Multimeric Hemoglobin of the Australian Brine Shrimp Parartemia

Michele Coleman, Charles M. Matthews and Clive N. A. TrotmanGo,

Department of Biochemistry, University of Otago, Dunedin, New Zealand


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion and Conclusions
 Supplementary Material
 Acknowledgements
 literature cited
 
The hemoglobin molecule of the commercially important brine shrimp Artemia sp. has been used extensively as a model for the study of molecular evolution. It consists of nine globin domains joined by short linker sequences, and these domains are believed to have originated through a series of duplications from an original globin gene. In addition, in Artemia, two different polymers of hemoglobin, called C and T, are found which differ by 11.7% at the amino acid level and are believed to have diverged about 60 MYA. This provides a set of data of 18 globin domain sequences that have evolved in the same organism. The pattern of amino acid substitution between these two polymers is unusual, with pairs of equivalent domains displaying differences of up to 2.7-fold in total amino acid substitution. Such differences would reflect a similar range of molecular-clock rates in what appear to be duplicate, structurally equivalent domains. In order to provide a reference outgroup, we sequenced the cDNA for a nine-domain hemoglobin (P) from another genus of brine shrimp, Parartemia zietziana, which differs morphologically and ecologically from Artemia and is endemic to Australia. Parartemia produces only one hundredth the amount of hemoglobin that Artemia produces and does not upregulate production in response to low oxygen partial pressure. Comparison of the globin domains at the amino acid and DNA levels suggests that the Artemia globin T gene has accumulated substitutions differently from the Parartemia P and Artemia C globin genes. We discuss the questions of accelerated evolution after duplication and possible functions for the Parartemia globin.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion and Conclusions
 Supplementary Material
 Acknowledgements
 literature cited
 
Previously, we described in detail the hemoglobin cDNA and gene from the brine shrimp Artemia (Manning, Trotman, and Tate 1990Citation ; Jellie, Tate, and Trotman 1996Citation ). The species of Artemia is probably franciscana, but owing to historical confusion, it is conventional not to use species names. The hemoglobin molecule consists of two polymers, C and T, which associate to form homodimers or the heterodimer and which differ from each other by 15.5% at the DNA level and by 11.7% at the amino acid level (Matthews, Vandenberg, and Trotman 1998Citation ). Each domain sequence can be aligned with the conventional globin fold (Trotman et al. 1991Citation ). The complete C and T sequences are identical in length and share numerous other characteristic features. On the basis of the sequence data, we previously suggested that the pair of nine domain molecules results from a number of gene duplication events occupying two phases (Jellie, Tate, and Trotman 1996Citation ; Matthews, Vandenberg, and Trotman 1998Citation ). The first (ancient) phase involved the multiplication from one to nine domains, possibly involving intermediate trimers. The second phase involved the duplication of the entire nine-domain gene to give rise to the C and T polymer genes. Because this happened relatively recently (nominally 60 MYA), and the alignment between C and T is exact, with the amino acid difference being a modest 11.7%, we would expect corresponding domains to be equally different from each other. In fact, some domains have changed up to 2.7- fold more than others (Matthews, Vandenberg, and Trotman 1998Citation ).

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 1963Citation ; Zuckerkandl and Pauling 1965Citation ). 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 1985Citation ; Vawter and Brown 1986Citation ; Li and Tanimura 1987Citation ; Li, Tanimura, and Sharp 1987Citation ; Ayala, Barrio, and Kwiatowski 1996Citation ; Gaut et al. 1996Citation ; Li et al. 1996Citation ).

Parartemia is another genus of brine shrimp, the members of which differ from Artemia in their larger size and other morphological features (Geddes 1980Citation ). 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 1980Citation ). 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 1941Citation ). Thus, the adaptation to salinity is reported to have occurred separately in two different lineages (Geddes 1975Citation ). 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 1981Citation ). 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 1955Citation ). Manwell (1978)Citation 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 1980Citation ). 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)Citation 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 1998Citation ). 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.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion and Conclusions
 Supplementary Material
 Acknowledgements
 literature cited
 
Sample Collection
Parartemia were collected from ponds at the Dry Creek Saltfields on the east coast of the Gulf of St. Vincent, near Adelaide, South Australia, where they occupy ponds of lower salinity (Mitchell and Geddes 1977Citation ). Mean salinity was 138 parts per thousand, mean oxygen content was 3.6 mg/liter, pH 8.04. Adults were collected by horizontally towing a 160-mm net in areas where they had been concentrated by wind action. Organisms were either kept alive to be used directly for DNA extraction or frozen in liquid nitrogen and then stored at -80°C for DNA, RNA, and protein extraction.

