/ . Embryol exp. Morph., Vol. 16, 2, pp. 259-270, October 1966
With 1 plate
Printed in Great Britain
259
Starch gel electrophoresis of the multiple haemoglobins of small and large larval Chironomus—
a developmental haemoglobin sequence
in an invertebrate
By CLYDE MANWELL 1
From the Laboratory of the Marine Biological Association of the U.K., Plymouth,
and the Department of Biological Science, Florida State University
INTRODUCTION
Two interrelated aspects of haemoglobin differentiation in vertebrates are
tissue specificity (muscle haemoglobin and blood haemoglobin) and time
specificity (embryonic, larval or foetal haemoglobin, and adult haemoglobin).
Tissue specificity of haemoglobin and of other respiratory proteins has been
found in invertebrates and has been shown to be the result of differences in
gene activity (Man well, 1963 a, 1964; Man well & Baker, 1966), as accounts for the
time and tissue specificity of vertebrate haemoglobins (Ingram, 1963; Manwell,
19636) and of vertebrate lactate dehydrogenases (Markert, 1963; Blanco,
Zinkham & Kupscyk, 1964).
Besides being the first report of a haemoglobin time-sequence during the
development of any invertebrate, the following data on the insect Chironomus
plumosus L. are important in view of the use of larval chironomids in studies on
changes in chromosomal morphology ('puffing') in relation to gene action and
to the control of formation and transport of specific proteins (Beerman, 1952;
Clever, 1964a, b; Laufer & Nakase, 1965).
MATERIALS AND METHODS
Numbers of a larval chironomid, tentatively identified as Chironomus plumosus
L., were collected from a slightly brackish pond on a rubbish tip adjacent to the
Saltram Estate, Plympton, Devon. Chironomus larvae contain haemoglobin in
solution in the haemolymph (reviewed: Buck, 1953; Weber, 1965) and this can
be obtained either by homogenization of pooled larvae (Braunitzer & Braun,
1965) or by bleeding isolated individuals with a capillary pipette. Haemoglobin
is the major soluble protein extracted from homogenized Chironomus larvae
1
Author's address: The Laboratory, Citadel Hill, Plymouth, Devon, England.
260
C. MANWELL
(Braunitzer & Braun, 1965) and, judging from the present electrophoretic
studies, comprises at least 90-95 % of the haemolymph protein. Haemoglobin
samples were usually converted to the carbon monoxide derivative for increased
stability, although as a check on the nature of the complex haemoglobin
heterogeneity, oxyhaemoglobin, methaemoglobin, and methaemoglobin-cyanide
derivatives were also studied as were CO-haemoglobin reacted with the following —SH and —S—S— reagents: reduced glutathione, oxidized glutathione,
o-iodosobenzoate, 7V-ethylmaleimide, iodoacetamide, sodium /7-hydroxymercuribenzoate, sodium p-chloromercuriphenylsulphonate, and mercaptoethanol.
These reagents were added directly to the haemoglobin in the approximate
ratio of 1 mg of reagent to 0-1 ml of 1-2% haemoglobin solution. In one
experiment mercaptoethanol at a final concentration of 1 % was added to the
discontinuous gel buffer used in electrophoresis.
Smithies' (1959) vertical starch-gel electrophoresis, modified in a variety of
minor ways (Manwell, 19636) and run at voltages of 400-650 V with air cooling
at 0-6 °C, was used with a variety of the standard buffer systems, including
Ferguson and Wallace's (1961) modification of Poulik's discontinuous buffer
(gel pH 8-0); Smithies' (1959) borate (pH 8-4-8-6); 'tris'-EDTA-borate (gel
pH 8-8); and potassium phosphate, ionic strength 0-02, pH 6-0, 6-7 and 7-5,
yielding gel pHs of 6-0, 6-7 and 7-2-7-3, respectively.
