Partial characterization of normal and Haemophilus influenzae-infected mucosal complementary DNA libraries in chinchilla middle ear mucosa

Abstract

Objectives: We sought to construct and partially characterize complementary DNA (cDNA) libraries prepared from the middle ear mucosa (MEM) of chinchillas to better understand pathogenic aspects of infection and inflammation, particularly with respect to leukotriene biogenesis and response.

Methods: Chinchilla MEM was harvested from controls and after middle ear inoculation with nontypeable Haemophilus influenzae. RNA was extracted to generate cDNA libraries. Randomly selected clones were subjected to sequence analysis to characterize the libraries and to provide DNA sequence for phylogenetic analyses. Reverse transcription-polymerase chain reaction of the RNA pools was used to generate cDNA sequences corresponding to genes associated with leukotriene biosynthesis and metabolism.

Results: Sequence analysis of 921 randomly selected clones from the uninfected MEM cDNA library produced approximately 250,000 nucleotides of almost entirely novel sequence data. Searches of the GenBank database with the Basic Local Alignment Search Tool provided for identification of 515 unique genes expressed in the MEM and not previously described in chinchillas. In almost all cases, the chinchilla cDNA sequences displayed much greater homology to human or other primate genes than with rodent species. Genes associated with leukotriene metabolism were present in both normal and infected MEM.

Conclusions: Based on both phylogenetic comparisons and gene expression similarities with humans, chinchilla MEM appears to be an excellent model for the study of middle ear inflammation and infection. The higher degree of sequence similarity between chinchillas and humans compared to chinchillas and rodents was unexpected. The cDNA libraries from normal and infected chinchilla MEM will serve as useful molecular tools in the study of otitis media and should yield important information with respect to middle ear pathogenesis.

Fig 1

Insert length of randomly selected clones from uninfected cDNA library. MW — molecular weight markers; Ch-MEM — chinchilla middle ear mucosa.

Fig 2

Images of agarose gels (1.5%) stained with EtBr. Expression of cysteine leukotriene (CysLT1R) and von Willebrand factor (vWF) genes in A) normal chinchilla middle ear mucosa (MEM) and B) Haemophilus influenzae–infected MEM. –T — no template RNA/DNA added to polymerase chain reaction (PCR); –RT — PCR performed without prior reverse transcriptase step in presence of template RNA; +T — template RNA plus reverse transcription (RT) before PCR. +T reaction using CysLT1R-specific primers from both normal and infected MEM yield predicted 211 bp PCR product, and +T reactions for vWF gene–specific primers yielded expected 266 bp PCR product.

Fig 3

RT-PCR expression of CysLT2r, LTA4 hydrolase, BLT1R, and LTC4 synthase genes.

Fig 4

Partial cDNA sequence of CysLT1R gene (GenBank accession No. AY462138).

Fig 5

Partial cDNA sequence of CysLT2R gene (GenBank accession No. AY462139).

Fig 6

Complete cDNA sequence of chinchilla LTA4 hydrolase gene (GenBank accession No. AY462137).

Fig 7

Phylogenetic trees demonstrate that chinchilla CysLT1R, CysLT2R, and LTA4 hydrolase genes have generally greater similarity to humans than other rodents.

Fig 8

Production of leukotrienes from arachidonic acid.,