From the Department of Zoology, University of Texas,
Austin, Texas 78712-1064 and the
Department of Biological
Sciences, Clemson University, Clemson, South Carolina 29631
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ABSTRACT |
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Hemoglobin (Hb) occurs in circulating red blood cells, neural tissue, and body wall muscle tissue of the nemertean worm, Cerebratulus lacteus. The neural and body wall tissue each express single major Hb components for which the amino acid sequences have been deduced from cDNA and genomic DNA. These 109-residue globins form the smallest stable Hbs known. The globin genes have three exons and two introns with splice sites in the highly conserved positions of most globin genes. Alignment of the sequences with those of other globins indicates that the A, B, and H helices are about one-half the typical length. Phylogenetic analysis indicates that shortening results in a small tendency of globins to group together regardless of their actual relationships. The neural and body wall Hbs in situ are half-saturated with O2 at 2.9 and 4.1 torr, respectively. The Hill coefficient for the neural Hb in situ, ~2.9, suggests that the neural Hb self-associates in the deoxy state at least to tetramers at the 2-3 mM (heme) concentration estimated in the cells. The Hb must dissociate upon oxygenation and dilution because the weight-average molecular mass of the HbO2 in vitro is only about 18 kDa at 2-3 µM heme concentration. Calculations suggest that the Hb can function as an O2 store capable of extending neuronal activity in an anoxic environment for 5-30 min.
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INTRODUCTION |
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Hemoglobin was first observed in neural tissue of invertebrates by Lankester (3) who noted the brilliant crimson color of the ganglia of the polychaetous annelid Aphrodite aculeata. Neural Hbs have since been recorded in or associated with nervous tissue of other annelids (4-7), molluscs (5, 8-12), arthropods (13, 14), and nemerteans (15, 16). The functional significance of Hb1 in neural tissue has been addressed in very few animals. Chalazonitis et al. (17) correlated the oxygenation state of neural Hb in Aplysia dipilans with the electrical activity of the neural ganglia and found that firing activity was proportional to the degree of oxygenation of the Hb. The neural tissue Hbs of the clams Tellina alternata and Spisula solidissima can extend the time of O2 delivery to nerves during anoxic periods by as much as 30 min by acting as an O2 store (12, 18).
Recently, DeWilde et al. (19) isolated and determined the amino acid sequence of the Hb from the neural tissue of A. aculeata. They found that the Hb was dimeric and had a relatively high O2 affinity and that the sequence and gene structure clearly showed it to be a member of the globin family.
Many nemertean worms express intracellular Hbs in red blood cells, body wall muscle tissue, and neural tissue. We report here the amino acid sequences, gene structure, and oxygen equilibria in situ for both the body wall and the neural tissue Hbs of the marine nemertean, Cerebratulus lacteus. We also propose a role of the neural hemoglobin as an oxygen store. We have used the amino acid sequences of the globins in maximum parsimony analyses to address possible phylogenetic relationships. These Hbs are very small, yet stable, unlike the artificially truncated mini-Mbs (20-22). This finding should make the C. lacteus Hbs particularly valuable for studies of folding and stability.
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EXPERIMENTAL PROCEDURES |
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Animals and Tissue Preparation
Eighty-four specimens of C. lacteus were purchased
from the Department of Marine Resources, Marine Biological
Laboratories, Woods Hole, MA, and maintained at 4 °C in the
laboratory in 0.45 µm of filtered seawater. Animals used for
oxygen-binding experiments were kept a maximum of 2 weeks. Tissue
samples needed for protein, RNA, and DNA experiments were dissected
from animals within 72 h of delivery, immediately frozen in a dry
ice/ethanol bath, and stored at 80 °C. Tissues were prepared for
microscopy by relaxing the animals in MgCl2 isotonic to
seawater and fixing the head and trunk segments in 2.5% glutaraldehyde
in 200 mM phosphate buffer, pH 7.4, made isotonic (0.58 M) by addition of NaCl. Segments were postfixed in 1.0%
osmium tetroxide in the same buffer, dehydrated in an ethanol series,
and embedded in Epon 812 resin. Ultrathin sections were stained with
methylene blue and examined by light microscopy.
