Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health
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Abstract |
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Introduction |
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Unexpectedly, a recent study revealed what appears to be diversifying selection acting on a tumor suppressor gene, BRCA1, in the evolution of humans and chimpanzees (Huttley et al. 2000)
. In this report, we describe yet another case of diversifying selection in a cancer-related gene, angiogenin (ANG). ANG, also known as RNase 5, is a member of the RNase A superfamily (reviewed in Riordan 1996
). ANG was originally isolated from human tumor cellconditioned medium based on its ability to stimulate the formation of new blood vessels (Fett et al. 1985
). This activity, as opposed to tumorigenesis, is a more relevant physiologic function from an evolutionary perspective. However, ANG expression is elevated in various tumors, and its activity is related to cancer progression (Shimoyama et al. 1996
; Montero et al. 1998
; Miyake et al. 1999
; Shimoyama et al. 1999
; Shimoyama and Kaminishi 2000
), with its antagonists having the ability to inhibit cancer growth in vivo (Olson et al. 1994, 1995
; Piccoli et al. 1998
). We begin by examining the phylogenetic relationships of the paralogous ANG genes of various mammalian species and then provide evidence for diversifying selection of the single-copy ANG gene in primates.
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Materials and Methods |
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Genotyping
The ANG coding region was amplified from genomic DNAs of 11 primate species by PCR with primers ANG-5 (5'-GTGTTGGAAGAGATGGTGATGGGC-3') and ANG-3 (5'-AGCACTTGACCAGGGGCCCGCTGGTTA-3'). The first four codons of the ANG gene were encoded in the ANG-5 primer and were excluded from subsequent analysis. The PCR products were cloned into a PCR II TA cloning vector (Invitrogen, San Diego, Calif.) and sequenced from both directions by the dideoxy chain termination method, with the Perkin-Elmer 377 automatic sequencer. The polymorphic alleles were obtained by PCR of 35 humans (8 Chinese, 5 Native Americans, 10 Europeans, and 12 African Americans) and 13 chimpanzees (5 obtained from San Diego Zoo, 1 from Coriell, and 7 from Yerkes), using the high fidelity Taq (Life Technologies, Rockville, Md.) with primers 253 (5'-GGTATGTCTTAATGTGCCTCAGG-3') and ANG-3. The PCR products were subject to direct sequencing, as described in Zhang and Rosenberg (2000)
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Evolutionary Analysis
Sequences of nonprimate ANGs were obtained from GenBank (accession numbers follow genus/species classificationrabbit Oryctolagus cuniculus: P31347; cattle Bos taurus: P10152; cattle2: P80929; pig Sus scrofa: P31346; mouse Mus musculus: NM_007447; mouse2: NM_007449; mouse3: U72672; chicken Gallus gallus: X61193). Two ANG pseudogenes and an EST from the mouse (reviewed in Strydom 1998
) were not included in the analysis. Phylogenetic trees were reconstructed by the neighbor-joining method (Saitou and Nei 1987
) with 1,000 bootstrap replications (Felsenstein 1985
). The MEGA2 program (Kumar et al. 2000
) was used for phylogenetic analysis. The numbers of synonymous (dS) and nonsynonymous (dN) nucleotide substitutions per site between ANG sequences were computed as described in Zhang, Rosenberg, and Nei (1998)
, with an estimated transition-transversion ratio (Kimura 1980
) of 1.2. The numbers of synonymous and nonsynonymous substitutions per site for each tree branch were estimated from the pairwise distances, using the least-squares method with a given tree topology (Zhang, Rosenberg, and Nei 1998
). The ancestral sequences were inferred by the distance-based Bayesian method (Zhang and Nei 1997
). Because the species concerned are closely related, the accuracy of the ancestral inference is greater than 96% for all nodes. The potential numbers of synonymous (S ), nonsynonymous (N ), conservative nonsynonymous (C ), and radical nonsynonymous (R) sites as well as the observed substitutions (s, n, c, r) at these sites for each branch were estimated (Zhang, Rosenberg, and Nei 1998
; Zhang 2000
). Binomial tests were used to test the substitution rate difference at various types of sites. For instance,
n is compared with the binomial distribution B(
s +
n, N/[S + N]) to test if the nonsynonymous substitution rate is significantly greater than the synonymous rate for the entire tree, where
s and
n are sums of s and n over all tree branches. In the present case, the binomial test is more appropriate and more conservative than the test described in Zhang, Kumar, and Nei (1997)
because multiple substitutions at individual sites can be taken into account. Following Zhang, Kumar, and Nei (1997)
, Fisher's test was used to test the difference in n/s among different branches (i.e., the episodic evolution hypothesis; Messier and Stewart 1997
). DnaSP (Rozas and Rozas 1999
) was used for estimating the nucleotide diversity
and its sampling error (Nei 1987
) and for performing the Tajima's (1989)
and Fu and Li's (1993)
tests of neutrality. The age of the most recent common ancestor of a sample of alleles was estimated following the method of Fu and Li (1996)
.
