Cynomolgus monkey testicular cDNAs for discovery of novel human genes in the human genome sequence

Progress in genome biology has revealed the complete genome sequences of many non-mammalian species, such as yeast, nematodes, and the fruit fly. In addition, the much larger and more complicated genome sequences of the mouse and the human will soon be made completely available. However, decoding the genome sequences, especially the human sequence will be a long process. In order to achieve a comprehensive understanding of how an organism is established by its genome sequence, we must identify the structure, function, and interaction of as many genes as possible. First, we should accumulate and compile many types of evidence from computational and empirical data. The immediate challenge is establishing a complete map of transcribed regions in the human genome. Current comprehensive studies predicting protein-coding genes from the human genome [1, 2] mainly employ three sources of information: empirical evidence provided by expressed sequence tags (ESTs) and cDNAs, nucleotide and protein sequence similarity to those of known genes, and statistical probability calculated by computer algorithms (ab initio prediction). All of these sources more or less lead to false-positive or false-negative types of errors. EST and cDNA sequences usually contain sequences that are not actually transcribed in vivo, i.e. artifacts arising from splicing intermediates, genomic DNA contamination, and transcription from nongenic regions [3, 4]. Moreover, rarely expressed genes that may represent only a small portion of all transcripts cannot be easily represented in cDNA libraries. Predictions based on nucleotide and protein sequence similarities to those of other gene families and organisms might misassign pseudo genes, and cannot identify evolutionarily diverged genes that have no sequence similarity to known genes. Ab initio prediction works well for some organisms, such as yeast, nematodes, and the fruit fly. However, the human genome makes ab initio prediction of protein-coding genes difficult because it generally consists of small exons separated by long introns. Ultimately, in order to make a complete catalog of human genes, it will be necessary to gather undiscovered evidence from experiments and discard spurious evidence.

Our strategy for finding novel genes is to perform cDNA analysis using an organism closely related to humans, the cynomolgus monkey (Macaca fascicularis). In previous studies, we accumulated a number of 5'-end sequences of many clones derived from the oligo-capped cDNA libraries of the brain with high mRNA complexity, and determined approximately 1,500 full insert sequences of the clones whose 5'-end sequences showed no significant similarity to sequences in the public databases [5, 6]. This method allowed us to identify many novel transcripts in the human genome sequence. Using fresh cynomolgus monkey tissues makes it possible to isolate rarely expressed genes, because mRNAs are so fragile that considerable portions of them degenerate during the usual construction of a cDNA library for humans. As an advantage of using cynomolgus monkey, evolutionary inspection can also provide information on gene function. If there are genes that evolved rapidly after the divergence of humans and cynomolgus monkeys, the function of the proteins and parts of the proteins might be important for human evolution. Moreover, biomedical interest in nonhuman primate genomes has been increased rapidly [7], especially in macaques, which also have been a material as transgenic primates [8], and thus genomic analysis of macaques will be important after the completion of human genome sequencing. In this study, we analyzed the cDNA library of the cynomolgus monkey testis. Analysis of testicular cDNA libraries has high potential for finding novel genes [9, 10], because the testis is an organ in which transcripts have high complexity and where important biological processes, such as cell differentiation and meiosis, occur. The genes expressed in the testis are also important for medical, evolutionary, and developmental research. It is ironic that one of the most attractive tissues for biology expresses a number of undiscovered genes. We anticipated that analysis of the testicular cDNA library would lead to the discovery of novel genes that would facilitate post-genomic studies to attempt to unravel the complex genomes of higher organisms. Further, we conducted an expression analysis of our full-sequenced cDNAs with cDNA microarray. DNA microarrays are a versatile tool for evaluating gene expression and sequence variation [11]. We used a cDNA microarray, to determine whether our putative genes were actually transcribed in cynomolgus monkey tissues and whether they were expressed dominantly in the cynomolgus monkey testis.

In this study we analyzed a cDNA library derived from a cynomolgus monkey testis. Although most of the human genome sequence has been determined, many unidentified genes remain, and a complete catalog of protein-coding genes is desired. Sequence similarity search of our full-sequenced cDNAs to the human draft genome sequence resulted in the assignment of 347 cDNA sequences to at least one human chromosome, indicating that most genes in the cynomolgus monkey have homologous regions in the human genome. The primary objective of this analysis was to find genes that have not been experimentally identified in the human genome. Among the 302 cDNAs carrying enough length of ORFs ( = 300 bp), we succeeded in identifying 89 putative genes that have no counterparts in the Ensembl 29,076-gene set. Another 89 genes that had highly similar sequences to Ensembl 'novel' genes were discovered in our full-sequenced cDNAs. The latter 89 genes strongly support the existence of predicted 'novel' cDNA sequences, which are relatively less accurate.

Many genes expressed in the testis cause male infertility in humans [22]. Since it is estimated that up to 11% of all genes in the fruit fly might lead to male sterility [23], in view of the complexity of the human genome, at least 4000 genes might be responsible for male infertility in humans and there must be many as yet unidentified genes that are related to male fertility. Functional analysis of 75 genes found to be highly expressed in the cynomolgus monkey testis may contribute such a medical interest about male infertility. A DNA microarray analysis is an appropriate method not only of annotating the pattern of expression of our full-length cDNAs, but of demonstrating that our strategy for finding novel gene works well. In the first set of the DNA microarray experiment, among the 199 genes that displayed two fold or more higher expression with the testicular probes than with the mixed probes, 67 (34%) were classified as the Ensembl 'known' genes, whereas among the 45 genes that showed ubiquitous pattern of expression (signal intensities within 1.5 fold of each other with both probes), 23 (51%) were classified as Ensembl 'known' genes. This finding indicated that the probability that transcripts overexpressed in testis are derived from unidentified novel genes is significantly higher than that of ubiquitous transcripts (p = 0.028: Fisher's exact test).

Evolutionary inspection is also important, especially for gene analysis of the testis, because genetic diversity in the male reproductive system is quite large, even among closely related species. Many reproductive proteins have evolved rapidly at the molecular level [24, 25]. We compared 117 sequences of cynomolgus monkey cDNA and the corresponding human genome sequences described above, and use of the cDNA microarray revealed that 79 of the 117 cDNAs were overexpressed in testis ( = 2.0 fold in testis) and 15 were ubiquitously expressed (within 1.5 fold of each other). We estimated the sequence divergence of putative coding sequences between humans and cynomolgus monkeys and found that the average non-synonymous nucleotide divergence of testis-dominantly expressed genes (0.024) was significantly greater than that of ubiquitously expressed genes (0.012; p value < 0.01), whereas divergence in synonymous sites were not different significantly (testis-dominant genes: 0.54, ubiquitous genes: 0.51). This finding is also highly consistent with a report that the proteins of genes expressed in a tissue-specific manner evolve an average of twice as fast as those that are ubiquitously expressed [26].

Although a number of full and partial sequences of human genes have been deposited in the public databases, many of the genes in the human genome have not yet to be discovered experimentally. Most of the undiscovered genes may be expressed very seldom or their expression may be restricted to certain tissues and developmental stages. The complete human genome will be available in 2003, and a search of the entire genome for novel genes by oligonucleotide-based microarray analysis is designed; i.e. an attempt to predict all candidate human genes from the human genome and experimentally confirm the transcript status of the predicted regions as well as the entire region by using a oligo-nucleotide-based microarray [27, 28]. However, it is difficult to overcome the problem of rarely or temporarily expressed genes for practical reasons. The transcriptional and genomic approaches will compensate for each other's blind spots, and many tissues, developmental stages, and other organisms should become experimental subjects for finding undiscovered genes to complete the human gene catalog.