GFScan: A Gene Family Search Tool at Genomic DNA Level

Neurotransmitter-Gated Ion-Channels (NGIC) Family

The human neurotransmitter-gated ion-channels family is a large family, whose members include GABA (gamma-aminobutyricacid) A receptors, glycine receptors, acetylcholine receptors, and 5-hydroxytryptamine-3 receptor. All members of the family have a common protein motif, called NEUROTR_ION_CHANNEL in the PROSITE database (ID: PS00236). Using the known 37 human genes of this family in the public database and the protein motif in PROSITE, a 45-bp intronless genomic DNA motif was constructed. We also found that one family member, CHRNB1, has an intron in the motif-matching genomic region, and the intron separates the 45-bp motif into two parts. An intron-containing genomic DNA motif was then constructed (see Methods). Both genomic DNA motifs were used to search the whole human genome. Of 37 known motif regions, 29 were found by GFScan. For the missed eight genes, all the genomic regions corresponding to the motifs fell into the gaps of the genome. Moreover, nine additional genomic regions were found. Three of them were duplications of the known genes. Among the remaining six novel genomic regions, one is located in the repeat region, and the other five were likely to be members of this gene family that are previously unidentified. Based on the human genome annotation in GoldenPath (http://genome.ucsc.edu/), these five regions were reported to be similar to mouse glycine receptor subunit α1, rat GABA A receptor subunit γ1, rat ρ3, mouse GABA A receptor subunit α3, and Gallus nicotinic acetylcholine subunit α8, respectively. With the exception of GABA A receptor α3, no mRNA or protein sequence has been known for the other four genes (see Table1).

Carbonic Anhydrases (CA) Family

Human carbonic anhydrases (CA) are zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide. There are 14 known members in the family. From the mRNAs of the known members, we first constructed a 57-bp cDNA motif based on the PROSITE protein motif (ID: PS00162). All of the genomic sequence regions corresponding to this cDNA motif contain one intron. The splice locations of the introns are identical among all members, but the lengths of the introns are different. We next constructed a genomic DNA motif from the cDNA motif incorporating information on the intron. By searching the whole human genome with the genomic DNA motif, 12 of 14 known genes were found, and the two genes that were missed had their motif-matching genomic region falling into the genomic gaps. Moreover, we found two additional genomic regions that match the motif: One was related to a non-CA family gene, PTPRG (protein tyrosine phosphatase, receptor type G) in Chromosome 3; the other was found in Chromosome 8, whose closest homologous gene was the mouse Car13 gene. It is worth noticing that the human CA13 gene has not been found before, and our finding may have shed light on this new member of the family (see Table 2).

Dbl Homology (DH) Domain Family

The Dbl homology (DH) domain is responsible for the guanine nucleotide exchange factor (GEF) catalytic activity (Zhu et al. 2001). Eight human genes belong to this family, and some of these genes are oncogenes, including DBL, Break Cluster Region(BCR) oncogene, VAV, VAV2, andVAV3. The protein sequences of all eight members share the DH domain (PROSITE ID: PS00741). From their mRNA sequences, a 78-bp cDNA motif was constructed. In the genomic regions corresponding to the motif, no intron was found for one of the family members,TIAM; two introns were found for ABR andBCR; and one intron was found for the remaining five members of the family. Based on above information on gene structure, we next constructed three genomic DNA motifs of this domain from the cDNA motif. Searching the whole human genome with the genomic DNA motifs revealed nine genomic regions that significantly match the motifs. Among the nine regions, seven contain known genes, one of the two new locations was the VAV gene's genomic DNA sequence duplication, and the other overlapped with the known VAV2's motif region (see Table 3). VAV3was the only known member of the family that was missed by the search, and this is because the genomic region matching the motif region was not available in the April 2001 Goldenpath freeze (it was found in the August 2001 freeze).

ETS-Domain Family

The ETS-domain gene family includes a group of proteins that function as transcription factors under physiologic conditions and, if aberrantly expressed, can cause cellular transformation (Karim et al. 1990). These proteins share a conserved domain, the ETS domain, which is involved in DNA binding. From the mRNAs of the 19 known members and a protein motif in the PROSITE database (ID: PS00346), a 48-bp cDNA motif was constructed. Four of these 19 genes have one intron in their genomic regions matching the motif, and the splice location of the intron is the same. Therefore, we constructed an intron-containing genomic DNA motif, and it is used to search the human genome together with the cDNA motif. Twenty-six genomic regions were found to match the motifs, which include 18 of the 19 known genes. ETV5's genomic DNA motif region was missed because the genomic DNA sequence around the motif-matching region was uncompleted. Out of the eight additional motif-matching regions, three were duplications of three known genes (i.e., GABP, ETV6, and ERF). The other five were related to unknown genes in human: one was in the FEVgene region, two were similar to mouse Ets-protein Spi-C (GenBank accession no. AF098863), and the last two were located in two genes predicted by Genscan and Ensembl. Both FEV and Spi-C are ETS-domain family members (Bemark et al. 1999). FEV was not listed in the PROSITE database because of the database-updating problem, and human Spi-C has not been found. Likely, these new motif-matching regions will provide experimental scientists with useful guidance to identify new members of the ETS-domain family in the human genome (see Table 4).