RNA Isolation and Analysis
Frozen shrimp (3–5 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 {lambda}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 {lambda}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 {lambda}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 1998Citation ). 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 1–4 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 1998Citation ).

Sequencing of Clones
The inserts of the {lambda}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)Citation . 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 1988Citation ).

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 1–9. Helices of the globin fold are identified, as is conventional, by the letters A–H 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.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion and Conclusions
 Supplementary Material
 Acknowledgements
 literature cited
 
Using cDNA probes from the Artemia hemoglobin sequence, clones were isolated from Parartemia libraries that contained sequence corresponding to the coding region of a 4,293-bp transcript and a 3' untranslated region of 87 bp, followed by a polyA tail of at least 29 bp. This transcript and its derived amino acid sequence were convincingly aligned with the existing Artemia sequences and shown to code for a molecule containing nine globin domains like Artemia hemoglobin (Supplementary Material). The derived molecular mass (Mr) of the molecule is about 160,000. The Parartemia sequence was assembled from two N-terminal clones with an overlap of 140 bp (positions 1–540 and 400–3179) and a C-terminal clone with an overlap of 409 bp (positions 2770–4357). Using the initial protocol, three libraries were made, none of which gave rise to 5' clones, although the protocol was designed to do this. As the protocol relies on PCR, it is possible that the 5' oligonucleotide can bind at another position in the sequence (although this is not evident from a homology search), thus giving rise to a shorter product which is preferentially amplified in the PCR reaction. Partial sequencing was conducted on six other clones, and no evidence for the existence of more than one hemoglobin gene was found. A small degree of polymorphism was observed (<2% at the DNA level).

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 7–9 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.


View this table:
[in this window]
[in a new window]
 
Table 1 Cross-Comparison of Parartemia Hemoglobin Domains and Linkers Showing Percentage Identity and Similarity in Terms of the Dayhoff Substitution Matrix

 
Each Parartemia domain contains a conserved sequence motif (consensus -HPEY-) in the first four residues of the C-helix, as do the Artemia sequences. In Parartemia domains P1–P7, this is identical to the corresponding motif in either T1–T7 or C1–C7 of Artemia, while in P8 it is -NPEY-, as distinct from -HPEY- (C8) or -HPDY- (T8), a conservative change. There is in domain P9, however, a crucial difference from Artemia. A peculiarity confined to domains C9 and T9 alone in Artemia is the deletion of one residue between residues C1 and C4 such that the consensus -HPEY- becomes -HGY-. This deletion is in the critical heme environment where the C2 Pro is practically invariant in vertebrates and invertebrates; its substitution is rare (Vinogradov et al. 1993Citation ; Kapp et al. 1995Citation ), and a deletion is possibly unique to Artemia. A single Gly could theoretically connect the C1 His to the C4 Tyr with minimal reconstruction of the C-helix (Trotman et al. 1991Citation ); however, it is not known whether the domain is rendered nonfunctional. The existence of so rare a deletion in both the T9 and the C9 domains of Artemia but not in any Parartemia domain powerfully suggests that the divergence of Artemia and Parartemia happened before the deletion. Parartemia domain P9 is itself unconventional in having a highly unusual C2 Ser substituted for the anticipated C2 Pro. Since the actual Ser codon is AGT, if a point mutation caused the change from Pro to Ser, it could not have been through a minimal first-base change from C to T (CCN to TCN), but would have required at least one and possibly three intermediate stages. It is possible that the subsequent deletion of the Ser altogether was, at worst, evolutionarily neutral.

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)Citation 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 1983Citation ), 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 1998Citation ), with the deletion of residue C3 therefore having happened between 80 MYA and 60 MYA if the molecular clock is applicable in this case.


View this table:
[in this window]
[in a new window]
 
Table 2 Analysis of Synonymous and Non synonymous Substitutions and Amino Acid Differences Between Pairs of Equivalent Domains

 
Assuming a constant rate of evolution for the globin genes, T and C should be equally different from P, but the actual amino acid differences are 17.7% between T and P and 13.9% between C and P (table 2 ). This implies that T and P are more diverged than C and P. Further evidence is provided by examination of the rates of nonsynonymous substitution (KA) at the DNA level as described below.

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 1998Citation ). 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 ).