Purified haemoglobin components were obtained by electrophoresis of pooled
haemolymph from twenty to thirty larvae of a given size. The isolated haemoglobins were removed from the starch gel by freezing and thawing under pure
carbon monoxide. Amounts of the different multiple haemoglobins of pooled
samples of large larvae were determined spectrophotometrically (Lemberg &
Legge, 1949). Purity of isolated haemoglobins was checked by both total
protein staining after electrophoresis and by spectrophotometry in the u.v. and
visible ranges. There are a number of minor non-haemoglobin proteins in
Chironomus haemolymph; however, with the exception of a few of the most
rapidly moving trace haemoglobin components, these do not overlap with the
haemoglobins.
Globin was prepared by treatment of electrophoretically isolated single
haemoglobin components with the usual technique involving 1 % HC1 in
acetone (Lemberg and Legge, 1949). To ascertain if purified haemoglobin components were made up of more than one kind of polypeptide chain type,
electrophoresis of the globins was carried out in starch gels made with HC1
and formic acid, pH 5 (Muller, 1960; Dozy, Reynolds, Still & Huisman, 1964),
or with 8 M urea, pH 8-0-8-6 (Baglioni & Sparks, 1963; Chernoff & Pettit, 1964a).
Such electrophoretic methods have been more successful than column chromatographic procedures in separation of polypeptide chain types of non-mammalian
haemoglobins (Elzinga, 1964; Manwell, 1966 and unpublished studies on
annelid, sea-cucumber and fish haemoglobins). In these studies on globin
electrophoresis, human globin, which separates into a and /? chains (Muller,
Ontogeny of Chironomus haemoglobin
261
1960; Chernoffand Pettit, 1964a), was used as a control; in addition, mercaptoethanol, 4-10 ml/1., was added to some of the acid and 8 M urea gels to investigate the possibility of disulphide bonds contributing to any quaternary structure.
Although it is desirable to have additional data, e.g. N- and C-terminal amino
acids, peptide subunit 'fingerprints', and molecular weights, in deciding the
very difficult question of the polypeptide chain types in a protein molecule,
electrophoretic methods are the only practical method in dealing with haemoglobins from individuals of so small an animal as larval chironomids (1-11 mg.
each). Electrophoretic methods do not require elaborate or expensive equipment,
and have provided the greatest insight into the complex heterogeneity of L and
H chain types in antibodies (Cohen & Porter, 1964).
5
10
4 3
2 1
Text-fig. 1. Diagrammatic representation of the results of discontinuous starch-gel
electrophoresis at pH 80 of the multiple haemoglobins from small larvae (lower part
of figure) and large larvae (upper part of figure) of Chironomus plumosus. Compare
with Plate. The haemoglobin zones are arbitrarily numbered 1 to 10 in the order
from most anodal to most cathodal: this order is preserved in other alkaline buffers.
In some experiments a very minor zone of o-dianisidine peroxidase activity occurs
between zones 7 and 8; whether this is a trace haemoglobin or a true peroxidase
is not yet settled; thus, there may be eleven haemoglobins in large larvae and ten are
consistently observed at present. The ten haemoglobins occur in sufficient concentration as to be directly visualized by the characteristic red colour in starch gels and
the eluted zones have the characteristic absorption spectrum of haemoglobin.
RESULTS
Electrophoresis of the haemoglobins from small and large larvae
The electrophoretic resolution of haemoglobin from small (1-4 mg each) and
large (8-11 mg each) larval Chironomus plumosus is shown diagrammatically in
Text-fig. 1 and by a photograph of the anodal portion of a starch gel in the
Plate. The latter was stained for total protein and emphasizes that almost all of
the haemolymph protein is haemoglobin. A minimum of eight haemoglobins
can be resolved by electrophoresis of haemolymph of large larval Chironomus in
the various buffers (pH 6-9) mentioned earlier. Two of the minor anodal
haemoglobins are further resolved into additional zones to give a total of ten
262
C. MANWELL
haemoglobin components in the discontinuous buffer of Ferguson & Wallace
(1961) (Text-fig. 1 and the Plate). The relative amounts of the multiple haemoglobins in large larvae are: minor anodal haemoglobins (zone 1,8%; zone 2,
5 % ; zones 3 + 4 together, 8%; zone 5, 5%); major haemoglobins (zone 6,
22 %; zone 7, 21 %; zone 8, 22 %), minor haemoglobin (zone 9, 6 %), and minor
cathodal haemoglobin (zone 10, 3-5 %). Small larvae consistently lack two of the
three major haemoglobins (zones 6 and 8) and a minor haemoglobin (zone 9).
