MRC Human Genetics Unit, Western General Hospital, Edinburgh, Scotland
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: adaptive evolution ß-defensin positive selection Mus musculus
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Four human ß-defensins have been functionally analyzed, and all of them have displayed antimicrobial activity in vitro (Lehrer and Ganz 2002). Interestingly, several human ß-defensins also display chemoattractant activity for immature dendritic cells and memory T-cells, suggesting that these peptides may also function as an important link between the innate and adaptive immune system (Yang et al. 1999; Garcia et al. 2001). Until recently, only eight murine ß-defensin genes had been identified, all mapping to a region of conserved synteny with the human locus on chromosome 8 (Huttner, Kozak, and Bevins 1997; Bals, Goldman, and Wilson 1998; Morrison et al. 1998; Morrison, Davidson, and Dorin 1999; Jia et al. 2000; Bauer et al. 2001; Yamaguchi et al. 2001). Synthetic murine ß-defensin peptides also possess antimicrobial activity (Bals, Goldman, and Wilson 1998; Morrison et al. 1998; Bals et al. 1999; Bauer et al. 2001; Yamaguchi et al. 2001; Morrison et al. 2002) demonstrating the functional conservation of these peptides between mouse and man. However, dramatic differences are evident within the murine ß-defensin gene family in terms of their tissue distribution and responsiveness to inflammatory stimuli (Lehrer and Ganz 2002). We have also reported functional differences within members of the murine ß-defensin gene family in terms of their ability to kill specific pathogens (Morrison et al. 1998, 2002). These results suggest that the murine ß-defensins may have evolved under positive selection to have different profiles of antimicrobial activity and thus maximize the host's ability to fight opportunistic infections.
Here we report the further characterization of the murine ß-defensin gene cluster and investigate the molecular mechanisms responsible for the divergence between these molecules.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
BAC Sequencing
BACs were sequenced as part of the UK mouse sequencing program (http://mrcseq.har.mrc.ac.uk), and the sequence is freely available.
BAC Sequence Analysis
Sequence generated from the BAC 353A15 and 357B4 was analyzed using NIX, a Web tool designed to allow the viewing of multiple DNA analysis programs (http://www.hgmp.mrc.ac.uk). The sequence was also analyzed for the presence of novel ß-defensins using a calibrated HMMER model (version 2.1.1). The HMMER model was constructed from a ClustalW (version 1.82 with default settings) (Higgins, Thompson, and Gibson 1996) multiple sequence alignment of ß-defensin sequences retrieved from GenBank.
Tissue Expression of Novel Murine ß-Defensin Genes
The RACE protocol was adapted from (Townley et al. 1997), as previously described (Morrison, Davidson, and Dorin 1999), and RT-PCR was carried out as described (Morrison, Davidson, and Dorin 1999). Primer sequences are available in supplementary information.
Phylogenetic Inference and Evolutionary Analysis
Sequences were aligned using ClustalW (version 1.82) (Higgins, Thompson, and Gibson 1996), and gapped positions were omitted from subsequent analyses. The phylogenetic tree was constructed by neighbor-joining (Saitou and Nei 1987) based on the proportion of different amino acid sites and was rooted with chicken Gallinacin 1; its reliability was assessed with 1,000 bootstrap replications using MEGA (version 2.1) (Kumar et al. 2001). In pairwise comparisons between nucleotide sequences within subgroups, the number of synonymous substitutions per synonymous site (dS) and the number of nonsynonymous substitutions per nonsynonymous site (dN) were estimated using the Nei-Gojobori test. Standard errors for dS and dN were calculated using 1,000 bootstrap replicates. In addition, the Jukes-Cantor correction (Jukes and Cantor 1969) was applied to account for multiple substitutions at the same site. A one-tailed Fisher's exact test was used to test the null hypothesis that the proportions of synonymous and nonsynonymous differences are the same (Zhang, Kumar, and Nei 1997). All distance calculations and codon-based tests of selection were carried out using MEGA (version 2.1). Estimates of the proportions of radical and conservative nonsynonymous substitutions were made according to the methods of Zhang (2000) using the HON-NEW program. The radical or conservative nature of nonsynonymous substitutions was assessed with respect to charge and to the polarity and volume of the amino acids (the Miyata-Yasunaga amino acid classification) as defined by Miyata, Miyazawa, and Yasunaga (1979). Intron sequences for Defb11, Defb10, and Defb2 were aligned using DIALIGN (version 2.1) (Morgenstern 1999). The numbers of substitutions between the introns were estimated using Kimura's two-parameter method (Kimura 1980).