Recombinant ANGs and the Angiogenic Activity Assay
The ANG genes of the human and owl monkey were subcloned into the bacterial expression vector pFLAG CTS (Kodak, New Haven, Conn.) and were verified by sequencing. The vector adds the octapeptide DYKDDDDK (FLAG) to the recombinant protein, which facilitates its purification and detection with M2 anti-FLAG monoclonal antibody. Recombinant proteins were isolated, purified, and quantified as described in Rosenberg and Dyer (1995)
. The angiogenic activity of the recombinant ANGs was tested using the chicken embryo chorioallantoic membrane (CAM) assay (Fett et al. 1985
; Gho, Kleinman, and Sosne 1999
). That is, salt-free aqueous solution containing recombinant ANGs was loaded onto a Thermonox disc (Nunc, Naperville, Ill.). The sample was dried under sterile air before being applied to the CAM of a chicken embryo aged 10 days. After 72-h incubation at 37°C, positive (appearance of a spokewheel pattern) or negative responses were assessed under a microscope. Each sample was tested in at least two independent experiments. The numbers of positive and negative responses for each sample from multiple experiments were combined, and a G-test was used to test the difference in angiogenic activity between samples. Water was used as the negative control.
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Results |
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Angiogenic Activity of Recombinant ANGs
As mentioned earlier, diversifying selection often widens the spectrum of the recognizable ligands but rarely changes the main function of the protein. To examine if this is the case for ANG, we made recombinant ANG proteins from the human and owl monkey, a New World monkey, and compared their angiogenic activities, using the chicken embryo CAM assay (Table 1
). We found that both ANGs are angiogenic (P < 0.01, G-test), with no statistically significant difference in activity (P > 0.4, G-test). This result suggests that, despite rapid evolution, primate ANG has maintained its ability to stimulate blood vessel formation. This finding provides further evidence suggesting that the positive selection acting on ANG is unlikely to be directional selection.
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Discussion |
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There are a number of intriguing similarities in the evolutionary histories of ANG and those of the breast cancer suppressor gene, BRCA1 (Huttley et al. 2000
). Similar to our results with ANG, comparisons of BRCA1 among humans, chimpanzees, and several other primate lineages yield dN/dS ratios either higher than or close to 1. This finding of positive selection operating in many lineages is suggestive of diversifying selection and is similar to that reported here for primate ANG genes. Similarly, the most variable sites in BRCA1 reside in a specific protein-protein interaction domain. These common features characterizing the positive selection of both BRCA1 and ANG raise the intriguing possibility of unusual selective pressures on specific cancer-related genes in primates. It is noteworthy that all these genes have normal physiological functions apart from their roles in cancer promotion or suppression, and their adverse effects may be related to a side effect of physiological function, random mutations, or alterations in expression. Because most tumors form after reproductive age, tumor promotion or suppression itself is not likely to be subject to natural selection, and the selective pressures acting on ANG are most likely related to another, more physiological role for this protein. Several authors have discussed a role for ANG during pregnancy in tissue vascularization of the developing embryo (Hayashi, Yanagihara, and Hata 2000
; Koga et al. 2000
; Malamitsi-Puchner et al. 2000
). The diversifying selection on ANG may be related to physiologic issues of conflict of interests between mothers and fetuses, as suggested by Haig (1993)
in explaining the origin of genetic imprinting. It is intriguing to consider the possibility that the natural selection that has improved ANG's fitness for prereproductive, physiologic events yielded tumor promotion and cancer progression as undesirable, albeit postreproductive results.
