The nucleotide sequence and in situ localization of a gene for a dimeric haemoglobin from the midge Chironomus thummi piger

297

Gerw, 64 (1988) 297-304 Elsevier GEN 02348

Short Communications The nucleotide sequence and in situ localization Chironomus thummi piger (Recombinant

DNA;

bacteriophage

of a gene for a dimeric haemoglobin from the midge

2; genomic library;

haemoglobin

gene evolution;

Chironomidae;

Insecta)

Thomas Hankeln, Peter Rozynek and Erwin R. Schmidt Ruhr-Utliverxitiit

Bochum.

Rccelved

25 August

Revised

12 December

Imtitut

I&

Genetik.

D-4630

Bochum

I (F.R.G.)

Tel. (0234)

7003839

1987 1987

Accepted

30 December

Received

by publisher

19X7 20 February

1988

SUMMARY

A cluster containing at least four globin genes was isolated by screening an 3,EMBL3 genomic DNA library of the midge Chimnomus thummipiger (Ctp) with a heterologous haemoglobin (Hb) gene IV (HbW) probe from Chironomus thummi thummi (Ctt). This globin gene cluster was localized by in situ hybridization to chromosome II. One globin gene together with its 5’- and 3’-flanking regions has been sequenced. It can be deduced from the sequence that it is a new member of the dimeric HbVIIB family. The Ctp HbVZIB-5 gene displays 91.8% nucleotide sequence homology to a HhVIIB cDNA sequence, reported previously. There is no evidence for intron/exon structure in the Ctp HbVIIB-5 gene.

The aquatic larvae of non-biting midges (Chironomidae, Diptera) contain at least twelve different Hb proteins in their haemolymph, which allow survival in an anoxic environment (Braun et al., 1968). The amino acid sequences of twelve Hb variants of the subspecies Ctt have been published

(Goodman et al., 1983), five of them are monomeric and seven are dimeric proteins. The evolutionary relationships between the different Hb genes have been deduced from the primary structure of the protein variants (Goodman et al., 1983). Furthermore, the existence of two different chromosomal loci, one for the ‘monomeric’ and one for the ‘dimeric’ Hb genes has been predicted (H. Tichy, unpublished

(‘orrespo~zdence 10: Dr. E.R. Schmidt,

complemcntarq

INTRODUCTION

Institut

t’tir Genetik,

(F.R.G.)

Tel. (0234)

Abbreviations:

037X-I

Postfach

Ruhr-Universitat

102148,

D-4630

Bochum. Bochum

7004979.

aa. amino acid(s); bp, base pair(s); cDNA,

l39/88,$03.50

I

0

19X8 Elsevier

Saence

Publishers

B.V.

DNA

(Biomedml

C‘hirommus

to RNA; thummi

Crp. Chirwxww

thummi;

Hb,

rhurw~i pi&r.

haemoglobin:

globulin; kb, 1000 bp; nt.nucleotide(s);

PBS.phosphate-buffered

saline;

(150 mM NaCI.

Na,

Division)

SSC, standard citrate,

saline

citrate

pH 7.6); u, unit(s).

C‘u.

I&, immunoI5 mM

298

data; cited in Goodman sequences

et al., 1983). The nucleotide

of the ‘monomeric’

genes determined

previously

Hblll

(Antoine

and

by the method of Benton

HblY

probe

and Niessing,

do not contain

Here, we report gene cluster

which

gene on Southern

relaxed stringency

introns.

the isolation

cluster from the subspecies

hybridization

of a globin

gene

We isolated

Ctp. One region of this

hybridized

readily

with HbIV

blots was sequenced.