Sequence Analysis of Exon 2 Within the Rodentia Genus
The exon 2 sequences of nine murine ß-defensin genes, representing members of both subgroups, were amplified from genomic DNA from the inbred Mus musculus mouse lab stains C57Bl/6N, 129/Ola, CBA, and BALB/cJ; from the Mus musculus species Mus musculus domesticus, Mus musculus musculus, Mus musculus castaneous, and Mus musculus hortulanus; from the Mus subspecies Mus spretus, Mus caroli, and Mus pahari; and from the non-Mus species Apodemus sylvaticus and Rattus norvegicus. DNA was purchased from the Jackson Laboratories. The primer sequences are detailed in supplementary information. Genomic samples that failed to generate a PCR product were amplified with at least one set of alternative primers designed specifically towards the murine gene exon 2 sequence. PCR products were sequenced as previously described (Morrison, Davidson, and Dorin 1999) and the resulting sequence compared with that obtained from C57Bl/6 DNA. Isoelectric points (pI) were calculated from predicted mature peptide protein primary sequence using the ExPasy compute pI tool (http://us.expasy.org/tools/pi_tool.html).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Expression Profiles of the Novel Murine ß-Defensin Genes Defb10, Defb11, Defb9, Defb15, Defb13, and Defb35
Full-length transcripts for two of the six sequences were present in the murine EST database. Defb11 was present in a neonate cerebellum and an adult testis expression library and Defb9 was present in an adult hippocampus library. 5' RACE analysis from trachea, testis, and brain tissue resulted in the identification of full-length transcripts for four of these sequences, Defb10, Defb11, Defb15, and Defb13.
Defb10 and Defb11 were found to be expressed in both adult and neonate brain (fig. 2) with weak expression being detected from kidneys, epididymis, and testis after increased numbers of cycles of PCR (data not shown). Defb15 and Defb13 are expressed in the testis and to a lesser extent in the epididymis (fig. 2). Weak expression of Defb15 and Defb13 was detected in the kidneys, and Defb15 was also found to be expressed at low levels in the colon (data not shown). Expression of Defb9 was only detected from brain cDNA after 35 cycles of RT-PCR followed by hybridization of the PCR product with a radiolabeled internal oligonucleotide probe. RT-PCR analysis of Defb35 using primers specific to the putative exon 2 sequence indicated Defb35 to be expressed in the testis and epididymis (fig. 2). RT-PCR using primers from exon 1 to 2 indicated expression also in kidney and neonatal and adult brain (data not shown). The predicted peptide sequences of the genes contained in the two separate contigs are shown in figure 3.
|
|
|
|
|
|
We aligned the introns of the genes, which showed the evidence for positive selection (see Supplementary Material). The rates of substitution between the introns from Defb2 and Defb11 and between those from Defb2 and Defb10 were estimated as 0.111 ± 0.013 and 0.090 ± 0.012, respectively. The rates of substitution between the introns from Defb7 and Defb5, introns from Defb7 and Defr1, and introns from Defb5 and Defr1 were estimated as 0.104 ± 0.009, 0.240 ± 0.015, and 0.237 ± 0.014, respectively. These values (except for Defr1 and Defb7 comparison) are not significantly different from the rates of synonymous substitution estimated between the second exons of the same pairs of genes given in table 1. Thus the high dN/dS ratio observed between these pairs of exons is not attributable to artificially low dS estimates as a result of sampling error, but is caused by a real increase in dN relative to dS, which is the pattern expected to be generated by positive selection.
