Analysis of 10,000 ESTs from lymphocytes of the cynomolgus monkey to improve our understanding of its immune system

Abstract

Background: The cynomolgus monkey (Macaca fascicularis) is one of the most widely used surrogate animal models for an increasing number of human diseases and vaccines, especially immune-system-related ones. Towards a better understanding of the gene expression background upon its immunogenetics, we constructed a cDNA library from Epstein-Barr virus (EBV)-transformed B lymphocytes of a cynomolgus monkey and sequenced 10,000 randomly picked clones.

Results: After processing, 8,312 high-quality expressed sequence tags (ESTs) were generated and assembled into 3,728 unigenes. Annotations of these uniquely expressed transcripts demonstrated that out of the 2,524 open reading frame (ORF) positive unigenes (mitochondrial and ribosomal sequences were not included), 98.8% shared significant similarities (E-value less than 1e-10) with the NCBI nucleotide (nt) database, while only 67.7% (E-value less than 1e-5) did so with the NCBI non-redundant protein (nr) database. Further analysis revealed that 90.0% of the unigenes that shared no similarities to the nr database could be assigned to human chromosomes, in which 75 did not match significantly to any cynomolgus monkey and human ESTs. The mapping regions to known human genes on the human genome were described in detail. The protein family and domain analysis revealed that the first, second and fourth of the most abundantly expressed protein families were all assigned to immunoglobulin and major histocompatibility complex (MHC)-related proteins. The expression profiles of these genes were compared with that of homologous genes in human blood, lymph nodes and a RAMOS cell line, which demonstrated expression changes after transformation with EBV. The degree of sequence similarity of the MHC class I and II genes to the human reference sequences was evaluated. The results indicated that class I molecules showed weak amino acid identities (<90%), while class II showed slightly higher ones.

Conclusion: These results indicated that the genes expressed in the cynomolgus monkey could be used to identify novel protein-coding genes and revise those incomplete or incorrect annotations in the human genome by comparative methods, since the old world monkeys and humans share high similarities at the molecular level, especially within coding regions. The identification of multiple genes involved in the immune response, their sequence variations to the human homologues, and their responses to EBV infection could provide useful information to improve our understanding of the cynomolgus monkey immune system.

Figure 1

Statistics of ESTs obtained from the cynomolgus monkey cDNA library. The length distributions of initial ESTs (A) and putative ORFs of unigenes after assembling (B).

Figure 2

Comparison of results of gene-oriented clustering (blast human mRNA) and non-gene-oriented clustering (direct phrap) clustering. The two clustering methods produced similar numbers of EST clusters, the distributions of the two sets of EST clusters were also similar.

Figure 3

The Gene Ontology (GO) categories of genes from the cynomolgus monkey lymphocyte cDNA library (CYLA) and the RAMOS cell line (RAMOS). The genes were functionally categorized according to the Gene Ontology Consortium and level two of the assignment results were plotted here. In this ontology, "biological process", "cellular component" and "molecular function" are categorized independently. 57% (2,124 of total 3,728 unigenes) unigenes from the cynomolgus monkey cDNA library and 63.9% unigenes from the RAMOS cell line were classified by GO.

Figure 4

The level 3 GO classification of genes from the cynomolgus monkey lymphocyte cDNA library (CYLA) and the RAMOS cell line (RAMOS). The "biological process" (A) and "molecular function" (B) are shown here.

Figure 5

Comparisons of gene expression profiles of MHC class I, II and III molecules. For the comparison purpose, the expression data of MHC genes in the human blood and lymph node were downloaded from the NCBI UNIGENE database and compared with that of the RAMOS cell line and our cDNA library of cynomolgus monkey B lymphocytes (CYLA). The gene expression abundance was indicated as "transcripts per million" for the comparison convenience.

Figure 6

The gene expression abundance of Clusters of Differentiation of lymphocytes in our cDNA library (CYLA).