The nucle-

Hind111 fragment

of clone

iG1

(Antoine and Niessing, 1984), which contains the ‘monomeric’ HbIV gene of Ctt. Hybridization was at

1984; Antoine et al., 1987), showed that the Chironomid globin genes - unlike all other known globin genes -

the 0.9-kb

and Davis (1977) using as

(3 x SSC, 55°C) to allow cross-

between

different

one recombinant

at least four globin

genes

Hb gene variants.

clone which contains according

to Southern

hybridization. One region of this Ctp clone, containing

otide sequence shows that it contains a globin gene belonging to the ‘dimeric’ group. The complete

homologous

cluster could be localized in situ to one chromosomal

tion Fig. this ture

technique

band on chromosome II, a position which is different from the one hybridizing predominantly with the ‘monomeric’ globin genes lFIhl1Z and HblV.

sequences,

HbIV-

has been sequenced

by the

of Maxam and Gilbert (1980). The restric-

map and the sequencing strategy are shown in 1. From the nucleotide sequence it is clear that region contains an Hb gene. The primary strucof this Ctp Hb gene and its flanking DNA is

shown in Fig. 2. (b) The coding region EXPERIMENTAL

AND

DISCUSSION

(a) Cloning and sequencing

The entire open reading frame consists of 483 nt corresponding to 161 aa. Alignment to the published sequences of the Ctt HbIV gene (Antoine and Niessing, 1984) and a ~h~~~~ornus thummi HbVIiB cDNA (HbVffB-3; Saffarini et al., 1985) reveals a

of a ‘dimeric’ Hb gene

A genomic library of Ctp DNA prepared in JEMBL3 (Frischauf et al., 1983), has been screened

2 I

I

I

4 1

I

6 I

I

10

8 I

I

I

I

12kb I

I

I

-I

I

-D

100 bp Fig. I. Restriction the method (marked Niessing,

map and nucleotide

sequencing

of Smith and Birnstiel(1976)

by heavy dots) have been determined 1984) which contains

are the IEMBL3 the direction

the isolated

vector arms. The sequenced

and extent of sequencing

strategy.

The restriction

or by double digestions by Southern

Cff HbIV

hybridization

map of the 13-kb insert of clone @iHbl restriction

S, S&961;

fragments. sequence

and the coding region is represented H, HindHI;

The approximate

using the 0.9-kb Hind111 fragment

gene as probe, and by preliminary

part is expanded,

(E, EcoRl;

of isolated

Hf, Hid;

X, XbaI;

was established locations

of J.Gl (Antoine

data. The hatched

by

of genes and

boxes at the ends

by a shaded box. The arrows indicate Sa, SalI).

299

.

.

.

.

5rp90

,

.

.

.

TTAAACTAATCTTT6TTTGTGACTGTATT6T6ATTTTTT66CAAAATTAATT6TATAAAA6CCACAATAATTTTCAAA6TAATTCA6TTT CT

T C6

T A

TTT6A6

6A6AT

C AAA6

CTTCTT

A

6A6

T

TC

. . . START -signal pcptidc TCBATTT6ACTTCAATTCAATTACTAA-CTIGLCAAAAT6AAATTCTTC6CT6TTCTC6CTCTCT6CATC6TT66A6CTATT6CCTCCCC l 7 6 CT A TC 6 C . . . , . ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ T

.

.

.

.

.

.

179 T T

. C C

C C

.

2b9

. T T

T T

.

359

.

CTACCCAGACATCCAAAACAAATTCTCACAITTCGCT66AAA66ACCTC6CTTCAATCAA66ATACT66T6CATTC6CCACACAC6CCAC T 66CT 6 C C T 6CT 6 C

.

.

.

.

.

.

.

.

AA6AATTGTTTCATTCTTGTCAGAAGTCATCTCTC666AAACACCTCAAAC6CT6CC6CA6TCAACTCACTC6TCTCAAA6TT666 T 6AA TT T T 6 T6

.

.

.

.

.

A A

.

C T C

A66

.

. . . . . . 666T6ACAAT~TT6CT6CT6CCT66AACAAA6CTCTTGACAACACCTTC6CCATC6TT6TCCCAC6TCTTTAAATTATTT-----AACTT AT C C T T 6 C T A6 T6 C T6 T AC .

.

.

.

.

.

.

.

.

.

TC TC

539

AACC AACC

C C . 624

TOP A6ATA T----

CT

I .

.

A

.

