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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.

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Artemia has evolved three distinct hemoglobins formed by the association of two nine-domain globin polymers. Sequence analysis of cDNA clones corresponding to two polymers, named T and C, indicates that their genes are the products of a duplication event some 60 million years ago. The present study indicates the presence of 22 introns in each of the T and C polymer genes. The 22 introns are classified into two groups: 17 correspond to positions within globin domains, and 5 correspond to interdomain linkers (or N- and C-terminal extensions). Intron position and reading frame phase are precisely conserved between T and C polymers for all 22 introns, but within each gene the position and phase are not always consistent from domain to domain or from linker to linker. The discordance of Artemia hemoglobin introns is discussed in terms of different model mechanisms and constraints: intron sliding, intron loss or gain, and the exon definition model of primary transcript RNA splicing. The results suggest that constraints of pre-mRNA processing should be considered when considering intron positional changes in homologous genes.

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The Artemia hemoglobin is a dimer comprising two nine-domain covalent polymers in quaternary association. Each polymer is encoded by a gene representing nine successive globin domains which have different sequences and are presumed to have been copied originally from a single-domain gene. Two different polymers exist as the result of a complete duplication of the nine-domain gene, allowing the formation of either homodimers or the heterodimer. The total population size of 18 domains comprising nine corresponding pairs, coupled with the probability that they reflect several hundred million years of evolution in the same lineage, provides a unique model in which the process of gene multiplication can be analyzed. The outcome has important implications for the reliability of local molecular clocks. The two polymers differ from each other at 11.7% of amino acid sites; however when corresponding individual domains are compared between polymers, amino acid substitution fluctuates by a factor of 2.7-fold from lowest to highest. This variation is not obvious at the DNA level: Domain pair identity values fluctuate by 1. 3-fold. Identity values are, however, uncorrected for multiple substitutions, and both silent and nonsilent changes are pooled. Therefore, to determine the variability in relative substitution rates at the DNA level, we have used the method of Li (1993, J Mol Evol 36:96-99) to determine estimates of nonsynonymous (KA) and synonymous (KS) substitutions per site for the nine pairs of domains. As expected, the overall level of silent substitutions (KS of 56. 9%) far exceeded nonsilent substitutions (KA of 6.7%); however, for corresponding domain pairs, KA fluctuates by 2.3-fold and KS by 1. 7-fold. The large discrepancies reflected in the expressed protein have accrued within a single lineage and the implication is that divergence dates of different genera based on amino acid sequences, even with well-studied proteins of reasonable size, can be wrong by a factor well in excess of 2.

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The Artemia hemoglobin contains two subunits that are similar or different chains of nine globin domains. The domains are ancestrally related and are presumed to be derived from copies of an original single-domain parent gene. Since the gene copies have remained in the same environment for several hundred million years they provide an excellent model for the investigation of intron stability. The cDNA for one of the two types of nine-domain subunit (domains T1-T9) has been sequenced. Comparison with the corresponding genomic DNA reveals a total of 17 intradomain introns. Fourteen of the introns are in locations on the protein that are conventional in globins of other species. In eight of the nine domains an intron corresponds to the B helix, amino acid B12, following the second nucleotide (phase 2), and in six domains a G-helix intron is located between G6 and G7 (phase 0). The consistency of this pattern is supportive of the introns having been inherited from a single-domain parent gene. The remaining three introns are in unconventional locations. Two occur in the F helix, either in amino acid F3 (phase 1) in domain T3, or between F2 and F3 (phase 0) in domain T6. The two F introns strengthen an interpretation of intron inheritance since globin F introns are rare, and in domains T3 and T6 they replace rather than supplement the conventional G introns, as though displacement from G to F occurred before that part of the gene became duplicated. It is inferred that one of the F introns subsequently moved by one nucleotide. Similarly, the third unconventional intron location is the G intron in domain T4 which is in G6, phase 2, one nucleotide earlier than the other G introns. Domain T4 is also unusual in lacking a B intron. The pattern of introns in the Artemia globin gene supports a concept of general positional stability but the exceptions, where introns have moved out of reading frame, or have moved by several codons, or have been deleted, suggest that intron displacements can occur after inheritance from an ancient source.

