Department of Biochemistry, University of Otago, Dunedin, New Zealand
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Abstract |
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Introduction |
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The basis of the theory of the evolutionary molecular clock is that particular proteins and nucleic acids evolve at a characteristic and constant rate per unit time within lineages (Margoliash 1963
; Zuckerkandl and Pauling 1965
). This rate may be influenced by mutation rate, generation time, and the fidelity of the DNA repair system. The reliability of a molecular clock has been questioned before (Wu and Li 1985
; Vawter and Brown 1986
; Li and Tanimura 1987
; Li, Tanimura, and Sharp 1987
; Ayala, Barrio, and Kwiatowski 1996
; Gaut et al. 1996
; Li et al. 1996
).
Parartemia is another genus of brine shrimp, the members of which differ from Artemia in their larger size and other morphological features (Geddes 1980
). Parartemia is endemic to Australia, where it is found in both inland lakes and coastal locations. Artemia, however, has a worldwide distribution and in Australia is found only in coastal, man-made salt lakes, suggesting that it has been introduced (Geddes 1980
). According to traditional phylogeny, the two anostracan branchiopod genera are distinct, with Artemia occupying a monogeneric family, the Artemiidae, while Parartemia is grouped with the family Branchipodidae (Linder 1941
). Thus, the adaptation to salinity is reported to have occurred separately in two different lineages (Geddes 1975
). Unlike the cysts of Artemia, those of Parartemia do not float, but sink to the bottom, where they combine with sediments, thus greatly reducing their availability for dispersal. This feature, together with the migration of water bodies with climatic changes in Australia, is believed to have influenced the evolution of Parartemia (Geddes 1981
). The two genera do coexist in some coastal salt lakes, usually in ponds of intermediate salinity, but are more often found separately, with Artemia occupying the ponds of higher salinity.
Artemia responds to decreased oxygen partial pressure by hemoglobin synthesis, which accounts for up to 2% of hemolymph protein and gives its species their distinctive red color (Gilchrist 1955
). Manwell (1978)
showed that Parartemia contains a putative hemoglobin molecule that is much less abundant than in Artemia, accounting for only 0.01%0.02% of hemolymph proteins. The fact that Parartemia cannot tolerate such high salinities as Artemia and never attains a red color suggests that it is unable to upregulate hemoglobin production under conditions of low oxygen partial pressure (Geddes 1980
). There is no experimental evidence concerning the function of hemoglobin in Parartemia, and the necessity for an oxygen carrier in such a small organism is puzzling. Manwell (1978)
suggested that the Parartemia hemoglobin molecule exists in such small amounts as to make its contribution as an oxygen carrier questionable and that its continued existence in minimal amounts was necessary for heme transport.
Previously, we described one domain from a putative Parartemia hemoglobin (Coleman, Geddes, and Trotman 1998
). In the present paper, we describe the cDNA and derived amino acid sequences for the entire Parartemia hemoglobin molecule. The sequence is one internal codon longer than that for Artemia and codes for nine domains, and many conserved features of the translated sequence have allowed an exact alignment between the two genera. The single-codon difference was in the crucial heme-binding region of Parartemia domain 9 (P9; nomenclature is described in Materials and Methods), where the codons for amino acids C2 and C3 have been retained, unlike both C and T polymers of Artemia, for which in domain 9 the two codons are contracted to one (Supplementary Material). Thus, where the Parartemia sequence from C1 to C4 translates to -HSEY-, Artemia C and T in the same region have -HGY-. This supports the proposal that the divergence of Parartemia and Artemia predated the loss of this codon and the divergence of the Artemia C and T polymers, assuming the lack of gene conversion events between T and C. Because the divergence of Parartemia and Artemia appears to predate the formation of the paralogous T and C polymers in Artemia, the distance between P and C should equal that between P and T, assuming T and C have evolved at the same rate. We examined the patterns of substitution at synonymous and nonsynonymous sites and asked whether discrepancies in evolutionary rate could be traced to particular domains or genes of the three genes available (two Artemia genes and one Parartemia gene). It was found that discrepancies were associated with the Artemia T gene and could be localized to particular domains.