Isolation and Purification of Hemoglobins
Hb-containing tissues were ground in liquid N2, then
transferred to 50 mM Tris acetate, 10 mM EDTA,
pH 7.5, at 0 °C, followed by centrifugation at 12,000 × g for 10 min at 4 °C. Brain and lateral nerve tissue
extracts are referred to as neural Hb, and body wall muscle extracts
are referred to as body wall Hb. Tissue extracts were stored at 0 °C
if used immediately or refrozen in liquid N2 and stored at
80 °C. Hbs were purified first by size-exclusion chromatography
with G-75-SF Sephadex (Sigma) in a column (12 × 450 mm)
equilibrated at 4 °C with 50 mM Tris acetate, 10 mM EDTA, pH 7.4 (measured at room temperature).
Hb-containing fractions from this column were pooled, dialyzed against
10 mM Tris acetate, pH 8.0, and concentrated. The Hb was
further purified by HPLC anion-exchange chromatography on a Synchropak
AX300 column (4.6 × 250 mm), (SynChrom, Inc., Lafayette, IN),
initially equilibrated with 10 mM Tris acetate, pH 8.0, at
room temperature. Hbs were eluted from the column with a linear
gradient from 0 to 50% of 10 mM Tris acetate, 500 mM sodium acetate, pH 8.0, at a flow rate of 0.5 ml/min. Hb
fractions, identified by absorbance at 415 nm, were pooled, dialyzed
against 10 mM Tris, pH 8.0, and lyophilized. These Hb
samples were resuspended in 0.1% trifluoroacetic acid in water and
further purified by RP-HPLC on a Synchropak RP300 C-18 column (4.6 × 250 mm) equilibrated with 0.1% trifluoroacetic acid in water at
room temperature. Globins were eluted by establishing a series of
linear gradients from 0 to 80% acetonitrile containing 0.1%
trifluoroacetic acid over 70 min at a flow rate of 0.8 ml/min.
Neural tissue extracts from four animals were analyzed on a
size-exclusion column, TSK-30 (4.6 × 300 mm, Bio-Rad), on an LDC Milton Roy CCM automated HPLC system. Extracts and molecular size standards (Sigma, catalog number MW-GF-70) were run at room temperature on the column equilibrated with 50 mM Tris acetate, pH 7.5, over 15 min at a flow rate of 1.0 ml/min. The standards were bovine serum albumin, ovalbumin, -lactoglobulin, horse heart myoglobin, and
cytochrome c. Protein standards and Hb samples were
monitored at 235 and 415 nm, respectively.
Amino Acid Composition and Sequence Analyses
Purified globins were hydrolyzed in 6 N HCl, sealed in vacuo in glass tubes, and hydrolyzed at 110 °C for 24 or 72 h. Amino acid compositions were determined with a Beckman model 121MB analyzer. Amino-terminal sequences of the globins were determined with a model 477A protein sequencer (Applied Biosystems, Inc.). Both instruments are at the University of Texas Microanalysis Facility.
Preparation and Amplification of Neural Globin mRNA
Neural tissue was ground in liquid N2 and dissolved
in a solution containing 3 M LiCl, 6 M urea,
and 0.2% sodium dodecyl sulfate at 0 °C. Brain homogenate was
extracted with phenol/chloroform/isoamyl alcohol (25:24:1 v/v/v), and
the RNA was precipitated with ethanol and resuspended in
diethylpyrocarbonate-treated, sterile, deionized distilled water and
stored at 80 °C. Poly(A)+ RNA was isolated from the
total RNA by oligo(dT) affinity chromatography and reverse-transcribed
using Moloney-murine leukemia virus reverse transcriptase (Promega).
This was followed by extraction with phenol/chloroform/isoamyl alcohol,
ethanol precipitation of the cDNA, resuspension in
diethylpyrocarbonate-treated water, and storage at
80 °C. The
cDNA was used as a template to amplify globin cDNA in the
polymerase chain reaction (PCRTM, Hoffmann-La Roche).
