Institute of Molecular Evolutionary Genetics and Department of Biology, Pennsylvania State University
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Abstract |
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Introduction |
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Protamine P1 is a small protein but contains a large proportion of positively charged arginine residues that allow it to tightly bind and condense sperm DNA. Interestingly, the total number of amino acids in protamine P1 and the positions of the arginine residues vary considerably with taxonomic group, yet the proportion of arginine residues remains nearly the same (fig. 1
). This indicates that arginine positions in protamine P1 are subject to evolutionary change and that the conservation of a high proportion of arginines is maintained at the protein level rather than at the amino acid site level. This pattern of conservation of amino acids (arginines) is different from that of most proteins, in which a particular set of amino acids are conserved by purifying selection at a given set of amino acid positions that are usually functionally important (Kimura 1983
; Nei 1987
). This suggests that protamine P1 is subject to an unusual form of selection. The purpose of this paper is to investigate the pattern of selection involved in this unusually arginine-rich protein.
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Materials and Methods |
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Results |
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It is apparent that nucleotide compositions are different between the intron and the coding regions (fig. 2 ). For example, in marsupials, the overall frequency of G in the coding regions is 37% considering all three codon positions combined, but it is 25% in the intron (fig. 2 ). Arginine residues are encoded by the six codons AGA, AGG, CGA, CGC, CGG, and CGT, which are used in the proportion of 0.45:0.27:0.01:0.08:0.11:0.08 in marsupial protamine P1 sequences. Because these codons contain G, selection for arginines in protamine P1 is expected to increase the frequency of G in the coding regions overall. To study this problem in more detail, we computed the nucleotide frequencies at each codon position separately. An increase in G due to selection for arginine should be most apparent at second codon positions, since most amino acid changes occur at second positions. We found that the differences in nucleotide frequency among the three codon positions of the protamine P1 gene are enormous (fig. 2 ). For example, the frequencies of G are only 7% and 26% at the first and third codon positions, respectively, in marsupial protamine P1, while the frequency of G at second positions is 77%. Essentially the same results are obtained for monotremes and placentals. These observations suggest that the selection pressure to maintain a large proportion of arginines in protamine P1 has changed the nucleotide frequency of the coding regions.
Arginine-rich Selection
Although arginine content is nearly the same (about 50%) for all taxonomic groups, the arginine positions in the sequence vary among them (fig. 1
). This suggests that the level of nonsynonymous substitutions would not be very low compared with that of synonymous substitution despite the purifying selection. To examine this problem, we computed dS and dN for different pairs of sequences. However, we computed these values only for the sequences within each of the monotreme, marsupial, and placental species groups, because the sequence lengths of the three species groups were quite different, and the sequence alignments among them were not very reliable. In this computation, we eliminated all alignment gaps, and the average values (S and N) of dS and dN for each group of pairwise comparisons were computed.
The results obtained (table 1
) show that N/S is less than 1.0 in all mammalian groups, but the ratio is not as low as that for many other genes. For example, Zhang (2000)
computed S and N for 47 genes evolving at moderate rates in primates and artiodactyls. They were S = 0.325 and N = 0.090. Therefore, N/S is 0.28. Of the 47 genes examined, the interleukin genes 6 and 7 showed N/S ratios of 0.90 and 0.66, respectively, but in all other genes, N/S was usually about 0.3 or less. These comparisons indicate that the N/S in protamine P1 is quite high despite the action of a special type of purifying selection, as predicted earlier.
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Discussion |
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One of the driving forces for arginine-rich selection on mammalian protamine P1 is probably the sperm DNAbinding function of this protein. However, if selection is driven solely by the need to maintain the basic charge of the protein, then perhaps lysine should also have a high frequency in protamine P1, since lysine is another basic amino acid. Yet, this pattern is not observed (fig. 1
). Why is lysine not used? The answer to this question appears to be the special function arginine has at the time of sperm-egg fertilization. Ohtsuki et al. (1996)
found that protamine P1, by way of polyarginine clusters, is able to activate casein kinase II (CK-II) in fertilized eggs, while oligopolylysine clusters cannot. CK-II is a serine/threonine protein kinase that is responsible for cellular metabolic alteration through phosphorylation of more than 50 different cellular polypeptides. Specifically, arginine residues were found to interact with an acidic amino acid motif of the regulatory ß subunit of CK-II (Ohtsuki et al. 1996
). Therefore, the high frequency of arginines in protamine P1 is apparently caused by the requirement of the amino acid for binding of sperm DNA and for activating an important regulatory protein in the fertilized egg.
So far, we have discussed only mammalian protamine P1. However, protamine genes also exist in other vertebrate classes, although the structure of these genes is somewhat different from those of mammals. The most notable difference is the fact that only mammals have an intron in their protamine P1 gene. Among the nonmammalian vertebrates, birds are most similar to mammals in terms of protamine gene organization (Oliva and Dixon 1989
). However, unlike mammals and birds, a multigene family of about 1520 protamine genes exists in rainbow trout, a teleost fish species (Dixon et al. 1986
). Unfortunately, the genomic organization of the protamine genes of amphibians and reptiles has not been studied. The only invertebrate animal so far studied is an insect, the boll weevil (Anthonomus grandis).
The protamine sequences from nonmammalian animals cannot be aligned reliably because the sequence length varies with species group and the sequence divergence is high. However, arginine content is very high in all animals, although the content for the boll weevil is somewhat lower (table 2 ). The relative nucleotide frequencies in the first, second, and third positions are also similar to those of mammalian protamine P1 genes (fig. 3 ). Therefore, the same type of purifying selection for maintaining a high arginine content is occurring in all animal species in which protamines are found. Of course, the animal species groups that have been studied so far are quite limited. It is therefore unclear whether the above unusual type of purifying selection is operating on all or most animal protamine genes.
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Acknowledgements |
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Footnotes |
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1 Present address: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.
2 Keywords: protamine
arginine
selection
nucleotide composition bias
3 Address for correspondence and reprints: Alejandro P. Rooney, Institute of Molecular Evolutionary Genetics and Department of Biology, 328 Mueller Laboratory, Pennsylvania State University, University Park, Pennsylvania 16802. E-mail: apr3{at}psu.edu
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