Department of Biochemistry, University of Otago, Dunedin, New Zealand
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hb constitutes up to 2% of the Artemia hemolymph protein (Gilchrist 1955
). The three dimers have different oxygen affinities and kinetic properties that may be of physiological importance through differential expression in response to salinity and oxygen tension (Heip et al. 1978
; Decleir et al. 1980
; Spicer and El-Gamal 1999
). How differential expression is regulated is not transparent because the distribution of T and C subunits among the three polymers present in the hemolymph often does not approximate the obvious 1:2:1 ratio of TT:CT:CC.
The present work describes the finding that each of the subunits, T and C, is coded on replicate but slightly variant genes so that different forms T1, T2, and C1, C2 are expressed. However, the variants are not freely interchangeable in the formation of dimers, and the three physiologically recognized Hbs, Hb I, Hb II, and Hb III, are composed of, respectively, C1C2, C1T2, and T1T2. This provides for an additional level of regulation by enabling, for instance, the expression of T1 to control the level of Hb III, whereas the expression of T2 could control the level of Hb II.
The aligned amino acid sequences (cDNA translations) of Artemia C1 and T1 globins have been published previously (Matthews, Vandenberg, and Trotman 1998
), and their alignment with Parartemia globin has been published in the Molecular Biology and Evolution website (see Coleman, Matthews, and Trotman 2001
).
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DNA Sequencing
Polymer-specific primers were used to amplify selected regions of positive clones using the polymerase chain reaction (PCR). All sequencing was performed on uncloned PCR products after purification using a QIAquick PCR purification kit (QIAGEN). Sequencing was performed using dRhodamine terminator chemistry and an ABI Prism 377 automated sequencer.
Southern Analysis
Genomic DNA (10 µg) was digested with HindIII and fractionated by electrophoresis at 30 V for 12 h on a 1% agarose, TAE gel. Before transfer, agarose gels were pretreated (depurination, denaturation, and neutralization) according to the instructions provided with the Hybond-N membrane (Amersham). The DNA was then Southern blotted to the membrane and fixed by baking at 80°C for 12 h. The Southern blots were hybridized with radiolabeled PCR products at 60°C for 12 h in 4x SSPE, 0.1% tetrasodium pyrophosphate, 0.5 mg/ml heparin, and 0.5% SDS. Membranes were washed twice with 2x SSPE, 0.5% SDS for 10 min each, then with 1x SSPE, 0.1% SDS for 15 min. Before a second round of hybridization, the membranes were stripped by pouring boiling 0.1% SDS onto the Hybond-N membrane and allowing the solution to cool to room temperature. This step was repeated if necessary.
Artemia Hb II
Artemia Hb II was kindly provided by C. Marshall (Department of Biochemistry, University of Otago). This Hb had been purified by elution from nondissociating gels as detailed previously (Marshall et al. 1986
).
Cleavage of Hb II
Cleavage reactions were performed in siliconized tubes. Artemia Hb II (100 µg) in 70% formic acid was incubated at 37°C for 20 h. The reaction mixture was dried in a Speed Vac, resuspended in SDS-PAGE loading buffer, and neutralized by adding 1 M Tris base until the sample turned from yellow to blue. The cleavage products were then separated by SDS-PAGE (Kolbe et al. 1984
).
Protein Sequencing
The protein to be sequenced was run on an SDS-PAGE gel, transferred to the PVDF membrane, stained with Coomassie Blue (0.025% Coomassie Blue in 40% methanol) for 3060 s, and destained (50% methanol) for 2 h. The protein bands were excised and amino-terminals sequenced using an Applied Biosystems gas phase peptide sequencer.
Protein and DNA Sequence Compilation and Analysis
Translation functions were performed using the program NLDNA (Stockwell 1987
). The homology of new DNA sequences was determined using HOMED (Stockwell 1988
) or GCG (Genetics Computer Group Inc.) sequence analysis package, version 7.3.
