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Physiol. Genomics 30: 213-222, 2007. First published April 24, 2007; doi:10.1152/physiolgenomics.00263.2006
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Received 30 November 2006; accepted in final form 16 April 2007.
Physiological Genomics 30:213-222 (2007)
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Call For Papers: Comparative Genomics

Comparative genomic analysis of a mammalian ß-defensin gene cluster

Yashwanth Radhakrishnan1, Mario A. Fares2, Frank S. French1 and Susan H. Hall1

1 Laboratories for Reproductive Biology, Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina
2 Evolutionary Genetics and Bioinformatics Laboratory, Department of Genetics, Smurfit Institute, University of Dublin, Trinity College, Dublin, Ireland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Comparative genomic analyses have yielded valuable insights into conserved and divergent aspects of gene function, regulation, and evolution. Herein, we describe the characterization of a mouse ß-defensin gene cluster locus on chromosome 2F6. In addition, we present the evolutionary analysis of this cluster and its human, rhesus, and rat orthologs. Expression analysis in mouse revealed the occurrence of defensin cluster transcripts in multiple tissues, with the highest abundance in the urogenital tract. Molecular evolutionary analysis suggests that this cluster originated by a series of duplication events, and by positive selection occurring even after the rodent-primate split. In addition, the constraints analysis showed higher positive selection in rodents than in primates, especially distal to the six-cysteine array. Positive selection in the evolution of these defensins may relate not only to the evolving enhancement of ancestral host defense but also to functional innovations in reproduction. The multiplicity of defensins and their preferential overexpression in the urogenital tract indicate that defensins function in the protection and maintenance of fertility.

gene expression; reproduction; immunity; antimicrobial; evolution; gene duplication; positive selection


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
COMPARATIVE GENOMIC ANALYSES across taxonomic groups from viruses to humans have driven considerable progress in gene discovery, functional genomics, and our understanding of the evolutionary forces that sculpt genomic architecture. Comparative genomics provides valuable insights into reproductive and developmental functions and disease defense mechanisms that have protected us from extinction (52). Freely available human, mouse, and rat genomic sequences have stimulated numerous comparative studies (6, 24, 30, 42, 43). These studies develop our understanding of the concept of "unity in diversity," the underlying commonalities shared by evolutionarily distinct genomes. Examples of this unity are seen in the conservation of intron/exon structures in rodent and primate genes and in the similar numbers of genes in these taxa, the majority persisting without deletion or duplication since the last common ancestor. Recent comparative analyses strikingly reveal that genes involved in two major functions, host-pathogen interactions and reproduction, are under positive selection (44a, 54a, 70). Albeit rare, positive Darwinian selection at the molecular level is critical for adaptation and for the "invention" of new functions that are crucial for speciation (32).

Plasticity, an intrinsic property of immune system gene evolution, supports the arms race-like evolutionary dynamics of host-pathogen interactions (69). While the pathogen is under constant pressure to devise new strategies for gaining access to the host, the host is under selection to deny access to the pathogen. Genes involved in reproduction that are under positive selection include the human sperm proteome (68, 70, 72). Competition among spermatozoa from different males results in the rapid divergence of sperm-associated proteins. It is therefore expected that sperm-expressed genes as a group may show greater divergence because of intersexual selective pressures (64, 65).

ß-Defensins are multifunctional [reviewed by Lehrer (35)], belonging to two functional classes evolving under positive selection. They are antimicrobial agents [reviewed by Ganz (15)] and fertility promoters (83, 86). More than 40 ß-defensins identified in humans are organized on three different chromosomes in five syntenic clusters (53, 54, 55, 58, 60). Orthologs of ß-defensins were identified in other vertebrates including rodents, dog, ruminants, and primates by computational predictions and experimental evidence (27–29, 44, 73, 74, 85). ß-Defensins are potent microbicidal agents against a wide variety of pathogens including gram-positive and gram-negative bacteria, fungi, viruses, and parasites (15, 19, 35, 76, 80). In addition, defensins are involved in human disorders including cystic fibrosis (8, 18) and psoriasis (22, 57). Furthermore, ß-defensins bridge innate and adaptive immune systems through chemoattraction of dendritic cells, memory T cells (77), and human neutrophils (51). ß-Defensins are increasingly recognized as mediators of reproductive functions, a model predicted by their expression primarily in the male reproductive tract. DEFB126, a sperm-bound ß-defensin, blocks sperm-zona recognition and binding (67) and, before its release during capacitation (83), protects spermatozoa from immunorecognition and attachment of anti-sperm antibodies (84). The rat ß-defensin-like SPAG11E is involved in motility acquisition during sperm maturation (86).

A major source of new biological functions, gene duplication occurred repeatedly during genomic evolution (39). The rearrangement of genomic segments has also occurred repeatedly during the evolution of eukaryotes (12), giving rise to many gene families distributed among different chromosomes. Alternative mRNA splicing represents yet another distinct evolutionary mechanism that generates new molecular diversity. An inverse correlation between the size of a gene family and its production of alternatively spliced mRNA variants (33) suggests that these represent alternative genetic innovation responses to changing environmental pressures. Which of these two responses predominates may depend on the relative advantages of evolving a variety of expressed sequences regulated through multiple promoters, as in the case of gene duplication, vs. the advantages of novel exonic combinations. All of these mechanisms have contributed to defensin diversity.