CTA6CTAAATTAATTTATAATTAT-----6-T6TT6AA6AATTAATAAACATCAATAATTCTCT6T6ATTTTTTAACATCTAAATACAAA 6 C AAAAT AAA A T -6 AGTTAA AATT6AA6 TA-- ---A-CT A6T C AC C TA ATC AT AA ATCAAT66

.

AAT

.

AGATGACCACAAAGCTCGTGGAGTTTCIGCCGCTCAATTC66A6AATTCA6AACC6CTCTC6TT6CCTACCTCCAA6CTAAT6TTTCAT6 T T T TCA T C CT A

.

449

.

706

. CC6

A CC

CC6+ TGCTATTT

.

.

790

.

.

880

.

.

976

CATATTTTATTATTTTTTTGAA6TTACGTTTTCAGCTTACTTATAAATTCATCACTTCAAA6TTTTCATTAAAAA6AATATTTC6AT

.

.

.

.

.

.

.

TGATTTATCTTGATCCTTTCTATCA6AAATAAATTTT66A6TCTA6AT6CAAATAC6TTTTAATTTTTTAAATTTATTCATAAAT6AC6T

.

.

.

.

.

.

.

TTATTTAAAAATGTTTTC66ATGTTTT66AACTCTTTTCTTTTA66TCACTAAACTATT6AATTAAAAATAAATTAATAAAAATAAAA6A6 Fig. 2. Nucleotide 127)/step

and the polyadenylation are compared

the potential

(-

capping

site (nt 86) and the signal peptide

site (nt 662) are underlined.

to the nucleotide

insertions/deletions homology.

of the Ctp HbVIIB-5 gene and its 5’- and 3’-flanking

sequence

(nt 611) codons,

sequence

) are necessary

ofHbVIIB-3 to assume

The nucleotide

sequence

region. The coding

region

region are marked.

A putative

is boxed. The start (nt TATA-box

of the coding region and its 3’. and 5’-flanking

(nt 54)

sequences

(2nd line; Saffarini et al., 1985) and HbVIIB-4 (3rd line; Trewitt et al., 1987). Some in the 5’- and the 3’-nontranslated

The 5’ and 3’ end of the HbVIIB-3 sequence

are marked

by asterisks.

regions

to allow an alignment

with optimal

300

leader sequence of 16 aa and a length of 145 aa for the mature protein. According to its nucleotide and

and HbVIIB-3 differ at 9 aa (Fig. 3). A slightly higher divergence is calculated if Ctp Hb VIIB-5 is compared

amino acid sequences, the Ctp globin gene is a new member of the dimeric Hb y;l;lB subfamily. We, there-

to C~iro~ornu~ t~~rnrn~HbVIIB-4 gene (Trewitt et al., 1987). There are 53 nt substitutions, 33 (62%) of

fore, refer to it as Ctp HbVIIB-5 Goodman

1985: HbVUB-4, The 91.87;

Trewitt

nucleotide

and -2,

Saffarini

them

et al.,

of C@ HbVIIB-.5

to the sequence

region,

HbVIIB-5

22

acid

substitutions.

is under selective pressure

at the protein

When comparing

Ctp HbVIIB-5

to HbVIIB-3

AMAPLSADQVALVKASWKTVKHNEVDI S T EAS RS A S KFFAVLfJLCIVGAIA*S T EAS BS A S MKFFAVLALC I VGA I A*S EAS m3 A s T

# ## # ## # LYAVFKAYPDIMAKFPQFAGKDLDSIKDTADFAVHAGHIVGFFSEVIALMGNAANM~AI~TL A A A GA T T SL Q A A A GA T T SL A A BN 5 A GA T T SL

S S

## # # ## # # LNELAASHKARGITKABFDEF~ASLVAYL~GHINWNDAIEAAWD~ALDNIFSVIFNALEG DK. GDP) VSA G TA C!A VS GNNVA SK: VSt::: GDD VSA G TA SN VS G NVA Nk: VSK. GDD VSA G TA QANVS G NVA NK