![]() |
Results and Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Southern analysis of Hind IIIdigested genomic DNA produced five well-separated bands (fig. 1 ) that have been interpreted as corresponding to five genes: T1, T2, C1, C2, and one T polymer pseudogene. The probe corresponded to the ninth exons of both the T1 and C1 polymer genes, which contain no HindIII sites. The different intensities of the bands presumably reflects the level of mismatch between the probe and target sequence. Thus, it appears that there are five distinct globin genes in Artemia, four functional and one nonfunctional. However, there is the possibility that two or more positive genomic fragments of similar size are running at the same position.
|
The coding regions of the T1 and C1 polymer genes show on average 84% identity at the DNA level. However, no obvious homology exits between the intron sequences of these genes. Partial sequences from all 22 introns of the T1 and C1 polymers were compared and the identity found to range from 26% to 38%, with much of the observed match likely to be contributed to by the high AT content of the introns (61%79%). In contrast, the identity between the T1 and T2 introns is much greater, 84%95%. At the coding level the T1 and T2 polymers are only about 3% different; therefore, the introns, with an average change of 10.7 substitutions per 100 sites, have a substitution rate about 3.5 times that of the coding sequences.
Protein Sequencing
Sequencing of the native Artemia Hbs indicates there are limitations on which two polymers can associate. Amino-terminal sequencing of Hb I showed that it is composed of two forms, one having arginine at the fourth position whereas the other has leucine. These two sequences were present in equal quantities and correspond to the C1 and C2 polymers. Only one sequence is obtained by this method for Hb III because the T1 and T2 polymers are identical for the first 148 residues. Interestingly, amino-terminal sequencing of the heterodimer, Hb II, gave only the C1 polymer sequence and the T polymer sequence (as for Hb III). The C2 polymer is not involved in the formation of Hb II. The cleavage of Hb II followed by sequencing of the fragments allowed the T polymer composition to be determined. Of the eight sequences generated five were C1, two were T2, and one was not polymer specific. The assignment of the sequences obtained by Marshall (1988)
from cyanogen bromide and trypsin fragments of Hb II gives similar results: four C1, one T2, three not specific. No sequences have been identified that are unique to T1; however, because not all the fragments have been sequenced, the involvement of T1 in Hb II has not been completely ruled out. These results indicate that Hb II is composed of C1 and T2.
The existence of four distinct polymers and their restricted pairing to form the three Artemia Hbs may resolve the question of how Artemia achieves both the developmental and environmental expression patterns that are observed. If only the polymer combinations C1C2 (Hb I), C1T2 (Hb II), and T1T2 (Hb III) exist, then the Hbs can be independently regulated by polymer expression alone. If the affinities between the two T polymers and between the two C polymers exceed that between C1 and T2, then the level of Hb I can be controlled by the expression of C2 and Hb III by T1. Production of Hb II is then dependent on excess C1 and T2. Under hypoxic conditions all three Hbs are present, which would not be possible if the affinity between C1 and T2 were greater than between T1 and T2 and between C1 and C2. However, we cannot completely rule out the possibility that under some developmental stages other combinations of polymers form. Similarly, we cannot exclude the possibility of the other uncharacterized globin genes being functional in the brine shrimp Artemia.
Globin Gene Expression
The developmental expression pattern of Artemia Hb, seen in figure 2
, can now be described in terms of the induction and inactivation of specific genes. The first genes to be expressed posthatching are C1 and T2, resulting in the appearance of Hb II (Heip et al. 1978
). A few hours later T1 is induced, and Hb III can then be detected. Not until 79 days posthatching does the induction of C2 occur, which is seen by the Hb I expression. The level of T1, and therefore Hb III, decreases at 2025 days because this gene is inactive in adult shrimp under normal conditions. Under hypoxic conditions all the globin genes are upregulated as seen by an increase in the level of all three Hbs (Heip, Moens, and Kondo 1978
). The greatest increase observed is for Hb III, which is induced in adult shrimp lacking Hb III, i.e., T1 is induced. Differential up-regulation of Hb polymers by hypoxia has been observed in Daphnia magna (Kimura et al. 1999
). A cluster of four Hb genes has been identified in D. magna with putative hypoxia responsive elements (HREs) located in the intergenic regions (Kimura et al. 1999
). The analysis of the genomic sequence of the Artemia globin genes is expected also to reveal putative HREs.