Gene Alignments Reveal That the Region of Adaptive Change Is Confined to a Small Portion of Exon 2
ClustalW lineups of Defb2, Defb10, and Defb11 reveal that exon 1 only has one nucleotide change between the three sequences, and the first 200 bp of the intron sequence 3' to exon 1 has 95% identity where all three sequences are present (see Supplementary Material). There are many examples of small deletions from the sequences, and Defb11 and Defb2 have independent deletions relative to Defb10. All three gene have small regions of dinucleotide expansions. Defb10 and Defb11 are recent duplications as shown by the peptide sequence identity and cladogram in figure 3. Within the intron, Defb10 and Defb11 have 98% identity where Defb10 has sequence to compare. In the coding sequence, there are only two amino acid changes in the first 44 residues of the peptide. This is up to the second cysteine of the mature peptide. From that point, however, to the end of the peptide, the DNA identity drops from 90% to only 30%. The amino acid identity between the two genes drops to only 50% in the region that encodes the 15 amino acids between cysteine 2 and 4 in the mature peptides. The dN/dS ratio of the second exon of Defb10 and Defb11 is 1.76 but does not reach significance for positive selection in Fisher's exact test. The intron sequence just 5' to exon 2, where all three sequences align, has 85% identity. Within the first 50 nucleotides of exon 2, the identity across the three genes falls to 66%, (although Defb10 and Defb11 are still 98% identical at the nucleotide level). Thus, the high similarity of these genes in both exon 1, the intron, and 3'and 5' of the first and second exons shows that these genes have recently duplicated.
The ClustalW lineups of Defr1, Defb5, and Defb7 are given in supplementary information and again it is clear that these genes have arisen by recent duplication. The DNA surrounding the exons and exon 1 are 85% identical between the three genes, whereas the exon 2 DNA are only 54% identical.
The ß-Defensin Genes Have Evolved At an Accelerated Rate Since Mouse/Rat Divergence
Southern blot hybridization experiments with mouse and rat genomic DNA indicate the existence of an excess of mouse ß-defensinlike sequences over rat sequences (data not shown), indicative of a high rate of species specific divergence. In order to examine ß-defensin evolution within Rodentia in more detail, we used PCR primers designed to intron sequence and specific for the murine ß-defensin genes Defb1 to Defb6, Defb10, Defb11, and r1 to amplify the corresponding genomic regions from animals of the Mus genus and the non-Mus species (table 3). A negative signal implies the gene is either not present or too divergent to detect using this assay. Based on exon 2 sequence, it appears that Def b1 and Defb4 arose before rat/mouse divergence. The subgroup 1 and 2 genes then arose from these as a consequence of gene duplication in an ancestral species. The genes in subgroup 2, Defb10 and Defb11 arose before Mus divergence from an ancestral species around 8 to10 MYA and Defb2 from a duplication of one of these genes in the common ancestor of M. hortulanus and M. musculus. In subfamily 1, Defb3, Defb4, Defb5, and Defb6 arose by dramatic duplication of an ancestral Defb4 gene before the divergence of M. caroli and other Mus species 4 to 6 MYA. The inability to detect genes in more ancient speciesfor example, Defb10 being present in M. pahari but not M. caroliis either due to their divergence rendering them undetectable by PCR or due to the loss of a functional gene in a particular species.
|
|
|
We recently identified and characterized a murine ß-defensinlike gene named Defensin-related 1 (Defr1) because it does not contain the first of the canonical six cysteine residues characteristic of the ß-defensin family (GenBank accession number AJ344114), however it does display very potent antimicrobial activity in vitro (Morrison et al. 2002). PCR analysis amplified Defr1 exon 2 sequence from C57BL/6 genomic DNA, but no signal was obtained from any of the other genomic samples. The lack of amplification in any other of the DNAs except C57B/6 was also obtained using two other sets of primers designed towards the Defr1 sequence. A product was also produced from C57BL/6 DNA using primers that had 100% identity to the very similar (98% identity) Defb8 gene described by Bauer et al. (2001) but contained mismatches to the Defr1 sequence. However, when this product was sequenced, it had 100% identity to Defr1 and not Defb8. We are forced to conclude that this novel gene arose by gene duplication very recently within the C57Bl/6 inbred strain, after duplication from an ancestral Defb7 gene.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The high similarity these peptides share over their first exons (particularly within subgroups 1 and 2), and dissimilarity in their second exons, prompted us to investigate the sequence similarities and disparities of these peptides in more detail.