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Materials and Methods |
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RNA Isolation and Analysis
Frozen shrimp (35 g wet weight) were thawed at 37°C, and total RNA was extracted using TRIzol (Gibco-BRL) according to the manufacturer's instructions, taking precautions to prevent nuclease contamination. RNA integrity was checked by running samples on a 1.1% formaldehyde gel, and transcript size was examined by Northern blotting using an Artemia hemoglobin cDNA probe.
cDNA Library Construction
Total RNA was used to generate cDNA using a Capfinder cDNA kit (Clontech) according to the manufacturer's instructions. This involved using a modified oligo-dT primer, MMLV reverse transcriptase, and a patented generic oligonucleotide to the eukaryotic 5' cap to produce first-strand cDNA. By using PCR primers, the cDNA was amplified before addition of adapter linkers and cloning into the bacteriophage vector gt11. As screening of this library only yielded clones containing the C-terminal part of the globin transcript, sequence from these clones was used to construct a 5' enriched cDNA library. The same procedure was followed, except that the oligo-dT primer was replaced with a Parartemia hemoglobin-specific oligonucleotide, NL10A, which corresponds to the peptide sequence in domain 7. Primer sequences were as follows. For the first library first-strand synthesis, the 5' primer was CapSwitch (5'- TACGGCTGCGAGAAGACGACAGAAGGG-3'); for PCR, the 5' primer was Clontech (5'-TACGCTGCGAGAAGACGACAGAA-3'); and the 3' primer was Oligo(dT)30N-1N (N = A, G, C, or T; N-1 = A, G, or C). The second library was handled in an identical fashion except for substitution of the primer NL10A (5'- GCATGTTCTTGACACGG-3') as the 3' primer.
PCR Conditions
PCR conditions were as follows: dNTPs 200 nM, 1 x KlenTaq polymerase, 1 x KlenTaq buffer, oligonucleotides 200 nM, 1:50 dilution of first-strand cDNA (starting RNA 1 µg); 95°C for 1 min, and 20 cycles of 95°C for 15 s and 68°C for 5 min.
Cloning into gt11
cDNA products were blunt-ended, had EcoR1- Not1-Sal1 adaptors added to the ends, and were phosphorylated and size fractionated before an overnight 16°C ligation into gt11 according to the manufacturer's instructions (Clontech User manual PT3000-1).
Screening of cDNA Library
Initial screening of the first library of 106 cDNA clones was performed with a mixture of two cDNA probes: a 1.3-kb section corresponding to parts of domains 6 and 7 from polymer T of the Artemia hemoglobin, and a 2-kb section corresponding to domains one to four of polymer C of the Artemia hemoglobin (Matthews, Vandenberg, and Trotman 1998
). Permissive conditions (hybridization and washes at 58°C) were used to allow for the expected mismatching between the sequences. For the second library, the screening of 200,000 clones was performed with a mixture of two 2- kb cDNA probes corresponding to domains 14 from both the T and C polymers of the Artemia hemoglobin, and a further domain 1 polymer C probe was used to confirm the presence of the 5' end (Matthews, Vandenberg, and Trotman 1998
).
Sequencing of Clones
The inserts of the gt11 clones were amplified by PCR using oligonucleotides designed to flanking vector sequences. The products were directly sequenced using an ABI automated sequencer. Sequence fidelity was verified by sequencing in both directions and using different PCR products as templates.
Sequence and Data Analysis
The GCG (Genetics Computer Group Inc.) sequence analysis package, version 7.3, was used to obtain amino acid sequence by translation of the cDNA sequence, to make alignments, and to determine DNA homologies. Data on substitution at synonymous and nonsynonymous sites (KS and KA values) were generated using the method of Li (1993)
. This method allows for differences in substitution rates between transitions and transversions and treats nondegenerate, twofold-degenerate and fourfold-degenerate sites separately. Some similarities were computed using HOMED (Stockwell 1988
).
Nomenclature
The globin proteins and their genes are known as T and C in Artemia and as P in Parartemia, followed where appropriate by the domain numbers 19. Helices of the globin fold are identified, as is conventional, by the letters AH and by NA at the N-terminus and HC at the C-terminus, followed by the amino acid number within the motif. Linker sequence between an H helix and the following A helix is designated HA, and the whole linker is named according to the domains linked, e.g., L4/5, or L-/1 and L9/- at the N- and C-termini, respectively.
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Results |
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Structural Interpretation of the Amino Acid Sequence
The derived amino acid sequence of each Parartemia domain reflects Artemia globin's conformity with the conventional globin fold while for the most part sharing idiosyncrasies characteristic of the Artemia globins. Sequence features that key the Parartemia sequence into the generic globin alignment center on the mandatory F8 proximal His. Also notable are the widely conserved A12 Trp, the B6 Gly which may assist close packing against the E helix, the E7 distal His, and the H8 Trp. Tryptophan, being large and rarely substituted, provides important alignment landmarks.