With the findings of Hughes and Yeager (26) that positive selection for changes at the amino acid level are driving the evolution of mammalian defensin genes, several hypotheses have emerged to explain structure-function relationships and the evolutionary divergence of these multiclustered genes (4, 5, 11, 40, 44, 48, 53, 54, 59, 60, 73). Previously, we described the epididymal sperm binding protein DEFB118, a potent antibacterial agent and lead member of the ß-defensin gene cluster on human chromosome 20q (38, 54, 81). Here, we report the discovery and characterization of the orthologous cluster in mouse. Furthermore, we present a comparative genomic analysis of the cluster in human, rhesus, mouse, and rat that yields insights into the evolution of their conserved structural and divergent functional properties.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Primate ß-defensin protein sequences derived from chromosome 20q and previously published (54) were used to search (basic local alignment search tool; BLAST) the public databases (2) to identify novel murine orthologous genomic sequences and expressed sequence tags (ESTs). Of the five related sequences identified, four were located in a cluster on chromosome 2 with the fifth on chromosome 14. The rat ß-defensin orthologs were similarly obtained. However, only second exon products were found by this method. To obtain the first exons of Defb21, -27, -30, and -36, upstream sequences were retrieved from the mouse genomic database (http://www.ncbi.nlm.nih.gov/genome/seq). These were translated in silico in three reading frames and analyzed for hydrophobic amino acid groups preceded by a methionine. These regions selected were then scanned for classical exon-intron boundaries. Primers were designed in predicted exons 1 and 2, and with the use of murine epididymis mRNA as a template, RT-PCR was performed. The specific products were sequenced, establishing the actual exons 1 and 2, and the corresponding exon-intron boundaries were assessed by alignment of amplified cDNAs with the genomic sequences. The predicted proteins were found to contain putative signal peptides using SignalP (49). Defb24 was previously investigated as testis-specific ß-defensin like (Tdl) (75) with an alternative name of Defb19 (GenBank accession no. NM_145157), and hence these published sequences were used for primer design and further analyses.

A panel of murine (Mus musculus) tissues was kindly provided by Dr. Peter Petrusz (Dept. of Cell and Developmental Biology, Univ. of North Carolina at Chapel Hill). Total RNA was extracted from these tissues, including brain, esophagus, salivary glands, heart, lungs, liver, spleen, adrenal, kidney, urinary bladder, testis, the epididymis regions caput, corpus and cauda, ovary, oviduct, uterus, cervix, and placenta, using Trizol reagent (Invitrogen, Carlsbad, CA). Total RNA (2 µg) was reverse transcribed using 50 U of Stratascript (Stratagene, La Jolla, CA) and 0.5 µg of oligo(dT) (Invitrogen) according to the supplier's instructions. Gene-specific intron-spanning primer sequences for Defb21, -24, -27, -36, and -30 and glyceraldehyde-3-phosphate dehydrogenase (Gapdh; NM_001001303) were as follows: Defb21, forward (F) 5'-GATGTCTGGATGTTCTACCTCCC and reverse (R) 5'-ACTCCTTCAACTATATTGCCCTAAG; Defb24, F5'-CCACTTACCACCTCCACCCTG and R5'-CTGGAAGTCTGTACACGGTGTG; Defb27, F5'-GATAGACACAAGCCAGCACCCTG and R5'-GGTCCAACTTCATTTCTGTGGCC; Defb36, F5'-GCTTCGCCTTGGGCCTTCTCC and R5'-CCATGGGGTCAACCTAGGACAGC; Defb30, F5'-GATGACTCTCTGCTCACTGGG and R5'-GGGGACAAAGGACTCGAGAGG; and Gapdh, F5'-CCGCATCTTCTTGTGCAGTGCC and R5'-GCCGTGAGTGGAGTCATACTGG.

PCR reactions were carried out using 2 µl of the resultant cDNA according to the following procedures: 94°C for 1 min followed by 24–35 cycles at 94°C for 30 s, 58°C for 30 s and 72°C for 1 min, and a final round of extension for 5 min. Linear amplification range for each gene was determined using the epididymal cDNA. The least expressed transcript, Defb36, required 35 cycles of PCR for detection in the linear range, while the others required 26–32 cycles. Amplification of Gapdh, which served as an internal control in this semiquantitative analysis, was carried out in parallel using similar conditions for 24 cycles. PCR-amplified products were analyzed by electrophoresis on 2% agarose gels. Gel-purified PCR products were sequenced at the University of North Carolina-Chapel Hill Genome Analysis Facility using an ABI PRISM model no. 377 DNA sequencer (PE Applied Biosystems, Foster City, CA) as described previously (20).

All evolutionary analyses were carried out using the sliding window analysis procedure to detect selective constraints (SWAPSC) (13) and molecular evolutionary genetics analysis (MEGA)3 (34). The amino acid sequences were obtained by in silico cDNA translation. The amino acid alignment employing only the expressed defensin genes for human, macaque, mouse, and rat (see Fig. 2) was constructed using the default options in Clustal W, embedded in MEGA3. Human DEFB122 was excluded from analysis because, as a pseudogene, it is not subjected to the same evolutionary pressures as the defensin protein encoding paralogs. The corresponding cDNA alignments were generated based on the protein sequence alignments. Most analyses were based on the nucleotide-aligned sequences.