In addition, the small larvae possess a major haemoglobin (designated 'zone X')
which migrates slightly but consistently more anodal than zone 6 in all alkaline
buffers, although it becomes clearly different only in the discontinuous buffer of
Ferguson & Wallace (1961) (Plate). It appears that a trace of 'zone X ' remains
in the haemolymph from large larvae although even with the sensitive odianisidine test for haemoglobin, small larvae lack completely haemoglobin
zones 6, 8 and 9.
Although the complex heterogeneity and the ontogenetic differences are consistent in all buffer systems, a number of other possible sources of error exist:
PLATE 1
Starch-gel electrophoresis of the multiple haemoglobins of small and large larval Chironomus
plumosus—consistency of the heterogeneity and ontogenetic change upon conversion of
haemoglobin to different derivatives. The seven samples are, from left to right: (1) methaemoglobin from large larvae; (2) methaemoglobin from small larvae; (3) oxyhaemoglobin from
large larvae; (4) oxyhaemoglobin from small larvae; (5) methaemoglobin-cyanide from large
larvae; (6) carbonmonoxyhaemoglobin from large larvae; (7) oxyhaemoglobin from large
larvae. Note:
(a) Small larvae lack the two slower haemoglobin zones (zones 8 and 9—see Text-fig. 1),
regardless of what haemoglobin derivative is studied.
(b) Small larvae have a major haemoglobin zone, zone X, marked by arrows, which
migrates slightly more rapidly than the second major haemoglobin, zone 6, in large larval
samples.
(c) Several of the individual haemoglobin zones have their electrophoretic mobility shifted
by formation of different derivatives, e.g. CO-haemoglobin of zones 7, 8, and 9 has a
slower electrophoretic mobility than either oxyhaemoglobin or cyanmethaemoglobin for these
zones. The methaemoglobin derivatives are especially altered in mobility, at least for zones
3, 4, 5 and 6, some of these migrating in the line of discontinuity at the top of the picture.
Nevertheless, the intrinsic heterogeneity and the difference between small and large larvae
remain.
Conditions of electrophoresis: The anode is at the top of the picture; the cathodal section,
including haemoglobin zone 10, has been removed to avoid reduction and loss of detail of the
anodal portion of the gel photograph. The starch gel was made with the pH 8 0 buffer of
Ferguson & Wallace (1961) and run at 450 V for 8 h. The gel was stained for total protein with
Amido Black lOB-nigrosin mixture; as a result it can be seen that nearly all of the haemolymph
protein is haemoglobin. Non-haemoglobin contaminants account for the heavy protein
staining at the line of discontinuity (uppermost part of the figure) and the light, sharp band,
just cathodal to the diffuse haemoglobin zone 9. The slight unevenness of the zones is the result
of use of solutions of ferricyanide and cyanide for formation of methaemoglobin and methaemoglobin-cyanide, with consequent dilution of the haemoglobin sample (thus zones are somewhat less intensely staining in samples 1, 2 and 5).
/. Embryol. exp. Morph., Vol. J6, Part 2
PLATE 1
C. MANWELL
facing p. 262
Ontogeny of Chironomus haemoglobin
263
mixing species, unusual individual variation, and chemical artifacts. Results
relevant to these three possible sources of error are presented below:
(1) Species differences. As small larvae raised in the laboratory acquire the
additional haemoglobin zones and lose the intensity of' zone X ' at approximately
5 mg size (3rd instar), the difference in haemoglobin patterns is not the result of
mixing species collected in the field—a source of confusion in other haemoglobin
studies (see Manwell & Baker, 1963).