Future studies on the evolution of ANG-binding molecules are likely to determine whether these molecules are also under diversifying selection and how they cope with the rapid evolution of ANG. RI is of particular interest in this regard because it is an antagonist of ANG and thus may play a regulatory role in one or more of the compatibility issues noted earlier. Consistent with this idea, RI is expressed in the placenta and other tissues, and our present work reveals charge-altering substitutions in nearly 40% of the RI-binding residues in primate ANGs.
Intraspecific polymorphisms of genes under diversifying selection vary greatly. For example, the accessory gland protein Acp26Aa of Drosophila mauritiana (Tsaur, Ting, and Wu 2001)
and human MHC loci (Nei and Hughes 1991
) have very high degrees of polymorphism, but human immunoglobulin (Li and Hood 1995
; Sasso, Buckner, and Suzuki 1995
), human protamine (Wyckoff, Wang, and Wu 2000
), and abalone sperm lysin loci (Metz, Robles-Sikisaka, and Vacquier 1998
) have quite low levels of variation. This dichotomy may reflect two different modes of diversifying selection, with one being an overdominant or frequency-dependent selection process that maintains polymorphism, and the other, a selection process that promotes interspecific changes by purging the intraspecific variations through selective sweeps. In the case of ANG, we detected a normal level of polymorphism in humans but no polymorphism in chimpanzees. Further analysis suggested the possibility of a selective sweep of a His23 allele replacing the Asp23 allele in chimpanzee evolution. Using the nucleotide differences between the human and chimpanzee ANG sequences (fig. 5
) and assumptions of a generation time of 20 years, an effective population size of 30,000, and a divergence time of 6 Myr between humans and chimpanzees (Kaessmann et al. 2001
), we estimated that the age of the most recent common ancestor of the 26 chimpanzee alleles examined here is Tmode = 915,200 years, which is about half of the corresponding age (1.9 Myr) estimated from noncoding regions (Kaessmann et al. 2001
). Although this comparison is consistent with the selective sweep hypothesis for the chimpanzee ANG gene, it has to be pointed out that T has a substantial stochastic variance. A larger sample of chimpanzees and inclusion of sequence from closely related bonobo species, together with an analysis of a larger region of gene sequence surrounding the ANG coding sequence, needs to be surveyed in order to time this potential selective sweep more precisely. Examination of pygmy chimpanzees may also help time the event. It is also possible that the selective sweep does not result from selection at ANG per se, but rather from a closely linked locus.
Although diversifying selection of ANG is detected in higher primates, it is unclear whether the same selection operates in other mammals. The evolutionary pattern of ANG may differ when multiple copies of ANG genes are present, as in the mouse and cattle. It is known that cattle ANG2 and mouse ANG3 are angiogenic (Strydom, Bond, and Vallee 1997
; Fu et al. 1999
), whereas mouse ANG2 is not (Nobile, Vallee, and Shapiro 1996
). It will be interesting to compare the selective pressures on and evolutionary rates of the angiogenic and nonangiogenic ANG genes because such information will be useful toward improving our understanding of the physiology of these proteins and may have an impact on future studies relating to ANG and cancer research.
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Acknowledgements |
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Footnotes |
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Present address: Departments of Ecology and Evolutionary Biology and Molecular, Cellular, and Developmental Biology, University of Michigan
Keywords: angiogenin
diversifying selection
evolution
ribonuclease
cancer
primates
Address for correspondence and reprints: Helene F. Rosenberg, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 11N104, 9000 Rockville Pike, Bethesda, Maryland 20892. hr2k{at}nih.gov
.
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