All phylogenetic trees were constructed using MEGA3 by the neighbor-joining (NJ) method (34, 56), using p-distance estimates, which is considered to be the most reliable method for calculating NJ trees of closely related sequences (66). To infer the evolutionary history, we used substitutions that are not likely to be under adaptive evolutionary forces. For these analyses, only third codon positions were used. Exon 1 of DEFB119 was excluded from analysis, since it also appears in DEFB120. A putative defensin sequence of orange-spotted grouper, reproduction regulator 1, was used to root the trees, and confidence limits were calculated by a bootstrap resampling method (500 replicates) as described previously (54).

To detect the presence of positive selection between nucleotide sequences in pairwise comparisons, the number of synonymous substitutions per synonymous site (dS) and the number of nonsynonymous substitutions per nonsynonymous site (dN) were estimated using the method of Nei and Gojobori (45). Additional analyses were carried out using a modified Nei and Gojobori method in MEGA3, and the Jukes-Cantor correction was applied to account for multiple substitutions at the same site. The selections operating in different regions of the sequences and within different branches of the phylogenetic tree under study were also estimated using SWAPSC with simulated data sets (13). This program uses the differences between the estimated and expected numbers of synonymous and nonsynonymous substitutions and the nonsynonymous-to-synonymous rate ratio ({omega}) to evaluate hypotheses. It seeks evidence for regions that have reached the saturation of synonymous sites and where the number of synonymous substitutions is significantly smaller than expected. Positive selection is inferred where the estimated number of nonsynonymous nucleotide substitutions is greater than expected by chance and where {omega} is significantly greater than 1. Where regions have an estimated number of nonsynonymous substitutions greater than expected but {omega} < 1, or where {omega} > 1 but there is evidence for saturation of synonymous sites, such regions are said to have accelerated rates of nonsynonymous substitutions. In some cases, despite high dN, it might not be considered positive selection, since the dS = 0 where there might be positive selection or, alternatively, relaxed functional or structural constraints in that particular codon and branch (covarion hypothesis). Thus SWAPSC seeks to avoid inferring positive selection where there is insufficient data to support it or where saturation may cause bias.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Prediction of genomic structures of ß-defensins.
Members of a gene family that evolved from a common ancestor as a result of duplication are paralogous, whereas orthologs are genes that diverged from a common ancestor during speciation. To obtain the six mouse and rat orthologs of human chromosome 20q ß-defensins (54), rhesus monkey ortholog protein sequences (accession nos. AY499406–AY499409 and AY500998) were used to query the mouse genomic database, Build 32.1, and the rat genomic database of the Rat Genome Sequencing Consortium, version 3.4 (79), in GenBank. Of the five genes identified, four are located in the distal region of mouse chromosome 2F6 and the fifth on chromosome 14 (Fig. 1). The four genes on chromosome 2, Defb21, -24, -27, and -36, are orthologs of DEFB118, -119, -122, and -123, respectively. The related gene Defb30 is located in a defensin cluster on chromosome 14 and was identified previously by TBLASTN as the ortholog of DEFB121 with 66% identity in the six-cysteine array. However, a more recent study showed that Defb30 and syntenic defensin genes are instead orthologs of a cluster on human chromosome 8p (53). In this 8p cluster, DEFB135 is the human ortholog of Defb30. The mouse DEFB30 protein in the six-cysteine motif is 47% identical to DEFB135 and 43% identical to DEFB121, while DEFB135 and DEFB121 are 51% identical to each other, an unusually close relationship between defensins on different chromosomes.


Figure 1
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  		Fig. 1. Mouse ß-defensin genes located on chromosome 2. An ideogram of chromosome 2 is shown at left; solid triangle indicates the location of the ß-defensin gene cluster at 2F6. The ß-defensin gene names are indicated in black next to the positions of exons that we determined, also in black. The Unigene accession nos. and Unigene-defined exons are indicated at right in blue. Arrows indicate direction of transcription of each gene. This figure is based on mouse genome Build 32.1, available at MapView (http://www.ncbi.nlm.nih.gov/mapview).

 
The human 20q defensin cluster spans 80 kb with a distance between DEFB119 and DEFB122 of 30 kb. In contrast, the mouse cluster evolved a more compact structure encompassing only 41 kb with a distance between Defb24 and Defb27 of only 11 kb. The species differences in these orthologous cluster sizes parallel the size differences of the human and mouse genomes (44a). The size of the human 20q cluster was not increased by additional exon recruitment, as shown by expressed sequence amplifications that revealed the two exon structure of these defensin orthologs in mouse (Fig. 1) to be conserved in humans (54). Instead, this cluster is enlarged by the presence of an additional defensin gene, DEFB121, and the 8p cluster is similarly enlarged by DEFB104 and DEFB108 arising by tandem gene duplication (60). Such gene duplication may be driven by demands for increased biological complexity of the defensin family.