Primers were oligo(dT) and a redundant oligonucleotide based on the
NH2-terminal residues of the globin (VNWAAV = 5'-GTXAA(T/C)TGGGCXGCXGT-3'). The
reaction conditions were 100 mM Tris-HCl (pH 8.3 at
25 °C), 50 mM KCl, 2 mM MgCl2, 1 µg/µl bovine serum albumin, 0.25 µM oligo(dT), 1 µM redundant primer, 200 µM dNTPs, and 2.5 units of AmpliTaq DNA polymerase (Perkin-Elmer) in a volume of 100 µl. Reactants were heated to 98 °C for 2 min prior to the addition
of enzyme, and amplification was performed for 30 cycles (94 °C, 1.5 min; 45 °C, 1 min; 72 °C, 1.5 min (cycles 1-10), and then 2.5 min (cycles 11-30)). PCR products were cloned into the pCR-1000
vector (TA Cloning System, Invitrogen, San Diego).
Genomic DNA Isolation and Sequencing
A genomic DNA library was constructed in the Lambda Dash II vector (Stratagene) from BamHI-restricted, size-selected genomic DNA that came from the sperm of a single animal. The library was screened with a 345-bp AccI fragment from the neural globin cDNA PCR product (see "Results"). A genomic clone containing a 25-kb insert that hybridized to the globin cDNA probe was isolated from the library, and several enzyme restriction fragments were subcloned into the SmaI site of pBluescript (Stratagene) for sequencing. Fig. 1 shows the positions of the various clones used to determine the organization and sequence of the genes for neural and body wall globins.
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Body Wall Globin-- The 6-kb HpaI clone (HpaI-6) contained 5.1 kb of a body wall globin gene that was truncated in the first intron of the gene and lacked exon I. Other clones (SphI-7, 7 kb; ClaI-3, 3 kb; and ClaI-7, 7 kb) provided additional sequence information (see Fig. 1). The missing exon I region was isolated from genomic DNA by the use of two PCR amplification protocols. The first utilized a redundant primer within exon I and a non-redundant primer from within intron I of the body wall globin gene to amplify the intervening sequence from a template of XhoI-digested genomic DNA yielding fragment TA9 (see Fig. 1). The reaction mixture consisted of 20 mM Tris-HCl (pH 8.8 at 25 °C), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 2.4 µM redundant primer, 0.2 µM non-redundant primer, 400 µM dNTPs, and 2.5 units of AmpliTaq DNA polymerase. The region from intron I into the region 5' to exon I of the body wall globin gene was amplified in a second PCR reaction using a set of two nested primers from intron I and two primers from the PCR in vitro Single Site Amplification and Cloning System (catalog number TAK R015, Panvera Corp., Madison, WI) in a reaction mix given by the manufacturer. HindIII-digested genomic DNA was used as the template for the reaction which yielded fragment p1-3 (see Fig. 1). All PCR products were subcloned into pUC19 or pCRTMII vector (TA cloning system, Invitrogen, San Diego, CA) for sequencing performed by the dideoxy method using Sequenase 2.0 (U. S. Biochemical Corp.).
Neural Globin-- A second screening of the genomic DNA library, using the 581-bp cDNA for neural globin, yielded a clone containing an HpaI digestion fragment of 4.5 kb that contained the complete sequence of the neural globin gene except for the first two codons of exon 1 and the adjoining 5' upstream sequence (clone p42, see Fig. 1). The missing region was obtained with the PCR in vitro Single Site Amplification and Cloning System with a pair of non-redundant, nested primers located in the region of exon 2 and a sample of PstI-digested genomic DNA yielding fragment pP8 (see Fig. 1). A second pair of non-redundant, nested primers from the exon 3 sequence was used to amplify exon 3 and intron 2 of the neuronal globin gene from a sample of XbaI-digested genomic DNA yielding fragment p4-7 (see Fig. 1). The PCR products and fragment p42 were cloned and sequenced as described above.