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Seven-hundred globin sequences, including 146 nonvertebrate sequences, were aligned on the basis of conservation of secondary structure and the avoidance of gap penalties. Of the 182 positions needed to accommodate all the globin sequences, only 84 are common to all, including the absolutely conserved PheCD1 and HisF8. The mean number of amino acid substitutions per position ranges from 8 to 13 for all globins and 5 to 9 for internal positions. Although the total sequence volumes have a variation approximately 2-3%, the variation in volume per position ranges from approximately 13% for the internal to approximately 21% for the surface positions. Plausible correlations exist between amino acid substitution and the variation in volume per position for the 84 common and the internal but not the surface positions. The amino acid substitution matrix derived from the 84 common positions was used to evaluate sequence similarity within the globins and between the globins and phycocyanins C and colicins A, via calculation of pairwise similarity scores. The scores for globin-globin comparisons over the 84 common positions overlap the globin-phycocyanin and globin-colicin scores, with the former being intermediate. For the subset of internal positions, overlap is minimal between the three groups of scores. These results imply a continuum of amino acid sequences able to assume the common three-on-three alpha-helical structure and suggest that the determinants of the latter include sites other than those inaccessible to solvent.

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Artemia has evolved the longest known concatenation of hemoglobin domains, the alpha subunit containing nine domains and the beta subunit having a similar size. Translation of the cDNA sequence of the alpha subunit reveals eight regions of inter-domain polypeptide linking together the nine heme-binding domains, together with partially analogous sequences preceding the first domain and following the last. Analysis of the structural possibilities of the linker sequences suggests how the domains may be organized in the subunit. The interdomain linker sequences were 14%-64% identical (62%-91% similar by Dayhoff substitution matrix) and approximately 14 residues in length including a consensus -Val-Asp-Pro-Val-Thr-Gly-Leu-. The linker composition resembled that of the 11 amino acid pre-A leader sequence of Petromyzon marinus (lamprey) hemoglobin V, the structure of which is known. Prediction of structure from the Artemia linker sequences indicated a nonhelical, turn-associated linker which could be modeled to the Petromyzon leader. Measurements confirmed that such a structure could support the packing of nine Artemia domains into a polymeric subunit of annular shape, two of which subunits (which can be similar or dissimilar) comprise the physiological molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

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1. The more than 140 amino acid sequences of non-vertebrate hemoglobins (Hbs) and myoglobins (Mbs) that are known at present, can be divided into several distinct groups: (1) single-chain globins, containing one heme-binding domain; (2) truncated, single-chain, one-domain globins; (3) chimeric, one-domain globins; (4) chimeric, two-domain globins; and (5) chimeric multi-domain globins. 2. The crystal structures of eight nonvertebrate Hbs and Mbs are known, all of them monomeric, one-domain globin chains. Although these molecules represent plants, prokaryotes and several metazoan groups, and although the inter-subunit interactions in the dimeric and tetrameric molecules differ from the ones observed in vertebrate Hbs, the secondary structures of all seven one-domain globins retain the characteristic vertebrate "myoglobin fold". No crystal structures of globins representing the other four groups have been determined. 3. Furthermore, a number of the one-, two- and multi-domain globin chains participate in a broad variety of quaternary structures, ranging from homo- and heterodimers to highly complex, multisubunit aggregates with M(r) > 3000 kDa (S. N. Vinogradov, Comp. Biochem. Physiol. 82B, 1-15, 1985). 4. (1) The single-chain, single-domain globins are comparable in size to the vertebrate globins and exhibit the widest distribution. (A) Intracellular Hbs include: (i) the monomeric and polymeric Hbs of the polychaete Glycera; (ii) the tetrameric Hb of the echiuran Urechis; (iii) the dimeric Hbs of echinoderms such as Paracaudina and Caudina; and (iv) the dimeric and tetrameric Hbs of molluscs, the bivalves Scapharca, Anadara, Barbatia and Calyptogena. (B) Extracellular Hbs include: (i) the multiple monomeric and dimeric Hbs of the larva of the insect Chironomus; (ii) the Hbs of nematodes such as Trichostrongylus and Caenorhabditis; (iii) the globin chains forming tetramers and dodecamers and comprising approximately 2/3 of the giant (approximately 3600 kDa), hexagonal bilayer (HBL) Hbs of annelids, e.g. the oligochaete Lumbricus and the polychaete Tylorrhynchus and of the vestimentiferan Lamellibrachia; and (iv) the globin chains comprising the ca 400 kDa Hbs of Lamellibrachia and the pogonophoran Oligobrachia. (C) Cytoplasmic Hbs include: (i) the Mbs of molluscs, the gastropods Aplysia, Bursatella, Cerithedea, Nassa and Dolabella and the chiton Liolophura; (ii) the three Hb of the symbiont-harboring bivalve Lucina; (iii) the dimeric Hb of the bacterium Vitreoscilla; and (iv) plant Hbs, including the Hbs of symbiont-containing legumes (Lgbs), the Hbs of symbiont-containing non-leguminous plants and the Hbs in the roots of symbiont-free plants.(ABSTRACT TRUNCATED AT 400 WORDS)