Multiple sequence alignment and isoelectric point.
The alignment of the mouse whole protein sequences with rat, human and monkey reveals conservation of the six-cysteine defensin signature motif (Fig. 2). The mouse paralogous genes are more divergent from one another than from their primate orthologs (data not shown), consistent with the ancient origins of the cluster before rodent-primate divergence. This relationship is also supported by the phylogenetic pattern with orthologs of mouse and primate clustered to the exclusion of mouse paralogs. Branch lengths and distance between mouse paralogs also suggest that no long-branch attraction (LBA) existed that could lead to an artifactual phylogeny. LBA is one of the most important factors hampering inference of phylogenetic trees, particularly when both orthologous and paralogous sequences are included in the analysis (7). Figure 2 reveals the high level of similarity among the predicted signal peptide amino acid sequences encoded by the first exons and the lower level of similarity of the mature peptides encoded by the second exons. In these comparisons, the degree of similarity declines only after the second cysteine in the mature peptide, in contrast to the defensin clusters on human chromosomes 8p and 20p where the dissimilarity is very high for the entire exon 2 (55, 60). Specific positions are highly conserved including the amino acid after the first cysteine, which is either methionine or tryptophan, in both primates and rodents except for rodent DEFB21. The glycine before the second cysteine and the glutamic acid between the third and fourth cysteines align in mouse, rat, rhesus, and human proteins as well as in the Epinephelus coioides (orange-spotted grouper) putative defensin sequence, suggesting a role in a function common to this ß-defensin lineage for over 450 million years (MYR) (54). The aligned proteins are all cationic with the sole exception of the monkey and human DEFB118s, which have long COOH-terminal anionic extensions. If this COOH-terminal extension is removed as a propiece as previously described (54), the remaining mature peptide is cationic. Interestingly, in the mouse and rat orthologs, DEFB21 proteins evolved differently to have shorter anionic extensions resulting in overall basic calculated isoelectric points (pIs).


Figure 2
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  		Fig. 2. Multiple sequence alignment of human, macaque, mouse, and rat ß-defensin protein sequences. The amino acids 100% conserved in this group are shaded in black, including the 6 cysteines. Less-conserved amino acids are shaded in grey. The sequences shown are human DEFB118 (AF347073), DEFB119 (AF479698), DEFB120 (AF479699), DEFB121 (AY501000), and DEFB123 (AY501002); macaque DEFB118 (AF207834), DEFB119 (AY499406), DEFB120 (AY499407), DEFB121 (AY499408), DEFB122 (AY499409), and DEFB123 (AY500998); mouse DEFB21 (DQ141308), DEFB24 (submitted as DEFB19; NM_145157), DEFB27 (AY591384), DEFB30 (DQ141309), and DEFB36 (AY591385); and rat DEFB21 (AY600147), DEFB24 (AY600148), DEFB27 (AY600149), DEFB30 (AY600146), DEFB36 (AY615297), and Epinephelus coioides (orange-spotted grouper) reproductive regulator 1 (rr1) (AY129305).

 
Tissue distribution of the cluster.
An important determinant of the types of environmental challenges these defensins are likely to encounter is location of expression. To establish whether these mouse defensins are predominantly expressed in the male reproductive tract as shown previously for their primate homologs (54), a systematic measurement of relative steady-state mRNA levels in 19 mouse tissues was performed. Interestingly, very few tissues showed expression of the ß-defensin genes (Fig. 3). Defb27 and Defb30 expression was restricted to testis and anterior regions of epididymis. Defb24 (also known as Defb19 or Tdl) was abundantly expressed in kidney, testis, and ovary and Defb36 in kidney, bladder, and ovary. Expression of Defb21, -24, and -36 in adrenal suggests that these genes may be hormonally regulated.


Figure 3
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  		Fig. 3. Expression of mouse ß-defensin genes in different tissues. Total RNA isolated from mouse brain (lane 1), esophagus (lane 2), salivary gland (lane 3), heart (lane 4), lungs (lane 5), liver (lane 6), spleen (lane 7), adrenal (lane 8), kidney (lane 9), bladder (lane 10), testis (lane 11), caput (lane 12), corpus (lane 13), cauda epididymis (lane 14), ovary (lane 15), oviduct (lane 16), uterus (lane 17), cervix (lane 18), and placenta (lane 19) was used for RT-PCR analysis. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was amplified as a control to check the quality and quantity of the template.

 
Comparative analysis of the expression and tissue distribution of these defensin genes shows wide variation in somatic and gonadal tissues of primates and rodents. Examples include expression of mouse defensins in female reproductive tissues and male and female urinary systems, in contrast to the male reproductive tract-specific expression of the orthologous primate defensins (54). Species-specific expression was also seen within the male tract where expression of Defb30 was high in rat cauda epididymis (53, 79) but undetected in mouse cauda epididymis (Fig. 3). Thus the expression of these rodent defensin genes, unlike that in primates, is not restricted to male reproductive tract tissues and therefore may have evolved to acquire additional functions, including defense against pathogens that colonize other tissues. In humans, we reported several alternatively spliced transcripts and a pseudogene for this cluster (54). Such alternatively spliced transcripts and pseudogenes are not found in primates and rodents. These properties, the transcript abundance in urogenital tract, lower expression in other tissues, and complete absence of pseudogenes and alternatively spliced transcripts, differentiate the mouse cluster from that of the human.