|
![]() |
Supplementary Material |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
Keywords: hemoglobin
Artemia
protein sequence
protein structure
evolution
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
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Coleman M., C. M. Matthews, C. N. A. Trotman, 2001 A multimeric hemoglobin of the Australian brine shrimp Parartemia Mol. Biol. Evol 18:570-576
Decleir W., J. Vos, F. Bernaerts, C. Van den Branden, 1980 The respiratory physiology of Artemia sp Pp. 137145. in G. Persoone, O. Roels, and E. Jaspers, eds. The brine shrimp Artemia, Vol. 2. Universa Press, Wetteren, Belgium
D'Hondt J., L. Moens, J. Heip, A. D'Hondt, M. Kondo, 1978 Oxygen-binding characteristics of three extracellular haemoglobins of Artemia salina Biochem. J 171:705-710[ISI][Medline]
Dickerson R. E., I. Geiss, 1983 Hemoglobin: structure, function, evolution, and pathology Benjamin Cummings, Menlo Park
Gilchrist B. M., 1955 Haemoglobin in Artemia Proc. R. Soc. B 143:136-146
Heip J., L. Moens, M. Joniau, M. Kondo, 1978 Ontogenetical studies on extracellular hemoglobins of Artemia salina Dev. Biol 64:73-81[ISI][Medline]
Heip J., L. Moens, M. Kondo, 1978 Effect of concentrations of salt and oxygen on the synthesis of extracellular hemoglobins during development of Artemia salina Dev. Biol 63:247-251[ISI][Medline]
Jellie A. M., W. P. Tate, C. N. A. Trotman, 1996 Evolutionary history of introns in a multidomain globin gene J. Mol. Evol 42:641-647[ISI][Medline]
Kimura S., S. Tokishita, T. Ohta, M. Kobayashi, H. Yamagata, 1999 Heterogeneity and differential expression under hypoxia of two-domain hemoglobin chains in the water flea, Daphnia magna J. Biol. Chem 274:10649-10653
Kolbe H. V., D. Costello, A. Wong, R. C. Lu, H. Wohlrab, 1984 Mitochondrial phosphate transport. Large scale isolation and characterization of the phosphate transport protein from beef heart mitochondria J. Biol. Chem 259:9115-9120
Manning A. M., R. J. Powell, C. N. A. Trotman, W. P. Tate, 1990 Developmental expression and cDNA cloning of globin genes from the brine shrimp, Artemia New Biol 2:77-83[Medline]
Manning A. M., C. N. A. Trotman, W. P. Tate, 1990 Evolution of a polymeric globin in the brine shrimp Artemia Nature 348:653-656[ISI][Medline]
Marshall C. J., 1988 Structures of the haemoglobins of the brine shrimp, Artemia: an examination of their properties and synthesis Doctoral dissertation, University of Otago, Dunedin, New Zealand
Marshall C. J., J. F. Cutfield, C. N. A. Trotman, W. P. Tate, 1986 Purification of the haemoglobins I and III from the brine shrimp Artemia Biochem. Int 12:693-700[ISI]
Matthews C. M., 1998 Evolutionary changes in the haemoglobin genes of Artemia Doctoral thesis, University of Otago, Dunedin, New Zealand.
Matthews C. M., C. N. A. Trotman, 1998 Ancient and recent intron stability in the Artemia hemoglobin gene J. Mol. Evol 47:763-771[ISI][Medline]
Matthews C. M., C. J. Vandenberg, C. N. A. Trotman, 1998 Variable substitution rates of the 18 domain sequences in Artemia hemoglobin J. Mol. Evol 46:729-733[ISI][Medline]
Moens L., 1982 The extracellular haemoglobins of Artemia sp: a biochemical and ontological study Meded. K. Acad. Wet. Lett. Schone Kunsten Belg. Kl. Wet 44:1-21
Ohno S., 1970 Evolution by gene duplication Springer-Verlag, Berlin
Ohta T., 1994 Further examples of evolution by gene duplication revealed through DNA sequence comparisons Genetics 138:1331-1337
Ridley M., 1993 Evolution Blackwell Scientific, Massachusetts
Spicer J. I., M. M. El-Gamal, 1999 Hypoxia accelerates the development of respiratory regulation in brine shrimpbut at a cost J. Exp. Biol 202:3637-3646
Stockwell P. A., 1987 DNA sequence analysis software Pp. 1945. in C. T. Rawlings and M. J. Bishop, eds. Nucleic acid and protein sequence analysis, a practical approach. IRL Press, Washington, D.C
. 1988 HOMED: a homologous sequence editor Trends Biochem. Sci 13:322-324[ISI][Medline]