Certain features characteristic of Artemia globin domains are confirmed in the Parartemia alignment. Invariant within all 18 Artemia domains, and reflected in Parartemia, are a Phe at B10; a Tyr at C4, where it is very unusual in globins; and a Gly at F5. Of even greater significance, since its irregular occurrence is replicated in domains 79 in both genera, is an unusual hydrophobic Leu at A14, along with a generally rare occurrence of a His at G4 in the same 7 out of 9 domains (domains 5 and 7 being the exceptions) in both genera.
The Parartemia globin has an N-terminal signal sequence identical in length to that of Artemia, coding for 17 amino acids. However, 10 of these 17 residues in Parartemia differ from the T or from the C signal sequence of Artemia, and 7 differ from both T and C. Conversely, domain P9 has a carboxy-terminal extension of 17 residues past the end of the H-helix that is identical in length and sequence to that of domain C9 and has only one conservative Ile-to-Val substitution compared with T9. Eight linker sequences, respectively, join the H-helix of a domain to the A-helix of the next. All linkers are 14 residues in length except for L4/5 (joining domains 4 and 5), which is 16 residues long, and L5/6, which is 13 residues long, with these lengths in Parartemia mirroring those in Artemia. The linkers show a range of identity between each other within the Parartemia polymer as high as 85.7% between L2/3 and L6/7, ranging down to 21.4% identity in three other instances (table 1 ) (excluding the N-terminal and C-terminal extensions L-/1 and L9/-). Similarities of character are substantially higher, ranging from 93.3% between L2/3 and L6/7 down to 62.7% between L4/5 and L5/6.
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Analysis of Rates of Protein Divergence
Sequence analysis leads to the conclusion that the polymeric construct of nine globin domains was achieved before the divergence of Parartemia and Artemia. Within the Parartemia polymer, the greatest amino acid difference between domains (excluding linkers) is 78.3% (21.7% identity; table 1
), which, as with Artemia, correlates with about 230 changes per 100 sites if the Dayhoff, Barker, and Hunt (1983)
empirical correction is applied. This places the divergence of the nine domains from a single domain very broadly around a billion years ago.
The divergence of Artemia and Parartemia is much more recent. That their globins diverged from an ancestral 9mer is evident from the duplication of unusual sequence motifs. The genera differ in globin sequence length only by the deletion of residue C3 in Artemia domains 9. At the amino acid level, the Parartemia polymer differs from Artemia T and C by 17.7% and 13.9%, respectively (table 2
). If the molecular-clock calibration of about 1% divergence at the amino acid level per 5 Myr commonly attributed to globins is adopted (Dickerson and Geiss 1983
), the average difference between Parartemia and Artemia of 15.8% would equate to a common ancestor nominally about 80 MYA. Multiple hits were not corrected for, since the correction of about 1% would be smaller than probable natural fluctuation. This date is not inconsistent with the more recent divergence of the two Artemia polymers about 60 MYA (Matthews, Vandenberg, and Trotman 1998
), with the deletion of residue C3 therefore having happened between 80 MYA and 60 MYA if the molecular clock is applicable in this case.
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Previous findings showed that equivalent pairs of domains in the C and T polymers had amino acid differences ranging up to 2.7-fold greater than those of others (T6 and C6, 17.3%; T7 and C7, 6.4%), showing that some domains appear to have diverged more than others (Matthews, Vandenberg, and Trotman 1998
). This variation is not as evident between the two genera, with the largest difference being 1.6-fold (T2 and P2, 21.4%; T3 and P3, 12.7%). Domain 3, however, does appear to have been more conserved in all three polymers; domains T3 and P3 are 12.7% different, whereas domains C3 and P3 are 10.2% different, both well below the respective means (17.4% for T and P, 13.3% for C and P, unweighted for domain length) (table 2
).
Analysis of Rates of DNA Divergence
At the DNA level, substitutions at nonsynonymous sites (KA) reflect the above pattern. The number of substitutions per 100 nonsynonymous sites between T and P (KATP) is 10.5 ± 0.8 (SE), while that between C and P (KACP) is only 8.2 ± 0.5, and the difference between these values, KATP - KACP = 2.3 ± 0.94, is statistically significant at the 5% level (table 2
). No reliable conclusion can be drawn about the rate of synonymous substitutions (KS), as in many cases these are approaching saturation. At the domain level, domain 3 again has a low KA value with respect to the mean (table 2
).