Developmental regulation of the cluster.
Age-dependent regulation of the expressed defensin genes was analyzed in the reproductive tract tissues, testis, caput epididymis, and ovary from mice at 10 and 100 days of age (Fig. 4). A unique pattern of developmental expression was observed for each defensin. Whereas in caput epididymis, all genes appeared upregulated during development, there were wide developmental differences in testis and ovary expression. In their role as protectors against sexually transmitted diseases, male tract defensins would be expected to reach full expression in sexually mature animals. Our observations that Defb24, -27, and -36 are highly expressed in testis and ovary at day 10 suggest possible roles in gonadal development as well as innate immunity. Similar abundant expression of these defensins in immature gonads was seen in rat (53, 79). In contrast, their orthologs in primate were expressed at low levels in immature and higher levels in mature epididymis (54). Future analyses to define the cell types expressing each defensin may help us to understand their functions at different ages. Because of the differences between the defensins in primates and rodents, we conducted an analysis of the selective constraints in these two evolutionary groups to correlate our results with specific evolutionary patterns at the sequence level.


Figure 4
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  		Fig. 4. Developmental regulation of mouse ß-defensin genes. Total RNA isolated from the testis, caput epididymis, and ovary of 10- and 100-day-old mice was used for RT-PCR analysis. RNA from 2 animals was analyzed for each tissue and age. Data are from 1 animal and are representative of similar results from both animals. Gapdh was amplified as a control to check the quality and quantity of the template.

 
Phylogenetic analysis.
To study the evolution of this multigene family, we first inferred the phylogenetic relationships between sequences using the pairwise Poisson-corrected amino acid distances method, which accounts for multiple substitutions per amino acid site, and NJ. In our analysis, amino acid site columns containing gaps were removed from subsequent analyses. The support for the different nodes of the tree was assessed by bootstrap using 500 pseudoreplicates. The paralogous genes in this cluster appear to have emerged in rapid succession by tandem duplication of individual genes rather than by whole cluster duplication, since most of the duplications are lineage specific (Fig. 5).


Figure 5
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  		Fig. 5. Phylogenetic tree of expressed human, macaque, mouse, and rat ß-defensin amino acid sequences. h, Human; m, monkey; m Defb, mouse defensin; r Defb, rat defensin. Neighbor-joining tree was rooted with a putative defensin of E. coioides reproductive regulator 1 (rr1) sequence using the pairwise Poisson-corrected amino acid distances method, which accounts for multiple substitutions per amino acid site with bootstrap confidence values of 500. The confidence values (expressed as %) obtained using bootstrap are indicated on nodes. Scale bar indicates the no. of substitutions per site.

 
The location of the Epinephelus sequence on a branch within the tree, not as an outgroup, indicates that the deepest splits in the cluster can be inferred to predate the fish-primate divergence ~450 MYR ago. Although ß-defensin evolution began before the fish-mammalian divergence, primate-specific defensins appear after the primate-rodent split 91 MYR ago (i.e., DEFB120 and DEFB121). Further differential gene expression giving rise to alternatively spliced transcripts and a pseudogene (DEFB122p) after the human-macaque split 23 MYR ago indicates the dynamic evolutionary history of this ß-defensin cluster.

Analysis of the number of nucleotide changes between two sequences reveals the type of evolution that has taken place. The rate of synonymous nucleotide change is considered independent of natural selection and is equated with the rate of neutral nucleotide substitution (46) unless the synonymous sites are saturated or under structural constraints at the RNA level. The mean nucleotide substitutions per synonymous site in our alignments estimated by the modified method of Nei and Gojobori (45) with the Jukes-Cantor correction was 0.825 ± 0.082, and hence we assume synonymous sites have not reached saturation. To measure the selective constraints operating in the defensin sequence alignments, we estimated the number of synonymous (dS) and nonsynonymous (dN) nucleotide substitutions per site using the modified method of Nei and Gojobori. To determine the intensity of selection, we measured the ratio between the two rates ({omega} = dN/dS) as previously suggested (1, 10, 31, 61, 78). This ratio helps to determine whether the gene has been fixing amino acid replacements neutrally ({omega} = 1), whether replacements have been removed by purifying selection ({omega} < 1), or whether mutations have been fixed by adaptive evolution ({omega} > 1). Comparison of dN and dS estimated values for exon 1 in the primate-rodent-based alignment (Fig. 6A) showed very small changes compared with exon 2 (Fig. 6B) and indicated that the rates of substitution were under neutral evolution. However, individual comparisons of the human-monkey and mouse-rat exon 1 sequences revealed subtle differences. The primate exon 1 sequences showed a slight excess of dN compared with dS (Fig. 6C), whereas none of the rodent exon 1 sequences showed an excess of dN over dS (Fig. 6D). However, the differences failed to reach statistical significance under the Fishers exact test, most likely because the sequences are only 20 amino acids in length. The total number of nonsynonymous sites (N), synonymous sites (S), nonsynonymous substitutions (n), synonymous substitutions (s), nonsynonymous substitutions per nonsynonymous site (dN), synonymous substitutions per synonymous site (dS), and dN – dS estimates for the first and second exon comparisons are available in Supplemental Data File 5 (supplemental data are available at the online version of this article).