(2) Individual variation. The eight haemoglobin components of small larvae,
including zone X, were observed not only in five sets of pooled individuals but
also in sixteen small larvae surveyed individually and in several different buffer
systems. The ten haemoglobin components of large larvae were observed in all
studies on eight sets of pooled material, plus all fifty-one large larvae screened
singly. Larvae of intermediate size are also intermediate in haemoglobin
pattern. There was no individual variation equivalent to a haemoglobin polymorphism of a genetic type among the C. plumosus larvae surveyed in this study,
although a complexity of esterase polymorphisms was observed among the same
individuals (Manwell & Baker, unpublished studies). The ten haemoglobins also
persist in pupae, though the quantity of haemoglobin is often reduced at the
end of pupation; adults usually lack haemoglobin, although some of the major
globin components remain, coupled with a greenish colour, presumably a bile
pigment.
(3) Chemical artifacts. It is difficult to test for all possible biochemical complications, especially with small samples and on an individual basis. However, a
number of known potential hazards in haemoglobin studies (Lemberg & Legge,
1949; Manwell, 19636, 1964; Riggs, 1965 a) have been checked out. The heterogeneity of Chironomus haemoglobin and the differences between small and large
larvae persist whether the haemoglobin is converted to oxyhaemoglobin, COhaemoglobin, methaemoglobin, or methaemoglobin cyanide (Plate). As would
be expected on the basis of configurational changes there are differences in the
electrophoretic mobility of certain of the haemoglobin components according
to whether combined with oxygen, carbon monoxide or cyanide, the greatest
differences being observed for the methaemoglobin derivatives (Plate) in agreement with studies on human haemoglobin (Chernoff & Pettit, 19646). However,
these alterations in electrophoretic mobility—even when oxygen and CO are
compared—change neither the fundamental heterogeneity nor the ontogenetic
difference. Thus, the observed developmental change is not the result of some
of the haemoglobin being in a different oxidation state or combined with
different ligands.
It has been shown recently that some of the heterogeneity of human, mouse,
turtle and frog haemoglobins is the result of chemical modification of the —SH
groups, in some instances involving glutathione (Sullivan & Riggs, 1964; Riggs,
Sullivan & Agee, 1964; Riggs, 19656; Trader & Frieden, 1966). Treatment of
Chironomus haemoglobin with the various —SH and —S—S— reagents
17
JEEM 16
264
C. MANWELL
enumerated in Materials and Methods is singularly without effect, although many
of these agents drastically alter the electrophoretic mobility and heterogeneity
of certain vertebrate haemoglobins. These observations prove that the heterogeneity of Chironomus haemoglobin does not involve chemical modification of
sulphydryl groups, as well as that there are no readily reactive —SH groups in
Chironomus haemoglobin. Similarly, Braunitzer & Braun (1965) report only
0-1 cysteine per chain for one of the multiple haemoglobins of Chironomus
thummi.
There is no differential retardation in electrophoretic mobility of any of the
Chironomus haemoglobin components by changes in the amount of starch in the
gels; this observation suggests (see Smithies, 1962; Ornstein, 1964) that none of
the haemoglobins are polymers of the others. Similarly, Weber (1965) reports
only a single peak for Chironomus plumosus haemoglobin in the analytical
ultracentrifuge. Thus, the heterogeneity of Chironomus haemoglobin is not the
result of different degrees of stabilized molecular aggregation.
Further evidence for the lack of interconvertibility of Chironomus multiple
haemoglobins and for the reality of the differences between small and large
larvae comes from studies on globins prepared from each of the isolated multiple
haemoglobins.
Electrophoresis of globins
Electrophoresis of the globins of the multiple haemoglobins of C. plumosus in
8 M urea starch gels, with or without mercaptoethanol, does not remove the
unique electrophoretic mobility of each component. The globins migrate as
single zones in 8 M urea, with or without mercaptoethanol, although traces of
protein, presumably denatured globin, remain at the sample insertion slots.