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Discussion and Conclusions |
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The Parartemia and Artemia globins, having average differences of 21.2% at the DNA level and 15.8% at the protein level, are close enough for divergences to be measured with certainty but distant enough for significant evolutionary events to have distinguished the two genera. Superficially, the amino acid differences suggest that protein P is related more closely to C (13.9% different) and less closely to T (17.7% different); therefore, a hypothetical variant of P, more like T, might exist; however, no evidence for the existence of such a protein or its gene can be found. The absence of the C3 deletion from Parartemia domain P9 is further evidence against the existence of two paralogous globin polymers in the common ancestor of Parartemia and Artemia, as the rare deletion event would need to have happened in exactly the same place in two different genes in the Artemia line, which is highly unlikely. Taking this evidence into account, we base the following conclusions about evolutionary rates on the existence of a single P gene.
The positive sign given by the KATP - KACP calculation implies that T has evolved more quickly than C. Instances of accelerated evolution after a duplication event have been noted (Czelusniak et al. 1982
; Tamechika et al. 1996
). Accelerated evolution usually occurs when a Darwinian positive selection pressure has applied and is sometimes associated with function switching (Wallis 1997
; Ohno et al. 1998
). Positive selection could have favored the formation of functionally distinct globin polymers, and its occurrence is conventionally determined by calculating the KA/KS ratio of a gene. Positive selection is normally invoked when the KA/KS ratio of a gene exceeds unity, but in no pairwise comparisons in the present study did it do so. However, the low generation time of the brine shrimp is likely to increase preferentially KS compared with KA. Furthermore, only a few sites may be influenced by positive selection, as only a few changes may be necessary for a functional change. Without knowledge of what sites are involved in functional differences, it would be difficult to confirm the actions of positive selection. The fact that accelerated rates of substitution may be short in duration is likely to limit detection of examples of positive selection using the criterion KA/KS > 1. It is not presently known which parts of the polymers interact to form the homo- and heterodimers; the extent of the divergence between the two polymers will also be subject to structural and functional constraints, and the conservation of domains T3 and C3 may reflect this. The chromosomal locations of the two genes are not known, and it is possible that they have become spatially separated and are now subject to differing mutation rates (Wolfe, Sharp, and Li 1989
; Wolfe and Sharp 1993
).
It is interesting to speculate on the biological consequences for Artemia and Parartemia arising out of the differences in their hemoglobins. If Parartemia has only one polymer, then only the one homodimer is possible. In Artemia, the different homodimers and the heterodimer are differentially expressed in relation to development. In Artemia, some selective advantage may have accrued from the ability to produce a heterodimeric molecule; indeed, it is the heterodimer that Artemia expresses at the highest concentrations for most of its life (Moens 1982
). However, it is the CC homodimer which has the greatest affinity for oxygen, and this is the polymer that is more like the Parartemia molecule.
Parartemia, although clearly able to osmoregulate in a hypersaline environment, cannot survive at the highest salt concentrations where Artemia can and does not upregulate its hemoglobin production. The exact function of its hemoglobin is unknown. As previously mentioned, Manwell (1978)
postulated that the minute amount of hemoglobin Parartemia does produce would not contribute significantly to carrying oxygen and suggested that it may exist purely as a heme transporter. The structure and sequence of the molecule has, however, been highly conserved between Parartemia and Artemia, and the functional differences between these molecules require further study. If the two genera adapted to a high-salinity environment independently (Geddes 1975
), Parartemia evidently developed some other method of dealing with low oxygen concentrations. It is unlikely that this method involves anaerobic metabolism, as Parartemia dies quickly under hypoxic conditions.
The characterization of a hemoglobin molecule from the brine shrimp Parartemia enabled us to localize previously noted discrepancies in the evolutionary rate of globins in the related genus Artemia. Gene duplication is a powerful method for the formation of novel gene families. However, once formed, the members of a gene family may acquire novel functions and interactions with other gene products. Differences in function and structural association with other protein species will limit the utility of molecular-clock assumptions to situations in which the long-term functional status of a given protein has previously been characterized. The hemoglobins are a good example. Those of vertebrates are generally tetrameric, whereas within invertebrate lineages, tetrameric globins are exceptional. The results of this study show that different globins, despite being evolutionarily related, can accumulate substitutions at distinct rates.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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1 Keywords: hemoglobin
Artemia
Parartemia
molecular clock
protein sequence
evolutionary rate
2 Address for correspondence and reprints: Clive N. A. Trotman, Biochemistry Department, University of Otago, Box 56, Dunedin, New Zealand. clive.trotman{at}stonebow.otago.ac.nz
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