamicum pheA gene, subcloned in both orientations with respect to the Escherichia coli vector pUC8, was able to complement an E. coli pheA auxotroph. The nucleotide sequence of the C. glutamicum pheA gene predicts a 315-residue protein product with a molecular weight of 33,740. The deduced protein product demonstrated sequence homology to the C-terminal two-thirds of the bifunctional E. coli enzyme chorismate mutase-P-prephenate dehydratase.

Cloning vector system for Corynebacterium glutamicum.

A protoplast transformation system has been developed for Corynebacterium glutamicum by using a C. glutamicum-Bacillus subtilis chimeric vector. The chimera was constructed by joining a 3.0-kilobase cryptic C. glutamicum plasmid and the B. subtilis plasmid pBD10. The neomycin resistance gene on the chimera, pHY416, was expressed in C. glutamicum, although the chloramphenicol resistance gene was not. The various parameters in the transformation protocol were analyzed separately and optimized. The resulting transformation system is simple and routinely yields 10(4) transformants per microgram of plasmid DNA.