    Discussion and Conclusions
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion and Conclusions
 Supplementary Material
 Acknowledgements
 literature cited
 
The erratic divergence of the Artemia globin domains since the recent duplication of their nine-domain parent gene, nominally 60 MYA, has previously been reported (Matthews, Vandenberg, and Trotman 1988Citation ), and it had to be assumed that both C and T proteins displayed past volatility. Introduction of Parartemia as an outgroup has revealed that the T gene has specifically evolved erratically.

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. 1982Citation ; Tamechika et al. 1996Citation ). Accelerated evolution usually occurs when a Darwinian positive selection pressure has applied and is sometimes associated with function switching (Wallis 1997Citation ; Ohno et al. 1998Citation ). 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 1989Citation ; Wolfe and Sharp 1993Citation ).

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 1982Citation ). 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)Citation 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 1975Citation ), 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.


    Supplementary Material
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion and Conclusions
 Supplementary Material
 Acknowledgements
 literature cited
 
The GenBank accession numbers of the hemoglobin cDNA sequences referred to are as follows: Parartemia, AF258616; Artemia globin C, AF104216; Artemia globin T, AF104217. An alignment of the Parartemia and Artemia T and C hemoglobin amino acid sequences is provided on the Molecular Biology and Evolution web site.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion and Conclusions
 Supplementary Material
 Acknowledgements
 literature cited
 
This work was funded by the Marsden Fund of The Royal Society of New Zealand. We are grateful to Professor Rob Saint for the provision of laboratory facilities at the Department of Genetics, and to Professor Bill Williams and Dr. Mike Geddes for their help in the Zoology Department, University of Adelaide.


    Footnotes
 
Claudia Kappen, Reviewing Editor

1 Keywords: hemoglobin Artemia Parartemia molecular clock protein sequence evolutionary rate Back

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 Back


    literature cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion and Conclusions
 Supplementary Material
 Acknowledgements
 literature cited
 

    Ayala, F. J., E. Barrio, and J. Kwiatowski. 1996. Molecular clock or erratic evolution? A tale of two genes. Proc. Natl. Acad. Sci. USA 93:11729–11734

    Coleman, M., M. C. Geddes, and C. N. A. Trotman. 1998. Divergence of Parartemia and Artemia haemoglobin genes. Int. J. Salt Lake Res. 7:171–180

    Czelusniak, J., M. Goodman, D. Hewett-Emmett, M. L. Weiss, P. J. Venta, and R. E. Tessin. 1982. Phylogenetic origins and adaptive evolution of avian and mammalian haemoglobin genes. Nature 298:297–300

    Dayhoff, M. O., W. C. Barker, and L. T. Hunt. 1983. Establishing homologies in protein sequences. Pp. 524–545 in C. H. W. Hirs and S. N. Timashef, eds. Methods in enzymology. Vol 91. Academic Press, New York

    Dickerson, R. E., and I. Geiss. 1983. Hemoglobin: structure, function, evolution, and pathology. Benjamin Cummings, Menlo Park, Calif

    Gaut, B. S., B. R. Morton, B. C. McCaig, and M. C. Clegg. 1996. Substitution rate comparisons between grasses and palms: synonymous rate differences at the nuclear gene Adh parallel rate differences at the plastid gene rbcL. Proc. Natl. Acad. Sci. USA 93:10274–10279

    Geddes, M. C. 1975. Studies on an Australian brine shrimp, Parartemia zietziana Sayce: salinity tolerance. Comp. Biochem. Physiol. 51A:553–559

    ———. 1980. The brine shrimps Artemia and Parartemia in Australia. Pp. 57–65 in G. Persoone, P. Sorgeloos, O. Roels, and E. Jaspers, eds. The brine shrimp Artemia, Vol. 3. Ecology, culturing, use in aquaculture. Universa Press, Wetteren, Belgium

    ———. 1981. The brine shrimp Artemia and Parartemia. Hydrobiologia 81:169–179

    Gilchrist, B. M. 1955. Haemoglobin in Artemia. Proc. R. Soc. Lond. B Biol. Sci. 143:136–146

    Jellie, A. M., W. P. Tate, and C. N. A. Trotman. 1996. Evolutionary history of introns in a multidomain globin gene. J. Mol. Evol. 42:641–647[ISI][Medline]

    Kapp, O. H., L. Moens, J. Vanfleteren, C. N. A. Trotman, T. Suzuki, and S. N. Vinogradov. 1995. Alignment of 700 globin sequences: extent of amino acid substitution and its correlation with variation in volume. Protein Sci. 4:2179– 2190[Abstract/Free Full Text]

    Li, W.-H. 1993. Unbiased estimation of the rates of synonymous and nonsynonymous substitution. J. Mol. Evol. 36: 96–99