Smudges of additional zones are often found in electrophoresis of the globins
from the fast anodal minor haemoglobins (zones 1-4 in Text-fig. 1); these probably represent contamination with small amounts of non-haemoglobin protein.
Electrophoresis of globins in pH 2 gels, with or without mercaptoethanol, yields
similar results to the 8 M urea electrophoresis, although globin from haemoglobin
zone 8 and from zone X yields two zones.
However, in contrast to the control separation of human and other vertebrate
globins into a and fi chains, which yields two zones of equal protein-staining
intensity consistently, the acid-gel electrophoretic splitting of globins from
Chironomus haemoglobins zone 8 and zone X is neither consistent nor, when
present, are the zones of equal intensity. It is known that additional variable
minor zones accompany electrophoresis of vertebrate a and /? chains (Baglioni
& Sparks, 1963; Dozy et al. 1964) and in some instances are the result of modification of—SH groups on the haemoglobin chains (Chernoff & Pettit, 1964 a;
Manwell, unpublished studies on fish haemoglobins). In the case of Chironomus
mercaptoethanol does not eliminate these additional zones in acid-gel electrophoresis. The lack of reproducibility and relative proportion suggests some
secondary alteration of the globin rather than additional heterogeneity of poly-
Ontogeny of Chironomus haemoglobin
265
peptide chain types. Thus, at present it appears as if most of the multiple
Chironomus haemoglobins possess a unique globin moiety and there may be a oneto-one correspondence between the multiple haemoglobins separated by mediumvoltage electrophoresis and polypeptide chain types. As the complex heterogeneity of Chironomus haemoglobin becomes visible only after one or two hours
of electrophoresis when certain broad bands of haemoglobin are resolved into
multiple components, and as there are near-stoichiometric equivalence for some
of the multiple haemoglobins, it is very likely that some degree of weak binding
between the different polypeptide chain types occurs, but, clearly, it is not as
strong as that maintaining human or certain other vertebrate haemoglobins into
the a2/?2 unit. Similarly, Weber (1965) observed very weak haem-haem interactions in the oxygen equilibrium of Chironomus plumosus haemoglobin (n =
1-05-1-30). Comparable electrophoretic and oxygen-binding data on lamprey
haemoglobins have been interpreted as indicating weak binding between polypeptide chains (Manwell, 19636; Rumen & Love, 1963; Briehl, 1963; Antonini
et al 1964).
DISCUSSION
Although such complex heterogeneity of haemoglobin in as small an animal as
Chironomus larvae may seem surprising, Braunitzer & Braun (1965) report five
haemoglobins separable by column chromatography of extracts of Chironomus
thummi. Furthermore, by additional chromatography or by counter-current
distribution, they separated two of these haemoglobins into additional components which were shown by amino acid composition, 'finger-printing' and
N- and C-terminal amino acids to be polypeptide chains of different primary
structure. They did not report any studies on single individuals of different
sizes and thus did not observe the developmental changes reported here. In both
C. thummi and C. plumosus there are at least three different major haemoglobin
chains, plus a number of minor polypeptide chain types. As different haemoglobin
polypeptide chains are the ultimate products of different genes (Ingram, 1963),
it is reasonable to suggest that the absence of haemoglobin components 6, 8 and
9 and the presence of large amounts of haemoglobin 'zone X ' in small C. plumosus larvae, and the presence of haemoglobin components 6, 8 and 9 and the
absence (or great reduction) of haemoglobin 'zone X ' in large C. plumosus
larvae, means a 'switchover' in the activity of certain haemoglobin genes in
C. plumosus accompanies normal development. In man the change from foetal
haemoglobin (Hbi 7 ) to adult haemoglobin (YLbA) involves a 'switchover' in
expression from the y-chain gene to the /?-chain gene (Ingram, 1963). A further
similarity between man and Chironomus is the constant expression of the a-chain
gene in human ontogeny and the persistence of the various trace anodal haemoglobins, one major haemoglobin (zone 7), and the cathodal minor haemoglobin (zone 10) throughout larval life of C. plumosus. Likewise during early
embryology of the chicken both the constant production of certain adult
17-2
266
C. MANWELL
haemoglobin chain types and a 'switchover' from embryonic to other adult
haemoglobin chain types are found (Manwell, Baker & Betz, 1966). That
haemoglobin differentiation in an insect, the chicken, and man have such features
in common is of interest in the application of genetic control systems to biochemical embryology (see Waddington, 1962). However, other patterns of
haemoglobin differentiation occur and these emphasize the complexity of
patterns that have been evolved, in several instances independently (Manwell &
Baker, 1966).