Figure 6
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  		Fig. 6. Rates of synonymous and nonsynonymous substitutions. A: graph for the first exons (which encode signal peptide) showing the no. of synonymous substitutions per synonymous site (dS; y-axis) against the no. of nonsynonymous substitutions per nonsynonymous site (dN; x-axis) for human, macaque, mouse, and rat orthologous ß-defensins. B: graph for the second exons (which encode the mature peptide). C: primate exon 1. D: rodent exon 1. E: primate exon 2. F: rodent exon 2. Diagonal line indicates dN = dS.

 
Although the mean dS for exon 2 (Fig. 6B, of all the aligned sequences) was higher than dN, the trend was different from that of exon 1 (Fig. 6A). In specific paralogous sequence comparisons, often dN exceeded dS significantly under a standard Z-test at the 10% level; however, these failed to reach statistical significance by the more rigorous Fishers test. Again, in separate comparisons among the primates and among the rodents, for the paralogous sequences of exon 2, the mouse-rat comparisons showed a greater excess of dN over dS than the human-monkey comparisons (Fig. 6, E and F). Interestingly, for those comparisons where dN exceeded dS, dN of exon 2 in the rat lineage was much higher than that in the mouse lineage when using the primate sequences as outgroups and thus showed stronger positive selection. In these rat-mouse comparisons, the modified Nei and Gojobori method (45) in MEGA3, taking into account the transition to transversion ratio, showed higher statistical significance for the excess dN, although the unmodified method did detect some positive selection (data not shown).

Human and monkey DEFB118 showed a dN – dS similar to their mouse paralog Defb21. Some of the substitution rate comparisons of the rat Defb21 with monkey DEFB122 and DEFB123, human DEFB123, rat Defb27, and mouse Defb27 showed higher dN and reached statistical significance with the modified Nei and Gojobori method (45) (data not shown). The human 20p cluster is very similar to the primate 20q cluster for most comparisons within this cluster. Taken together, these results show that in primates, with the similarity of the sequences, the lower dN, and the constant pI, it is apparent the clusters on chromosome 20 show neutral selection when compared with the 8p cluster, which shows bouts of negative selection.

Constraints analysis.
Traditional statistical tests based on pairwise comparisons involve calculating the rate of dN and dS within two full-length sequences, averaging over all codons. Alternative strategies for detecting positive selection utilize maximum likelihood and allow calculation of the estimate of {omega} at individual sites. However, several studies have shown that maximum likelihood methods are sensitive to the violation of assumptions made in models to detect adaptive evolution, and that false-positive results could be obtained under those conditions (63). Recently, Fares et al. (14), using the Kimura-based model of Li (37), developed a sliding window analysis procedure to detect selective constraints simultaneously at individual branches of a phylogeny and specific protein coding regions. This mathematical approach is based on the maximum parsimony method of Suzuki and Gojobori (62). The method of Fares et al. is implemented in the program SWAPSC (13), which has the advantage of estimating the statistically optimum window size for the analysis of a particular set of data, reducing the chances of detecting false-positive results. Several hypotheses regarding selective constraints can be tested using this procedure, including positive Darwinian selection, accelerated rates of amino acid substitutions where {omega} is <1, hot spots of nucleotide substitutions, and saturation of synonymous sites. We used a combined approach to detect the distances between the paralogous sequences by pairwise comparisons and SWAPSC for site-specific analysis (Fig. 7).


Figure 7
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  		Fig. 7. Substitution rates and selective constraints of the mammalian defensins. A: rodent Defb21 [21] substitutions compared with ancestral sequence [22]. B: primate DEFB118 substitution [20] compared with ancestral sequence [22]. Each graph shows nonsynonymous substitutions, Ka (•), synonymous substitutions, Ks ({circ}), and significant positive and negative selection across the sequence (midpoints of the window) encoded for both the first and second exon. Box indicates the codon site with a high {omega} value where P < 0.0001.

 
In this study, we obtained evidence that several amino acid sites in the active peptide of the mammalian ß-defensins are under positive Darwinian selection (Fig. 7). The optimum window size was two codons, as estimated by SWAPSC. When a two-codon window was slid throughout the sequence alignment, taking into account the phylogenetic tree, two branches were found to have undergone positive selection (Fig. 7, A and B). Both branches show the recent formation of DEFB120 and DEFB121 by duplication in primates. The most surprising observation by the SWAPSC analysis was that there is neutral selection throughout the six-cysteine motif and signal peptides. By contrast, the COOH-terminal extensions after the six-cysteine motif had the highest {omega}, indicating positive selection (P < 0.0001) (see Supplemental Fig. 7, C–I). Although SWAPSC does not impose or assume a molecular clock, most regions of these defensins show few or no changes, as expected over the 23 MYR since the human and macaque diverged from their common ancestor. The branches separating ancestral primate and ancestral rodent sequences show greater variation in dN and dS across the sequence, which is evidently due to the accelerated fixation of amino acid substitutions over the last 91 MYR. In addition, most of the rat sequences have greater fixation of dN over the common mouse-rat ancestor (41 MYR).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this mammalian ß-defensin comparative genomic analysis, we report the characterization and evolution of four paralogous mouse ß-defensin genes (Defb21, -24, -27, and -36) clustered on mouse chromosome 2F and orthologs in the human ß-defensin cluster at 20q11.1. Mouse chromosome 2 and human chromosome 20 demonstrate striking orthology over 40 Mb, including coding and noncoding sequences, maintenance of gene order, and position of ancient repetitive sequences (44a, 87). Despite this remarkable sequence and order conservation, no ortholog of primate DEFB121 exists in rodent, indicating the continuing evolution of the primate cluster. The high amino acid similarity of DEFB121, DEFB135, and mouse DEFB30 needs further study to understand fully the origin and evolution of the multiple ß-defensin clusters in the genome.