A phylogenetic tree constructed using all of the known mammalian ß-defensin genes showed species specific clustering (Hughes 1999). Many second exon fragments from known and novel mouse ß-defensins were recently reported by Schutte et al. (2002), but from these data it is impossible to tell which fragments are pseudogenes and which are functioning genes. Although we acknowledge the value of examining pseudogene sequence in determining "birth and death" mechanisms of evolution (Nei, Gu, and Sitnikova 1997), in the present study (because we are primarily interested in function), we restricted our attention to complete gene sequences. These are either previously known or determined from the analysis of the BAC clone based map of the region and verified as encoding real transcripts by RNA analysis.
The existence of independent, species-specific gene clusters suggests that multiple duplications and diversification events have occurred at these loci after mammalian radiation. The increased number of mouse hybridizing fragments compared with rat under reduced stringency conditions implies that gene duplications took place after mouse and rat divergence approximately 40 MYA (Hedges et al. 1996). Our cross-species PCRs indicates that only Defb4 and Defb1 can be found in the rat. The construction of a phylogenetic tree with only the Mus musculus C57Bl/6J sequences separated the genes into subgroups, and it became apparent that within subgroups 1 and 2, there were distinct regional constraints upon the divergence of these sequences. Within subgroups 1 and 2, the genes appear to have risen by recent duplication and the extremely high level of sequence identity in the first exons may be due to this and possibly some level of gene conversion after duplication. However, in marked contrast to exon 1, the exon 2 sequences show rapid divergence as a result of positive selection. Although statistical significance is not achieved for all of the genes showing dN > dS, only Defb1 and Defb9 show dS > dN. Intriguingly, primate ß-defensin 1 orthologs (Del Pero et al. 2002), in common with the mouse Defb1 gene, also demonstrate neutral rather than adaptive evolution.
In addition to the clear evidence we have for positive Darwinian selection in the M. musculus ß-defensin genes, there is also evidence that the subgroup evolved relatively recently by a "birth and death" mechanism (Nei, Gu, and Sitnikova 1997). Rapid "birth and death" and "gene sorting" has recently been described for the rodent eosinophil-associated RNase family. "Gene sorting" is defined as the process leading to differential retention of ancestral genes or gene lineages in different species, where "birth" implies gene duplication and "death" refers to gene deactivation (Zhang, Dyer, and Rosenberg 2000). We postulate that the inability to detect particular genes in certain species is as a result of this process. For example, Defb10 and Defb11 arose by duplication of an ancestral gene in an ancestor of M. pahari. Defb10 was then lost from M. caroli, but in other Mus species, the gene was functionally important and was retained. In addition, it appears that once a gene arises by duplication, rapid change occurs until optimal function arises and is selected so the gene sequence is stabilized and adaptive change is relaxed.
Hill and Hastie (1987) first demonstrated that certain regions of genes can demonstrate an excess of nonsynonymous nucleotide change. Subsequent research has shown that gene duplication and specialization by positive Darwinian selection is a hallmark of vertebrate host-defense, although a few other genes, including those involved in reproduction (Swanson and Vacquier 2002), are also known to be under strong positive selection. Interestingly, the subgroup 1 genes Defb15, Defb13, and Defb35 are strongly expressed in the testis and epididymis, and indeed expression in male testis/epididymis is seen for all the novel genes described here. Recently, a rat gene Bin1b that is exclusively expressed in the caput region of the rat epididymis, and which is responsible for sperm maturation, storage, and protection, was described. Bin1b exhibits structural characteristics and antimicrobial activity similar to that of ß-defensins. Bin1b appears to be a natural epididymis-specific antimicrobial peptide that plays a role in reproductive tract host defense and male fertility, and the mouse defensins we describe may also be involved in these processes (Li et al. 2001).