    Li, W.-H., D. L. Ellsworth, J. Krushkal, B.-H. J. Chang, and D. H. Emmet. 1996. Rates of nucleotide substitution in primates and rodents and the generation-time effect hypothesis. Mol. Phylogenet. Evol. 5:182–187[ISI][Medline]

    Li, W.-H., and M. Tanimura. 1987. The molecular clock runs more slowly in man than in apes and monkeys. Nature 326: 93–96

    Li, W.-H., M. Tanimura, and P. Sharp. 1987. An evaluation of the molecular clock hypothesis using mammalian DNA sequences. J. Mol. Evol. 25:330–342[ISI][Medline]

    Linder, F. 1941. Contributions to the morphology and the taxonomy of the Branchiopoda Anostraca. Zool. Bidr. Upps. 20:101–302.

    Manning, A. M., C. N. A. Trotman, and W. P. Tate. 1990. Evolution of a polymeric globin in the brine shrimp Artemia. Nature 348:166–168

    Manwell, C. 1978. Haemoglobin in the Australian anostracan Parartemia zietziana: evolutionary strategies of conformity vs regulation. Comp. Biochem. Physiol. 59A:37–44

    Margoliash, E. 1963. Primary structure and evolution of cytochrome c. Proc. Natl. Acad. Sci. USA 50:672–679

    Matthews, C. M., C. J. Vandenberg, and C. N. A. Trotman. 1998. Variable substitution rates of the 18 domain sequences in Artemia hemoglobin. J. Mol. Evol. 46:729– 733[ISI][Medline]

    Mitchell, B. D., and M. C. Geddes. 1977. Distribution of the brine shrimps Parartemia zietziana Sayce and Artemia salina L. along a salinity and oxygen gradient in a South Australian saltfield. Freshw. Biol. 7:461–467

    Moens, L. 1982. The extracellular haemoglobins of Artemia sp: a biochemical and ontological study. Mededelingen van de Koninklijke Academie voor Wetenschappen, Letteren en Schone Kunsten van Belgie, Klasse der Wetenschappen 44: 1–21

    Ohno, M., R. Menez, T. Ogawa et al. (12 co-authors). 1998. Molecular evolution of snake toxins: is the functional diversity of snake toxins associated with a mechanism of accelerated evolution? Prog. Nucleic Acid Res. Mol. Biol. 59: 307–364

    Stockwell, P. A. 1988. HOMED: a homologous sequence editor. Trends Biochem. Sci. 13:322–324[ISI][Medline]

    Tamechika, I., M. Itakura, Y. Saruta, M. Furukawa, and A. Kato. 1996. Accelerated evolution in inhibitor domains of porcine elafin family members. J. Biol. Chem. 271:7012– 7018[Abstract/Free Full Text]

    Trotman, C. N. A., A. M. Manning, L. Moens, and W. P. Tate. 1991. The polymeric hemoglobin molecule of Artemia. Interpretation of translated cDNA sequence of nine domains. J. Biol. Chem. 266:13789–13795[Abstract/Free Full Text]

    Vawter, L., and W. M. Brown. 1986. Nuclear and mitochondrial DNA comparisons reveal extreme rate variation in the molecular clock. Science 234:194–195

    Vinogradov, S. N., D. A. Walz, B. Pohajdak, L. Moens, O. H. Kapp, T. Suzuki, and C. N. A. Trotman. 1993. Adventitious variability? The amino acid sequences of nonvertebrate globins. Comp. Biochem. Physiol. B 106:1–26

    Wallis, M. 1997. Function switching as a basis for bursts of rapid change during the evolution of pituitary growth hormone. J. Mol. Biol. 44:348–350

    Wolfe, K. H., and P. M. Sharp. 1993. Mammalian gene evolution: nucleotide sequence divergence between mouse and rat. J. Mol. Evol. 37:441–456[ISI][Medline]

    Wolfe, K. H., P. M. Sharp, and W.-H. Li. 1989. Mutation rates differ among regions of the mammalian genome. Nature 337:283–285

    Wu, C.-I., and W.-H. Li. 1985. Evidence for higher rates of nucleotide substitution in rodents than in man. Proc. Natl. Acad. Sci. USA 82:1741–1745

    Zuckerkandl, E., and L. Pauling. 1965. Evolutionary divergence and convergence in proteins. Pp. 97–166 in V. Bryson and H. J. Vogel, eds. Evolving genes and proteins. Academic Press, New York

Accepted for publication December 13, 2000.