Comparison with the BLAST Results

The other common method to search for new members of a gene family is to run the BLAST program against the whole genome using known members' sequences as queries. We compared BLASTand GFScan on all four families. We searched the protein sequence of each known member of a given family in human genome usingTBLASTN. We also used the motif region of the mRNA sequence of each known member to search the human genome usingBLASTN. The results are listed in Table5.

Table 5 indicates that GFScan had less false positives than TBLASTN (except for the CA family under a lowE-value threshold, but the false positives ofTBLASTN were increased when the E-value threshold was increased). In the BLASTN search, even with a very high E-value threshold (e.g., E = 10), some known genes were still not found, especially the ones whose motifs contain introns. For those genes, the match of the motif region to the genomic sequence is rather poor. Meanwhile, very few new genomic regions were found in this case. In short, compared with BLAST,GFScan offers both higher sensitivity and higher specificity, especially in intron-containing cases.

Mouse Genome Searching with Two Human DNA Motifs

We searched Celera's mouse genome using the motif constructed from human genes. For the neurotransmitter-gated ion-channels family, 23 of 24 known mouse members in the NCBI LocusLink Database (http://www.ncbi.nlm.nih.gov/LocusLink/) were found byGFScan. For the one that was missed (NM_017369: 1824–1868), the genomic DNA sequence of this gene was incomplete in the database. At the same time, 13 new motif-matching genomic locations were found, which may correspond to 13 novel mouse members of this family.

The result was different for the CA family. For 13 known mouse CA members in the LocusLink Database, 11 had the genomic DNA sequence matches. Using GFScan and the motif constructed by human genes, we could only find five loci. The reason for missing the other six was that the motif segments in these mouse genes are different from the motif in human genes (Fig.1). Three of these six genes cannot even match the motif in human (NM_030558, mouse Car15; NM_009802, mouse Car6; NM_007608, mouse Car5a) at the protein level. However, two new genomic locations matching the human motif were still found, which may correspond to novel members in mouse.

Figure 1.

The mRNA regions of 11 members of the mouse CA family identified by searching the Celera Mouse genome using the motif constructed based on CA family members in human. The first five lines were regions found byGFScan as they matched the human motif in the middle lines (R = A or G, Y = T or C, K = G or T, M = A or C, S = G or C, W = A or T, B = G or T or C, D = G or A or T, H = A or C or T, V = G or C or A, N = A or T or G or C). The bottom six lines show the other regions of the gene family that were missed byGFScan. The unmatched sites are in bold fonts. The underlined lower-case triplet represents the amino acid code that did not even match the protein motif of this family.

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In summary, GFScan is capable of identifying all the true members of a family with very few false positives and requiring no gene prediction. It performs especially well with intron-containing motifs where most BLAST-based tools may fail. One should be cautioned when using GFScan for cross-species search, however, as the results may depend on the divergence among members of the family, as well as the evolutionary distance between the two species. By adding more mRNAs from different species or modifying a genomic motif to allow species-specific codon usages, further improvement on performance can be achieved. GFScan is implemented in a way that such customizations can be easily made (see Methods for more detail).

Same Species versus Cross-Species

As DNA sequences are usually less conserved than protein sequences in evolution, we recommend constructing motifs using known mRNAs in one species and then using the motif to search the genome of the same species. This will reduce false positives. For cross-species searches, this method sometimes worked well, as in neurotransmitter-gated ion-channels family; at other times it missed many true positives, as in the case of the CA family described above. As the program allows users to reconstruct motifs by adding more mRNAs from other species, it is easy to extend the search to the cross-species cases. One could also redefine the motif by relaxing on codon usage when searching related species or adding other conserved information into the motif.

Regular Expression Pattern Search and Weight Matrix Search

From the mRNA sequences and protein motifs of the known members of a given gene family, we could construct both a regular expression pattern and weight matrix for later searching. GFScan can use either of them to search the genomic DNA. Based on the matrix constructed, the scores of all known motif regions were calculated. When we chose the minimum score of the known motif regions as the threshold of matrix search to minimize false positives, we found that the genomic locations whose scores were higher than the threshold could all be found by a regular expression pattern search (Table6), whereas the latter saved a lot of CPU time, because searching with regular expressions is almost 15–20 times faster than searching with matrices. However, because matrix search has higher sensitivity (at the expense of specificity and CPU time), the genomic locations missed by a regular expression pattern search may be recovered by a matrix search, especially in the cross-species cases.

Motifs

In the present program, the motif length is taken as a constant; in other words, all the motif regions in the family should have the same length. For those families whose protein motifs have variable lengths, it is difficult to construct the DNA motif, and allowing gaps in the motif can be very CPU-expensive. We will address these issues in future work.

Although GFScan constructs the genomic motif automatically, it also accepts user-defined motifs as its input. This makes GFScan a very flexible tool for gene family analysis at the genomic level. In conjunction with gene prediction tools, it can be used for gene finding and gene structure prediction as well.

Availability

The program GFScan is available athttp://www.cshl.org/mzhanglab/.