Interestingly, for functional studies of the nonsynonymous changes seen for subgroup 2, there has been a tendency to change the charge but not the polarity and volume of the amino acids. However, this is not so strong for subgroup 1, where positive selection is observed between Defb7 (pI = 9.59), Defr1 (pI = 9.26), and Defb5 (pI = 9.03). In this subgroup, the ß-defensins appear to remain very cationic, but in subgroup 2, the charge varies from 7.8 to 9.13. Again, the signal peptide remains virtually unchanged between the paralogs. Inefficient secretion may be too damaging to the organism to allow amino acid change. However, we see no evidence for a coordinated change of charge in the prepropeptide, such as is observed with the -defensins (Hughes and Yeager 1997). The propeptide region is very small and does not balance the change in charge observed in the mature peptides. In the cross-species analysis, Defb11 shows very rapid accumulation of amino acid changes by positive selection. Independent changes occur within different species. Interestingly, one amino acid (leucine), which is nonpolar, is present in the Defb11 M. pahari sequence and in M. caroli and M. m. hortulanus but is phenylalanine (also nonpolar) in M. spretus and the other strains more closely related to the inbred lines. This amino acid is coded for by three different codons in the three species that have a leucine rather than a phenylalanine. The phenylalanine codon is TTT, and the leucine codons are TTA, TTG, and CTT, respectively, in M. m. hortulanus, M. caroli, and M. pahari. We conclude from these data that this amino acid change must be highly advantageous for functional change, as its change has been selected three independent times.
How have these genes evolved to rapidly accumulate nucleotide changes in the mature region of the peptide and to remain relatively unchanged in the signal peptide and intron? The rapid, accelerated evolution of the exon 2 domain is achieved by positive selection, and the striking sequence similarity between genes adjacent on the chromosome implies recent duplication. After duplication, both relaxation of purifying and the existence of strong positive selection may have had complementary roles in allowing the mature peptide region of duplicate genes to functionally diverge, as recently postulated for the evolution of the ribonuclease gene in a leaf-eating monkey (Zhang, Zhang, and Rosenberg 2002). Our cross-species analysis demonstrates that the duplications of all genes except Defb4 and Defb1 occurred after the divergence of Mus. Thus, the amino acid changes have occurred over a small time scale and imply a strong selective pressure. Bauer et al. (2001) recently showed that Defb7 and Defb8 (Defr1 variant) mature peptides are similar in tertiary structure, despite a high level of sequence change (58% identity). A high level of sequence change must be permissible without loosing antimicrobial function, and change is advantageous to confer pathogen specifity. The rapid evolution of these mouse-specific clades is reminiscent of the morpheus gene family recently described by Johnson et al. (2001), which arose after chromosomal duplication and positive selection during the emergence of humans and African apes.
Functional differences between murine ß-defensins identified to date have been observed (Lehrer and Ganz 2002). Our own data have demonstrated a species-specific antimicrobial action of Defb1 (Morrison et al. 1998; Morrison et al. 2002). We demonstrate here that positive selection acting on ß-defensins can account for the diversity observed between the mature peptide regions of paralogous and orthologous genes. Selection appears to promote changes in the charges of the residues encoded. This rapid adaptive evolution is in keeping with their proposed specialized role in innate immunity diversity and a necessity to respond to pathogen diversity.
![]() |
Supplementary Material |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Intron alignments, gene alignments, primer sequences, and nucleotide sequences from the cross Rodentia gene analysis are included in supplementary information available online only at the journal Web site.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
E-mail: julia{at}hgu.mrc.ac.uk.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bals, R., M. J. Goldman, and J. M. Wilson. 1998. Mouse ß-defensin 1 is a salt-sensitive antimicrobial peptide present in epithelia of the lung and urogenital tract. Infect. Immun. 66:1225-1232.