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Oligonucleotides related to parts of a globin-like sequence in the genome of Caenorhabditis elegans were used to probe a cDNA library from the same species. A complete globin-like sequence was found in the cDNA, showing that a globin gene appears to be expressed. The hypothetical protein was compatible with the conventional globin fold but may be truncated in the B helix, as in Chironomus globin III. An intron in the codon for residue E3 in the E helix was removed in expression. An initiation codon preceded the globin but the sequence upstream (extending for 30 nucleotides to the vector ligation site) had characteristics both of the code for a protein hydrophobic leader and of a trans-spliced RNA leader. The evidence indicates that C. elegans globin has a single domain, unlike some nematodes that express two tandem globin domains in a continuous translation product, and from its sequence may be predicted to have a high affinity for oxygen.

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The Caenorhabditis elegans and Artemia T4 globin sequences are highly homologous with other invertebrate globins. The intron/exon patterns of their genes display a single intron in the E and G helices respectively. Precoding introns in multirepeat globins are inserted in homologous positions. Comparison of the intron/exon patterns in the known globin gene sequences demonstrates that they are more diverse than first expected but nevertheless can be derived from an ancestral pattern having 3 introns and 4 exons.

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Translated cDNA for Artemia hemoglobin provided sequence data for almost nine domains, from the fourth residue of the A helix of one domain through 1405 residues to a stop codon after the ninth domain. The domain sequences were all different (homology between pairs 17-38%) but aligned well with each other and with conventional globins, satisfying the requirements for Phe at CD1, His at F8 and most other highly conserved features of globins including His at E7. Features found to be characteristic of Artemia globin and present in all nine domains were Phe at B10, Tyr at C4, Gly at F5, Phe at G5 and Gly at H22. Approximately 14 residues including a consensus -Val-Asp-Pro-Val-Thr-Gly-Leu- were available to form the linker between each pair of domains. The Artemia sequence data were compared with the crystal structures of Chironomus thummi thummi erythrocruorin III and sperm whale myoglobin in order to identify features of structural similarity and to examine the consequences of the differences. The Artemia sequences were compatible with the main helices and critical features of the globin fold. Possible modifications to the C helix, FG turn, and GH turn were studied in terms of molecular coordinates.

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There are two major domains of interaction between the Escherichia coli release factors (RF-1 and RF-2) and each subunit of the ribosome. RF-2 has a binding domain on the shoulder and lower head region of the small subunit at the small lobe distant from the decoding site. This is in close proximity to one of the domains on the large subunit which includes the body dimer of L7/L12 and L11. The other domains of interaction, at the decoding site on the small subunit, and at the peptidyltransferase centre of the large subunit of the ribosome, are some distance from the first two, although the evidence for direct contact with the ribosome is less comprehensive. The release factors may therefore have two distinct structural domains, and in support of this concept RF-1 and RF-2 can both be cleaved into two fragments by papain. Region-specific antibodies, and antibodies against defined peptide within the RF sequences have given an indication that a significant part of an interacting RF molecule is in close proximity to the ribosome surface, confirming an observation by immunoelectron microscopy which suggested that the RF penetrates deeply into the cleft between the two subunits. A region of highly conserved primary sequence between the two release factors from E coli is also conserved in those from B subtilis suggesting it forms an important structural or functional domain. Antibodies against peptides from the N-terminal end of this region strongly inhibit binding of the RF to the ribosome.