Phylogenetic Analysis of Globin Sequences
Alignment-- Amino acid sequences of globins were obtained from the literature. Sequences of 22 globins from vertebrate and invertebrate animals (including Cerebratulus), bacteria, protists, algae, plants, and fungi were aligned by eye in a series of steps as follows. First, the 84 conserved residue positions identified by Kapp et al. (23) as shared by all members of the globin family were used for a core alignment that included the conserved CD1 Phe and F8 His. The template of Vinogradov et al. (24) was used to facilitate the identification of conserved hydrophilic or hydrophobic residues at external and internal locations, respectively. This template is a version of template I of Bashford et al. (25) extended to include invertebrates. This core comprises the shared parts of helices A, B, C, E, F, G, and H. The second step was to fill in the interhelical regions by using known x-ray structures of individual globins where possible (10 out of 22 globins). The helical regions, as identified in the x-ray analyses, were aligned with a minimum number of insertions or deletions in order to maintain the overall tertiary structure. Gaps were confined almost entirely to the loops between helices. Finally, the external/buried/internal classification for individual residues of Fermi and Perutz (26), originally developed for vertebrate myoglobins and hemoglobins, was taken as an additional template. This template includes both helical and interhelical regions, unlike those of Bashford and Vinogradov (24, 25), which carry only helical information. The Fermi template helps to identify and preserve the chemical patterns such as the arrangement of polar and non-polar amino acids in interhelical loops. All interhelical regions were expanded just enough to accommodate the longest sequence in these regions. The resulting alignment needed a total of 179 positions for the 22 globins with ~110 positions common to a majority of sequences.
Analysis--
The aligned sequences were analyzed for
relationships by employing the maximum parsimony method. All programs
involved in this analysis are part of the PHYLIP Program Package,
version 3.5, of Felsenstein (27). The original sequences were
bootstrapped either 100 or 1,000 times using the program SEQBOOT (28).
The replicate data sets so obtained were analyzed by the program
PROTPARS which calculated a corresponding number of unrooted parsimony trees with the use of both the global rearrangement option and the
option for a randomized input order of sequences. Finally, the CONSENSE
program was used to generate a single majority-rule consensus tree from
that population of 100 or 1,000 parsimony trees, with calculated
bootstrap support values at each node. The human -globin sequence
was taken as outgroup for rooting the tree because the monophyly of
vertebrate myoglobins and hemoglobins is established.
Oxygen Equilibria
The oxygen binding properties of the neural and body wall Hbs
were determined in situ by thin layer microspectrophotometry at 15 °C (29, 30). Hb solutions for in vitro experiments
were prepared by homogenizing tissues on ice in 50 mM Tris
buffers at various pH values followed by centrifugation at 12,000 × g at 4 °C for 10 min to remove cellular debris. Tissue
for in situ experiments was prepared by cutting a piece of
the brain or body wall muscle and rinsing it in a solution of 0.45 µm
of filtered seawater at 0 °C buffered with 50 mM Tris at
the appropriate pH and containing 20 mM KCN to inhibit
cellular respiration. Thin sections were removed from the pieces with a
razor blade and placed in a fresh buffer at 0 °C. All oxygen binding
experiments were performed at 15 °C. All pH measurements were made
at room temperature. Absorbance data from 12 wavelength (nm) pairs
(410/420, 425, 430, 435, 440; 415/425, 430, 435, 440; 420/430, 435;
425/435) were used in a two-wavelength method for determining
fractional saturation values. The tissue slice pathlength, measured
optically, varied between 80 and 240 µm. Mean absorbance changes were
0.050 and 0.183 at 415 and 430 nm, respectively. Between 5 and 7 experiments were performed under each set of conditions. Typically,
40-60 data points were averaged for each of 6 oxygen pressures in each experiment. The combined data for the experiments under each set of
conditions were fitted by nonlinear least squares (31) directly to the
Hill equation, y = KPn/(1 + KPn) to
estimate n and P50 (= nth
root of K1). Comparison of the absorption spectra before
and after an experiment suggests that Met-Hb formation during an
experiment is no more than about 5%.
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RESULTS AND DISCUSSION |
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Hemoglobin Expression-- Examination of C. lacteus neural ganglia and lateral nerve cords in fresh tissue shows that the cells surrounding axon bundles are uniformly bright red (HC in Fig. 2, A and B). Microspectrophotometry of this tissue gives the characteristic spectrum of HbO2, which is not detectable within the axonal bundles (AX), although some Hb-containing cells appear to have processes that enter the axonal bundles. Cells within the body wall also express Hb, but at differing concentrations, being most concentrated in the trunk region surrounding the gonadal sacs where it may aid in the delivery of oxygen to the developing gametes.