Bals, R., X. Wang, R. L. Meegalla, S. Wattler, D. J. Weiner, M. C. Nehls, and J. M. Wilson. 1999. Mouse ß-defensin 3 is an inducible antimicrobial peptide expressed in the epithelia of multiple organs. Infect. Immun. 67:3542-3547.
Bauer, F., K. Schweimer, E. Kluver, J. R. Conejo-Garcia, W. G. Forssmann, P. Rosch, K. Adermann, and H. Sticht. 2001. Structure determination of human and murine ß-defensins reveals structural conservation in the absence of significant sequence similarity. Protein Sci. 10:2470-2479.
Del Pero, M., M. Boniotto, D. Zuccon, P. Cervella, A. Spano, A. Amoroso, and S. Crovella. 2002. ß-defensin 1 gene variability among non-human primates. Immunogenetics 53:907-913.[CrossRef][ISI][Medline]
Ganz, T., and R. I. Lehrer. 1994. Defensins. Curr. Opin. Immunol. 6:584-589.[CrossRef][ISI][Medline]
Garcia, J. R., F. Jaumann, and S. Schulz, et al. (13 co-authors). 2001. Identification of a novel, multifunctional ß-defensin (human ß-defensin 3) with specific antimicrobial activity. Its interaction with plasma membranes of Xenopus oocytes and the induction of macrophage chemoattraction. Cell Tissue Res. 306:257-264.[CrossRef][ISI][Medline]
Hao, H. N., J. Zhao, G. Lotoczky, W. E. Grever, and W. D. Lyman. 2001. Induction of human ß-defensin-2 expression in human astrocytes by lipopolysaccharide and cytokines. J. Neurochem. 77:1027-1035.[CrossRef][ISI][Medline]
Hedges, S. B., P. H. Parker, C. G. Sibley, and S. Kumar. 1996. Continental breakup and the ordinal diversification of birds and mammals. Nature 381:226-229.[CrossRef][ISI][Medline]
Higgins, D. G., J. D. Thompson, and T. J. Gibson. 1996. Using Clustal for multiple sequence alignments. Methods Enzymol. 266:383-402.[ISI][Medline]
Hill R. E., and N. D. Hastie. 1987. Accelerated evolution in the reactive centre regions of serine protease inhibitors. Nature 326:96-99.[CrossRef][ISI][Medline]
Hughes, A. L. 1999. Evolutionary diversification of the mammalian defensins. Cell Mol. Life Sci. 56:94-103.[CrossRef][ISI][Medline]
Hughes, A. L., and M. Yeager. 1997. Coordinated amino acid changes in the evolution of mammalian defensins. J. Mol. Evol. 44:675-682.[ISI][Medline]
Huttner, K. M., C. A. Kozak, and C. L. Bevins. 1997. The mouse genome encodes a single homolog of the antimicrobial peptide human ß-defensin 1. FEBS Lett. 413:45-49.[CrossRef][ISI][Medline]
Jia, H. P., S. A. Wowk, B. C. Schutte, S. K. Lee, A. Vivado, B. F. Tack, C. L. Bevins, and P. B. McCray, Jr. 2000. A novel murine ß-defensin expressed in tongue, esophagus, and trachea. J. Biol. Chem. 275:33314-33320.
Johnson M., L. Viggiano, J. Bailey, M. Abdul-Rauf, G. Goodwin, M. Rocchi, and E. Eichler. 2001. Positive selection of a gene family during the emergence of humans and African apes. Nature 413:514-519.[CrossRef][ISI][Medline]
Jukes T. H., and C. R. Cantor. 1969. Evolution of protein molecules. Pp. 21132 in H. N. Munro, ed. Mammalian protein metabolism. Academic Press, New York.
Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16:111-120.[ISI][Medline]
Kumar, S., K. Tamura, I. B. Jakobsen, and M. Nei. 2001. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244-1245.
Lehrer, R. I., and T. Ganz. 2002. Defensins of vertebrate animals. Curr. Opin. Immunol. 14:96-102.[CrossRef][ISI][Medline]
Li, P., H. Chan, B. He, S. So, Y. Chung, Q. Shang, Y. Zhang, and Y. Zhang. 2001. An antimicrobial peptide found in the male reproductive system of rats. Science 291:1783-1785.