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Several invertebrate species possess haemoglobins in which each polypeptide contains multiple haem-binding domains, possibly reflecting the fusion of multiple monomeric globin transcriptional units at the gene level. We have now analysed the transcript of such a polymeric globin gene from the brine shrimp Artemia, which expresses three polymeric haemoglobins, each of relative molecular mass 260,000 (Mr 260K). These are formed by the variable association of two different subunit types, alpha and beta (refs 2,3). Haemoglobins I and III are homodimers of alpha and beta subunit types, respectively, and haemoglobin II is a heterodimer (alpha beta). The individual globin chains are of similar size (Mr 130K), but the exact nature of the differences between the two subunit types is unclear. Analysis of complementary DNA clones encoding one of the subunits of the Artemia dimeric haemoglobin showed that the globin messenger RNA encodes nine myoglobin-like domains, connected by linking peptides. The residues in the linkers are characteristic of those found generally in such protein linkers, and include turn-promoting amino acids. Each domain also contains the conserved residues that are required for functional haem-binding, and from analysis of the sequences it was predicted that they all can adopt the classic myoglobin-like fold. Analysis of the derived amino-acid sequences indicated that the individual domains are duplicated monomers that fused to form the polymeric globin some 200 Myr ago. The fusion of multiple transcriptional units for the evolution of a polymeric globin gene may have been a general mechanism for the appearance of such polymeric haemoglobins in invertebrates.

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An increasing number of cases where tri-nucleotide stop codons do not signal the termination of protein synthesis are being reported. In order to identify what constitutes an efficient stop signal, we analysed the region around natural stop codons in genes from a wide variety of eukaryotic species and gene families. Certain stop codons and nucleotides following stop codons are over-represented, and this pattern is accentuated in highly expressed genes. For example, the preferred signal for Saccharomyces cerevisiae and Drosophila melanogaster highly expressed genes is UAAG, and generally the signals UAA(A/G) and UGA(A/G) are preferred in eukaryotes. The GC% of the organism or DNA region can affect whether there is A or G in the second or fourth positions. We suggest therefore, that the stop codon and the nucleotide following it comprise a tetra-nucleotide stop signal. A model is proposed in which the polypeptide chain release factor, a protein, recognises this sequence, but will tolerate some substitution, particularly A to G in the second or third positions.

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The sequences around the stop codons of 862 Escherichia coli genes have been analysed to identify any additional features which contribute to the signal for the termination of protein synthesis. Highly significant deviations from the expected nucleotide distribution were observed, both before and after the stop codon. Immediately prior to UAA stop codons in E. coli there is a preference for codons of the form NAR (any base, adenine, purine), and in particular those that code for glutamine or the basic amino acids. In contrast, codons for threonine or branched nonpolar amino acids were under-represented. Uridine was over-represented in the nucleotide position immediately following all three stop codons, whereas adenine and cytosine were under-represented. This pattern is accentuated in highly expressed genes, but is not as marked in either lowly expressed genes or those that terminate in UAG, the codon specifically recognised by polypeptide chain release factor-1. These observations suggest that for the efficient termination of protein synthesis in E. coli, the 'stop signal' may be a tetranucleotide, rather than simply a tri-nucleotide codon, and that polypeptide chain release factor-2 recognises this extended signal. The sequence following stop codons was analysed in genes from several other procaryotes and bacteriophages. Salmonella typhimurium, Bacillus subtilis, bacteriophages and the methanogenic archaebacteria showed a similar bias to E. coli.

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In protein synthesis Escherichia coli release factor-2 binds to 70 S ribosomes when the termination codon UAA or UGA appears at the decoding site. The weak interaction between factor and ribosome has been stabilized in vitro by chemical cross-linking. Factor so bound can still be recognized by a specific antibody to release factor-2. Examination of the resulting immuno-complexes by electron microscopy revealed 70 S ribosomes in different projection forms, and the occasional dissociated subunit labelled with antibody. The antibody-binding site was localized on previously characterized 70 S projection forms, and its three-dimensional localization on the 70 S model established. The release factor-2-binding site was found to be positioned at the ribosomal subunit interface, comprising the stalk-protuberance region of the large subunit and the head-neck region of the concave side of the small subunit.