Especially promising is the possibility of linking the developmental haemoglobin changes in Chironomus with the time-specific alterations in the 'puffing'
patterns of the giant chromosomes in the salivary glands and other tissues;
several striking 'puffing' changes occur in the middle of larval development
(beginning of the third instar) (Beerman, 1952; Clever, 1964a) which is when the
'switchover' from the small to the large larval haemoglobin pattern occurs,
but much remains to be learned concerning the site of globin synthesis in
Chironomus and the times that globin synthesis is programmed (transcription)
and activated (translation).
After this paper was accepted for publication electrophoretic data on the multiple haemoglobins of several Chironomus species were presented by P. E. Thompson and D. S. English
(1966) ('Multiplicity of hemoglobins in the Genus Chironomus (Tendipes)', Science, N.Y.
152,75-6). These authors reported that certain of the multiple haemoglobins appeared earlier
than others during larval development and compared the situation with the developmental
haemoglobin sequence in man. No data on polypeptide chain arrangement were presented but
it was inferred from the species differences that the multiple haemoglobins are the products of
different genes. Thus the question of the number of different haemoglobin chain types in
Chironomus multiple haemoglobins remains. It is possible that some of the multiple haemoglobins will be found to be made up of only one chain type and that others will be found to be
comprised of two similar chains. An example of the complexity of quaternary structure
organization has been provided by studies on the polypeptide chain composition of the
multiple haemoglobins of closely related holothurians (C. Manwell (1966), 'Sea cucumber
sibling species: polypeptide chain types and oxygen equilibrium of hemoglobin', Science,
N.Y. 152,1393-6). In two species four anodal haemoglobin components are made up ofbut one
electrophoretically demonstrable chain type; the equilibrium between the four components
can be altered by chemical modification. Thus, these four haemoglobins represent an interconvertible set of isomers or polymers based on only one polypeptide chain type. However,
the less acidic haemoglobin components in these species are composed either of equal amounts
of two chain types, or entirely of one chain type. One species has a total of three chain types
and has haemoglobin components composed of all possible single and pairwise combinations;
another species has the same number of electrophoretically distinguishable chain types but
has fewer multiple haemoglobins because certain of the chain combinations do not occur; a
third species has fewer multiple haemoglobins simply because it has lost one of the chain
types. It may well be that only by 'finger-printing' each of the multiple Chironomus haemoglobins, combined with studies on genetic haemoglobin polymorphism, will the precise
number of haemoglobin chain types (and thus haemoglobin cistrons) in Chironomus be
determined.
Ontogeny of Chironomus haemoglobin
267
SUMMARY
1. Small larvae (1-4 mg each) of Chironomus plumosus, whether surveyed in
pooled samples or individually, have eight different haemoglobins in the
haemolymph. Large larvae (8-11 mg each) of this species have ten different
haemoglobins. Seven of the haemoglobins are unchanged throughout larval
development. However, small larvae lack two of the major and one of the minor
haemoglobins found in all large larvae; furthermore, a unique major haemoglobin of small larvae occurs only in traces in large larvae.