Miyata, T., S. Miyazawa, and T. Yasunaga. 1979. Two types of amino acid substitutions in protein evolution. J. Mol. Evol. 12:219-236.[ISI][Medline]
Morgenstern, B. 1999. DIALIGN 2: improvement of the segment-to-segment approach to multiple sequence alignment. Bioinformatics 15:211-218.
Morrison, G. M., D. J. Davidson, and J. R. Dorin. 1999. A novel mouse ß defensin, Defb2, which is upregulated in the airways by lipopolysaccharide. FEBS Lett. 442:112-116.[CrossRef][ISI][Medline]
Morrison, G. M., D. J. Davidson, F. M. Kilanowski, D. W. Borthwick, K. Crook, A. I. Maxwell, J. R. Govan, and J. R. Dorin. 1998. Mouse ß defensin-1 is a functional homolog of human ß defensin-1. Mamm. Genome 9:453-457.[CrossRef][ISI][Medline]
Morrison G., M. Rolfe, F. Kilanowski, S. Cross, and J. R. Dorin. 2002. Identification and characterization of a novel murine ß-defensin related gene. Mamm. Genome 13:445-451.[CrossRef][ISI][Medline]
Nei, M., and T. Gojobori. 1986. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol. Biol. Evol. 3:418-426.[Abstract]
Nei, M., X. Gu, and T. Sitnikova. 1997. Evolution by the birth-and-death process in multigene families of the vertebrate immune system. Proc. Natl. Acad. Sci. USA 94:7799-7806.
Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425.[Abstract]
Schutte, B. C., J. P. Mitros, J. A. Bartlett, J. D. Walters, H. P. Jia, M. J. Welsh, T. L. Casavant, and P. B. McCray, Jr. 2002. Discovery of five conserved ß-defensin gene clusters using a computational search strategy. Proc. Natl. Acad. Sci. USA 99:2129-2133.
Swanson W., and V. Vacquier. 2002. The rapid evolution of reproductive proteins. Nat. Rev. Genet. 3:137-144.[CrossRef][ISI][Medline]
Townley D. J., B. Avery, B. Rosen, and W. C. Skarnes. 1997. Rapid Sequence analysis of gene trap integrations to generate a resource of insertional mutations in mice. Genome Res. 7:293-298.[Abstract]
Yamaguchi, Y., S. Fukuhara, T. Nagase, T. Tomita, S. Hitomi, S. Kimura, H. Kurihara, and Y. Ouchi. 2001. A novel mouse ß-defensin, mBD-6, predominantly expressed in skeletal muscle. J. Biol. Chem. 276:31510-31514.
Yang, D., O. Chertov, and S. N. Bykovskaia, et al. (11 co-authors). 1999. ß-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 286:525-528.
Zhang, J. 2000. Rates of conservative and radical nonsynonymous nucleotide substitutions in mammalian nuclear genes. J. Mol. Evol. 50:56-68.[ISI][Medline]
Zhang, J., K. D. Dyer, and H. F. Rosenberg. 2000. Evolution of the rodent eosinophil-associated RNase gene family by rapid gene sorting and positive selection. Proc. Natl. Acad. Sci. USA 97:4701-4706.
Zhang, J., S. Kumar, and M. Nei. 1997. Small-sample tests of episodic adaptive evolution: a case study of primate lysozymes. Mol. Biol. Evol. 14:1335-1338.
Zhang, J., H. F. Rosenberg, and M. Nei. 1998. Positive Darwinian selection after gene duplication in primate ribonuclease genes. Proc. Natl. Acad. Sci. USA 95:3708-3713.
Zhang J., Y. Zhang, and H. Rosenberg. 2002. Adaptive evolution of a duplicated pancreatic ribonuclease gene in a leaf-eating monkey. Nat. Genet. 30:411-415.[CrossRef][ISI][Medline]