Analyses to determine the age of defensin gene duplications are aided by our discoveries of the absence of DEFB120 and DEFB121 orthologs from the mouse and rat genomes and previous studies indicating evolution of species-specific ß-defensin genes (53, 60). These findings suggest that although some ß-defensins evolved before the divergence of mammals from fish, gene duplications and rodent- and primate-specific defensin genes appeared after the rodent-primate divergence 91 MYR ago (23, 53, 60). Our studies have not shown gene duplication subsequent to the separation of the macaque and human primate lines 23 MYR ago, long before human-chimp divergence (5 MYR ago).

Expression of the mouse 2F6 ß-defensin cluster in a variety of organs, as reported for other ß-defensins (9, 44, 74, 85), contrasts with the highly male-restricted expression of their primate orthologs (53, 54, 60). Expression in the urogenital tract of both sexes extends previous observations of gonadal and kidney expression of mouse Defb24 (Tdl) (41, 75). Our novel finding that each of the genes in this cluster is expressed in the adrenal gland raises questions regarding the function and regulation of these defensins in this environmentally and hormonally responsive organ. This differential expression in rodent and primate could be due to gain or loss of functions in different tissues. Recently, gene expression was found to evolve under natural selection (17), and since genes with altered expression and genes under selection correspond (50), the differential distribution and abundance of the defensin genes in rodents and primates may have led to differential selection and ultimately to changes in function. Taken together, differential expression reflects the dynamic and complex evolutionary forces operating on the ß-defensin gene clusters.

The birth-and-death model describes the evolution of several host defense multigene families, including the major histocompatibility complex (MHC) locus as well as the {alpha}- and ß-defensins (47). Our previous report (54) describing the human orthologs of this mouse cluster contains supporting evidence for the birth-and-death model, including alternatively spliced transcripts (DEFB119/120) and the pseudogene (DEFB122p). These observations contrast with the absence in mouse of orthologs of primate DEFB120 and DEFB121, showing that these genes were born in the primate line after divergence from rodents. The active expression of the mouse (Defb27) and macaque orthologs of DEFB122p suggests death of DEFB122 in humans subsequent to divergence from macaques (23 MYR ago). Similar to gene birth, alternative mRNA splicing increases gene diversity. The absence in mouse (82) of the majority of the 30,000 alternative splicing relationships in humans (43), including DEFB120, implies splice-variant acquisition in humans rather than loss in mouse (33). Since mice do not appear to be defense deficient, the relative genomic simplicity we report here in the mouse cluster could be interpreted as evidence that the human orthologs may be in the process of acquiring new functions and shedding previous functions, consistent with the birth-and-death evolutionary model.

The evolutionary dynamics of this defensin gene cluster containing species- and tissue-specific functions may be explained in terms of three processes. The modified duplicates may perform a new function as hypothesized by neofunctionalization, may improve on the ancestral function as explained by subfunctionalization, or may lose all function as in nonfunctionalization. Most defensins tested appear to have retained the ancestral function of host defense (36, 79), and in addition, some have gained reproductive functions, thereby corresponding to the subneofunctionalization hypothesis, i.e., Bin1b (86), DEFB126 (83, 84).

Molecular evolutionary analyses of genomic sequences reveal the sequence divergence, which might lead to functional diversity. There is a trend toward greater excess of nonsynonymous substitutions over synonymous substitutions in exon 2, coding for the mature peptide, compared with exon 1, encoding the signal peptide. We and others postulated that the divergence due to excess nonsynonymous substitutions in a defensin exon 2 may lead to functional divergence of the protein, possibly affecting microbe killing efficiency (3), target pathogen diversity, and host-parasite co-evolutionary processes (subfunctionalization). Alternatively, divergence might lead to acquisition of reproductive functions (neofunctionalization) (44, 53, 54, 60, 85). Analyses have shown that the ratio of nonsynonymous substitutions to synonymous substitutions is higher in the 8p cluster than in the human 20q and orthologous mouse 2q clusters evolving during same time (60). One contributor to this difference may be the inclusion in the 8p analyses of the pseudogene DEFB109p, which introduces a larger ratio of nonsynonymous to synonymous substitutions than actively expressed genes (44a), whereas human DEFB122p was excluded from our 20q evolutionary analysis. The properties of each ß-defensin and thus the evolutionary pressures operating on each gene in a cluster and between such clusters containing paralogous genes vary widely and could also explain differences in evolution of the defensin clusters across the genome.