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Structure of Globins and Globin Genes-- A single major globin protein fraction was isolated from neural tissue extracts in a three-step chromatographic procedure (Fig. 3, A-C). The body wall Hb also gave a single major peak in the first two chromatographic steps, but RP-HPLC of the Hb fraction showed several minor peaks (Fig. 3D), raising the possibility of additional Hbs in this tissue. NH2-terminal sequences of neural and body wall globins were determined (43 and 45 residues, respectively) and found to share 79% amino acid sequence identity (Fig. 4). The first eight residues of both globins are identical (VNWAAVVD), allowing us to construct a redundant oligonucleotide primer directed at both globin genes (see "Experimental Procedures"). Use of this primer and oligo(dT) permitted the amplification and cloning of a 581-bp cDNA fragment that encodes the complete sequence of a globin from the neural tissue (Fig. 5). Five clones were completely sequenced and found to be identical in the coding region, but they possessed some differences in the 3' non-coding region. The amino acid sequence determined by direct sequencing was DFY at residues 9-11 but was EL-STOP in the PCR-generated cDNA clones (Figs. 4 and 5). We conclude that the EL-STOP sequence is the result of errors in amplification and/or cloning because the neural globin genomic DNA sequence (Fig. 6) matches the direct determination of residues 1-42. An additional difference is that the cDNA sequence has Leu at position 56, whereas the genomic DNA gives Ile for this position.
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Protein Structure-- The chains of neural and body wall Hbs have only 109 residues which makes them the smallest naturally occurring Hbs known. Unlike the proteolytically generated mini-Mb of 108 residues (20-22), the mini-Hbs of C. lacteus are stable. Paramecium Hb (37) is slightly larger (116 residues) than the nemertean Hbs and autoxidizes (38) 10-fold faster at pH 8 than sperm whale Mb (153 residues). The C. lacteus Hbs are sufficiently stable to permit the HbO2 to survive the lengthy purification procedures and to make possible the measurement of O2 equilibria over a period of several hours. In contrast, the mini-myoglobins (20-22) are rapidly oxidized in air, and O2 equilibrium measurements are impossible. We examine here how the C. lacteus chains differ from those of other Hbs and how these chains may achieve stability despite their short length.
The Alignment Reveals Amino- and Carboxyl-terminal
Deletions--
The alignment of 22 globins (Fig.
8) shows an
impressive conservation of hydrophobic
residues at 37 internal positions throughout the globin family
(i.e. the 34 columns of residues with blue
backgrounds plus the CD1, E7, and F8 columns in red in Fig.
8) This pattern of 37 non-polar amino acids in globins at
solvent-inaccessible locations, identified by Gerstein et
al. (68), was applied to a sequence alignment by Kapp et
al. (23). It allows a much improved refinement of the alignment of
all globins. These residues at solvent-inaccessible positions are a
major factor for maintaining the universal globin fold. The C. lacteus sequences have 34 of these 37 positions retained and lack
only A8, B6, and H19 because of deletions (see Fig. 8). Kapp et
al. (23) have also identified 84 positions conserved in all 700 globin sequences examined (boxed regions in Fig. 8).
According to our alignment, the C. lacteus globins have all
of these conserved positions except for 7 residues deleted in regions
B5-B7 and H16-H19. Despite their shortness, the globins of this
nemertean have clearly retained most of the 37 solvent-inaccessible and
the 84 core residue positions. However, large deletions appear at both
ends of C. lacteus globins. The alignment shows that greater
parts of the A and B helix, as well as from the H helix, are missing.
Still, all six A-helical core residues are maintained in the nemertine
globins. A tryptophan occupying position 3 of this conserved core forms
a hydrophobic anchor that makes contacts with the E, G, and H helices.
This position, Trp-3 in C. lacteus globins, corresponds to
human chain residue Trp-14 (A12) which forms contacts with Ala-63
(E12), Leu-66 (E15), Thr-67 (E16), Val-70 (E19), Leu-105 (G12), and
Leu-109 (G16) (see Ref. 69). The alignment suggests that very similar contacts are likely in C. lacteus neural Hb: Val-49 (E12),
Ile-52 (E15), Asn-53 (E16), Leu-56 (E19), Leu-86 (G12), and Cys-90
(G16). The alignment preserves the propensity for a hydrogen
bond-forming residue at position E16 in 15/22 of the chains shown in
Fig. 8. The six-residue core of the A helix identified by Kapp et
al. (23) is part of the 13-residue core found in six high
resolution x-ray studies (70). The six-residue core is conserved in all globins including that of C. lacteus. We conclude that
C. lacteus globins have retained the minimal core of
residues in helix A required for the Mb fold.