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1. Polyclonal antibodies (pAb 1-73 and pAb 26-120) have been raised against both an N-terminal fragment of Escherichia coli ribosomal protein L7/L12 (amino acids 1-73), and a fragment lacking part of the N-terminal domain (amino acids 26-120). 2. Only pAb 26-120 inhibited release-factor-dependent in vitro termination functions on the ribosome. This antibody binds over the length of the stalk of the large subunit of the ribosome as determined by immune electron microscopy, thereby not distinguishing between the C-terminal domains of the two L7/L12 dimers, those in the stalk or those in the body of the subunit. 3. A monoclonal antibody against an epitope of the C-terminal two thirds of the protein (mAb 74-120), which binds both to the distal tip of the stalk as well as to a region at its base, reflecting the positions of the two dimers is strongly inhibitory of release factor function. 4. A monoclonal antibody against an epitope of the N-terminal fragment of L7/L12 (mAb 1-73), previously shown to remove the dimer of L7/L12 in the 50S subunit stalk but still bind to the body of the particle, partially inhibited release-factor-mediated events. 5. The mAb 74-120 inhibited in vitro termination with a similar profile when the stalk dimer of L7/L12 was removed with mAb 1-73, indicating that the body L7/L12 dimer, and in particular its C-terminal domains, are important for release factor/ribosome interaction. 6. The two release factors have subtle differences in their binding domains with respect to L7/L12.

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The brine shrimp, Artemia, is unusual in having large (130 kD) extracellular hemoglobins composed of two globin chains, each of which is a polymer of at least eight myoglobin-like domains. Hemoglobin first appears at the time of naupliar hatching apparently as a result of a physiological switch. Globin gene expression is induced at this time, indicating transcriptional control of hemoglobin synthesis during early development. From a globin partial cDNA clone constructed by a primer extension strategy, we have derived the amino acid sequence of a region that links two of the myoglobin-like domains together. The post H region of the myoglobin-like domain has adapted to accommodate the fusion of these domains. We used the partial cDNA to isolate two classes of large clones containing globin cDNA, and thus established that there are at least two distinct hemoglobin genes in Artemia.

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A translational frameshift is necessary in the synthesis of Escherichia coli release factor 2 (RF-2) to bypass an in-frame termination codon within the coding sequence. The nucleotide sequence preceding the in-phase stop codon within RF-2 mRNA is complementary to the 3' anti-(Shine-Dalgarno sequence) region found in prokaryotic 16S rRNA and Weiss et al. (1988) have concluded that this pairing triggers the frameshift event. In vitro production of RNA coding for RF-2, suitable for translation on eukaryotic ribosomes, has enabled testing of whether eukaryotic ribosomes can frameshift at this sequence. The 18S rRNA of eukaryotic ribosomes does not contain the 3' anti-(Shine-Dalgarno sequence) region. The prokaryotic RF-2 gene and the gene for the other release factor, RF-1, which does not contain an in-frame stop codon, were subcloned into transcription vectors such that the RNA transcripts produced in vitro would resemble a typical eukaryotic mRNA. These RF-1 and RF-2 RNAs both synthesized a major product of Mr approximately 45,000 when translated in vitro within reticulocyte lysate; the size expected for full length RF-1 and RF-2 molecules. The RF-2 product was immunoprecipitated by RF-2-specific antibodies, including those to regions of the protein encoded in the mRNA downstream from the frameshift site. The putative premature termination product, an oligopeptide of 25 amino acids, was not detected, but a chemically synthesized derivative was shown to be very unstable within the translation system. Although it was not possible therefore to calculate an absolute efficiency of frameshifting, the relative efficiency of the translation of RF-2 RNA was estimated to be 10-20% of that of RF-1 RNA in the reticulocyte system. This was similar to the relative synthesis of the two proteins in a plasmid-DNA-directed prokaryotic transcription/translation system. These results show that in vitro on eukaryotic ribosomes where the Shine-Dalgarno-type interaction is not possible, high efficiency frameshifting around the in-phase stop codon in the RF-2 mRNA can still occur.

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A domain of the 30S subunit of the Escherichia coli ribosome is in close contact with the release factor when it binds to the 70S particle during the termination of protein biosynthesis. This has been characterised using antibodies specific for the individual proteins of the small ribosomal subunit. Most antibodies do not affect the release factor-mediated reactions but those against S3, S4, S5 and S10 are inhibitory. These proteins are clustered on the lower head and the upper part of the small lobe of the subunit. The regions of these features which are near the interface between the two subunits in the 70S ribosome are known to be close to the base of the stalk of the 50S subunit.

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