NJ analyses show that rat exon 2 tends to accumulate more nonsynonymous substitutions than mouse or primate exon 2 and to exhibit more positive selection, an important trend in the dynamic evolution of murine and rat defensin genes after the rodent and primate divergence (59). The constraints analysis using SWAPSC provides further evidence for positive selection and more nonsynonymous substitutions in rats than primates, especially within the COOH-terminal propiece-like extensions, beyond the six-cysteine array. The COOH-terminal extensions in primate DEFB118 and mouse and rat orthologous DEFB21 are highly anionic. Since cationic properties of defensins are important in bacterial membrane disruption (21), and increased cationicity leads to increased killing efficiency (3), this divergent anionicity is likely to translate into functional diversification (neofunctionalization). The anionic COOH termini may have evolved to perform new functions mediated by interaction with positively charged host or pathogen molecules. Similarly, Semple et al. (59), in analyzing human chromosome 8p defensins and their rodent orthologs, concluded that the propiece region of the rodent is subjected to positive selection and thus functional diversification. In addition, often those sites neighboring the cysteines are subjected to selection, as evidenced in both the alignment analysis (glycine and glutamic acid aligned nearby the cysteines) and the SWAPSC analysis, corroborating previous findings (59). It is pertinent to note that, unlike the 8p cluster, which is under both positive and negative selection, the 20q cluster is more under positive selection.

Overarching concepts of gene function are developed through cross-species comparisons, which reveal how a gene has responded to evolutionary pressures in the varied environments of different genomes and habitats. Our comparative study highlights the hallmarks of molecular diversification of the mouse defensin cluster, the smaller genomic span, absence of alternative splicing, broader tissue distribution, and retention of cationicity throughout. The contrasting orthologous primate defensin cluster exhibits preferential expression in male reproductive tract tissue, formation of alternate transcripts, additional tandem duplications, accumulation of more nonsynonymous substitutions, and positive selection sites distal to the six-cysteine array. Additional analyses are needed to understand how these specializations are adaptations to the internal and external environments of different species. Broader tissue distribution in rodents may indicate adaptations to protect additional organs, while the COOH-terminal specializations and male tract-specific expression in primates may be related to acquisition of reproductive functions such as sperm maturation. These interpretations of rodent and primate gene function diversifications await experimental verification.

Evolutionary system biology will not only increase our understanding of how novel protein functions evolve, but it will also answer basic questions of why biological systems work the way they do (25). Determination of the in vivo functions of the duplicated genes is a prerequisite and must be the next target in understanding the range of evolutionary outcomes, not necessarily restricted to immune response and reproduction. Finally, the efficacy of ß-defensins as microbicides indicates their potential usefulness in drug development. Moreover, the demonstration of a role in reproduction establishes ß-defensins as possible fertility control targets.

Supplemental data.
The following supplemental data files are available for this article: the aligned human, macaque, mouse, and rat DNA sequences (Supplemental Data File 1); the aligned first exon human, macaque, mouse, and rat DNA sequences (Supplemental Data File 2); the aligned second exon human, macaque, mouse, and rat DNA sequences (Supplemental Data File 3); and the aligned human, macaque, mouse, and rat DNA sequences along with the E. coioides (rr1) sequence (Supplemental Data File 4). The data files are in ".meg" format and can be used with MEGA software, which can be downloaded from http://www.megasoftware.net. The full N, S, n, s, dN, dS, and dN – dS estimates for the first and second exon comparisons are given in Supplemental Data File 5. Supplemental Fig. 7, C–I, for the SWAPSC analysis of the defensins is given as Supplemental Data File 6. The data of the SWAPSC analysis are given in Supplemental Data File 7, and the phylogenetic tree used for numbering the branches of Supplemental Fig. 7, C–I, is given as Supplemental Data File 8. These supplemental data files are available at the online version of the article and also at http://www.med.unc.edu/lrb/shall.htm.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
We are grateful for financial support for this project (CIG-96-06-A) from the Consortium for Industrial Collaboration in Contraceptive Research (CICCR) Program of the Contraceptive Research and Development (CONRAD) Program, Eastern Virginia Medical School, Norfolk, VA. The views expressed by the authors do not necessarily reflect the views of CONRAD or CICCR. This work was also supported by grants from The Andrew W. Mellon Foundation, by National Heart, Lung, and Blood Institute Grants R37-HD-04466 and U54-HD-35041 as part of the Specialized Cooperative Centers Program in Reproduction Research, and by Fogarty International Center Training and Research in Population and Health Grant D43TW/HD00627.


   ACKNOWLEDGMENTS
 
We thank Katherine Hamil and Suresh Yenugu for support.


   FOOTNOTES
 
Address for reprint requests and other correspondence: S. H. Hall, Laboratories for Reproductive Biology, CB#7500, Dept. of Pediatrics, Univ. of North Carolina, Chapel Hill, NC 27599-7500 (e-mail: shh{at}med.unc.edu).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


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