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Heme Pocket--
The structural stability of Hbs depends strongly
on the affinity for heme. Ferrous Mbs and Hbs show little tendency to
lose heme because of the covalent bond between the proximal His and the
Fe2+ atom. This bond is much weaker in ferric Hb (Met-Hb).
The lower retention of the hemin in Met-Mb and Met-Hb then depends more on the hydrophobicity of the heme pocket and, in aquo-Met-Hb, on the
water molecule that is anchored to the iron and to distal residues
(72). Additional stabilization is contributed by electrostatic contacts
with the propionic acid groups on the heme. Table
II shows the residues of the heme pocket
in sperm whale Mb and in the chain of human Hb. Without exception,
all residues in the positions that are hydrophobic in both these chains
are also hydrophobic in C. lacteus globins.
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Hydrophilicity--
The hydrophilicity patterns of Fig.
9 show the close similarity of the body
wall and neural Hbs of C. lacteus. The neural Hb has,
however, substantially more hydrophilicity than the body wall Hb. This
difference can be quantified by examining the 24 positions where the
amino acid residues differ (Fig. 4). The sums of the hydrophilicity
values at these positions for body wall and neural Hbs are 7.9 and
39.4, respectively.2 Why
the Hbs should differ in this way is puzzling. Perhaps it reflects
differences in the nature of the subunit interfaces involved in
self-association.
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Phylogenetic Analysis of Globin Sequences-- The 22 aligned globin sequences (Fig. 8) were analyzed as described under "Experimental Procedures." Fig. 11 summarizes the results. Consensus tree A is based on 1,000 bootstrap replicates, and the consensus trees B-D each are based on 100 replicates of the original data set of the 22 sequences.
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Hemoglobin Function--
Data on oxygen equilibria measured
in situ by microspectrophotometry (Fig.
12, A and B, and
Table III) show that the neural tissue Hb
has a higher oxygen binding affinity (P50 2.9
torr) than that of the body wall Hb (P50
4.1
torr) at 15 °C. Oxygen binding by the neural Hb in situ
exhibits little or no dependence upon extracellular pH within the
narrow range of pH 7.3-7.9 (
log P50/
pH < 0.1). Intracellular pH was not
measured. Therefore, these results could mean that the intracellular pH
was relatively constant and independent of extracellular pH.
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ACKNOWLEDGEMENTS |
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We thank Peter Smith, Elizabeth Balser, and Pat Behrens for assistance with the experiments; Alfred Wheeler and Edward Ruppert for the use of equipment; and Janet Young, Gwen Gage, Kristina Schlegel, and David Green for assistance with graphics. We thank Sandra Smith of the University of Texas Microsequencing Facility for automated sequencing of proteins.
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FOOTNOTES |
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* This research was supported in part by Welch Foundation Grant F-213 (to A. F. R.), National Science Foundation Grants DMB-8600614 (to J. M. C.), MCB 9205764, 9511759 and 972385 (to A. F. R.), and National Institutes of Health Grant GM35847 (to A. F. R.). Preliminary accounts of this work have been presented (1, 2).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF033001 and AF033002.
§ Present address: Natural Sciences Division, Pepperdine University, 24255 Pacific Coast Hwy., Malibu, CA 90263. Tel.: 310-456-4326; Fax: 310-456-4758; E-mail: tvanderg{at}pepperdine.edu.
¶ Present address: Hematology-Oncology Division, LMRC-2, Brigham and Women's Hospital, 221 Longwood Ave., Boston, MA 02115.
** To whom correspondence should be addressed.
1 The abbreviations used are: Hb, hemoglobin; Mb, myoglobin; HPLC, high performance liquid chromatography; RP-HPLC, reverse phase HPLC; STPD, standard temperature and pressure, dry; PCR, polymerase chain reaction; bp, base pair; kb, kilobase pair.
2 We use the Kyte-Doolittle "hydropathic index" for these values. Hydrophilic residues are negative in this scale (73). The MacVector Program reverses the sign to provide a hydrophilicity scale (Fig. 9). The original Kyte-Doolittle scale (here called hydrophobicity) is used in Fig. 10.
3 Only the starred sequences are shortened. Bootstrap confidences are given as superscripts.
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REFERENCES |
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