2. The heterogeneity and the ontogenetic differences in Chironomus haemoglobin persist whether the haemoglobin is studied as oxyhaemoglobin, carbonmonoxyhaemoglobin, methaemoglobin or methaemoglobin cyanide, although
the electrophoretic mobilities of some of the haemoglobin components are
shifted by combination with different ligands and, especially, by oxidation to
methaemoglobin.
3. The heterogeneity and the ontogenetic differences are not altered by
treatment of the haemoglobin with —SH and —S—S— reagents, nor by
conversion of haemoglobin to globin by splitting off the haem with acidacetone.
4. Electrophoresis of globins prepared from each of the isolated major and
minor Chironomus haemoglobins suggests that, in contrast to typical vertebrate
haemoglobins, there is only a single polypeptide chain type in each of the
multiple Chironomus haemoglobins. However, each of the multiple haemoglobins probably has a unique haemoglobin chain type, suggesting that each
Chironomus haemoglobin component is coded from a different gene.
5. Thus the insect Chironomus resembles the human in possessing an ontogenetic haemoglobin sequence involving both constant expression of some
haemoglobin genes and modulation of the activity of others. As, in contrast to
the human, Chironomus possesses giant chromosomes in many of its tissues, this
discovery of an ontogenetic sequence to the major soluble protein present in
larval Chironomus presents an attractive system for studying the interrelations
between protein phenotype and the nuclear genotype during development.
RESUME
1. Qu'elles soient etudiees individuellement ou ensemble, les petites larves de
Chironomus plumosus (de 1 a 4 mg chacune) contiennent huit hemoglobines
differentes dans leur hemolymph. Les grandes larves de cet espece (de 8 a 11 mg
chacune) possedent 10 hemoglobines differentes. Sept restent inchanges pendant
tout le developpement larvaire. Toutefois, il manque aux jeunes larves certaines
des hemoglobines presentes dans toutes les larves de grande taille: c'est a dire,
deux des hemoglobines majeures et deux mineures; de plus, l'hemoglobine
principale des petites larves ne se trouve qu'a l'etat de trace dans les grandes
larves.
268
C. MANWELL
2. Une etude des formes oxy-, carbonmonoxy, methemoglobine ou cyanure
de methemoglobine a revele que l'heterogeneite et les variations ontogenetiques
persistent, quelque soit la forme sous laquelle les hemoglobines differentes se
trouvent; toutefois, les mobilites electrophoretiques de quelques uns des composants sont modifiees par rapport aux differentes ligandes, surtout apres oxidation
en methemoglobine.
3. L'heterogeneite et les differences ontogenetiques des hemoglobines ne
sont pas modifiees par un traitement aux reactifs des —SH ou des —SS, ni par la
conversion de l'hemoglobine en globine apres extraction de Theme par l'acetone
acide.
4. L'electrophorese des globines preparees a partir des hemoglobines majeures
et mineures de Chironomus suggere que, a l'encontre des vertebrates typiques,
chacune des hemoglobines multiples de Chironomus ne possedent qu'une seule
chaine polypeptidique. II est cependant probable que chacune des hemoglobines
ne possedent qu'un type unique de chaine; on peut done imaginer que chaque
type d'hemoglobine est l'expression d'un gene different.
5. L'insecte Chironomus possede, done, comme l'homme, une sequence
ontogenetique caracterisee non seulement par l'expression constante de certaines
genes pour l'hemoglobine, mais aussi par la modulation de l'activite des autres.
A la difference de l'homme, l'existence de chromosomes geants dans beaucoup
de tissus de Chironomus, ainsi que la decouverte d'une sequence ontogenetique
pour une proteine soluble majeure, rendent la larve de Chironomus particulierement interessante, pour l'etude des relations entre le phenotype proteique et le
genotype nucleaire au cours du developpement.
I am especially grateful to Miss C. M. Ann Baker for assistance in collection of Chironomus
larvae and in electrophoretic experiments. This research was done while the author was the
recipient of a United States Public Health Service Postdoctoral Fellowship (F3-AM-22232-01, 02). The research has been generously supported by the U.S. National Science
Foundation Grant GB-3037 to Florida State University.
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