T AIVVPR TYAIVVF’H T AIVVPR

of

Calculated

the

sature

homoloqies

in Ctt

Ctt

HbVIIE

HbVI

IB-3

Ctp

HbVIIB

4 anino

V G VNS VNS

* * +

#=haen-contacts

acids: HbVIIB-3

HbVIIB

cDNC\

cDNA

91

-_

5

91

94

(see Fig. 2) is compared the haem-contact

polypeptide,

S AA ES AS TS AA

E

Ctp

HbVI

IB-5

--

f ig. 3. .A comparison Lund CO ‘dimcric

and

peptide

signal

*=StartlStop

The

level.

so that Ctp HbVIIB-5

Di mer i c Consensus Ctt HbVI ID HbVI IEi-:2 cDNA Ctp HbVIIB-5

in amino

Steigemann and Weber, 1979) are devoid of changes (Fig. 3). This provides strong evidence that Ctp

Fig. 2). Ofthe

in the coding

(55”/,) are silent mutations,

is

of HbVIIB-3

et al., 1985; for comparison,

total 40 nt substitutions

resulting

amino acid substitutions are mainly clustered in two The haem-contact sites (according to regions.

et al., 1987).

sequence

homologous

(Saffarini

(HbVIIB-1

et al., 1983; HbVIIB-3,

of tlbVlI

amino acid sequences.

to the amino acid sequences

Hb’ consens~1s sequence

(Goodman

sites (cited after Goodman

‘I hc amino acid sequence ofIlhP’IIB-.?

(Saffarini

-deduced

et al.. 1983). The signal peptide

et al.. lYX3) arc indicated

from the nucleotide

sequence

of Crp Hh WiB-.T

ct al., 1985). Ccr Hb VlIB protein (Sladic-Simic by symbols

region.

the ends of the mature

* and # explained

in the figure.

et al.. lY77),

polypeptide

and

301

HbVZZB-4, nucleotide

one has to take into account

that the

and amino acid changes might partially be

HbVZZB-5 and HbVZZB-4 strongly suggests that these genes are non-allelic.

due to the fact that we have cloned the DNA from

ing regions

the subspecies

contrast

Ct_n (pure-bred

while the ZZbI/IIB-3 cDNA

laboratory

culture),

as well as the ZZbVZZZ?-4

sequence may have been derived from Ctt. In the description of the ZZbk’ZZIB-3 and HbVZZB-4 sequences the subspecies was not specified (Saffarini et al., 1985; Trewitt et al., 1987). An between

alternative

explanation

for the

differences

Ctp HbVZZB-5 and HbVZZB-314 is that Ctp

display

The 5’- as well as the 3’-flankmore than

to this finding

translated tance

part of the HbVZZB-3 cDNA

of about

homology

does

HbVZZB-5

and

not

necessarily

HbVZZB-3

Similarly high homologies

data of Sladic-Simic et al. (1977), who determined the primary structure of the HbVIIB protein in Ctt and published an amino acid sequence, which is slightly different from the amino acid sequences encoded in both Hb VZZB-314 and Ctp Hb VZZB-5 (for the pairwise comparison of the amino acid sequences, see Fig. 3). The assumption that there are at least five different HbVZZB genes can explain these data. As in the Ctt HbZZZ and HbZV genes (Antoine and Niessing, 1984; Antoine et al., 1987) and in the HbVZZB-4 gene (Trewitt et al., 1987) there is no evidence for intron/exon structure in the Ctp HbVZZB-5 gene. This is in agreement with the view that the monomeric ones (Goodman

Hb’s are ancestors

of the dimeric

et al., 1983).

(c) The 5’- and 3’4lanking

regions

In the 5’- and 3’-flanking regions of the gene Ctp HbVZZB-5 putative regulatory sequences necessary for gene function could be identified (Fig. 2). The transcriptional start point was deduced 41 nt upstream from the start codon by alignment to the 5’-flanking region of the Ctt HbZV gene (Antoine and Niessing, 1984). In the 5’-flanking DNA a typical TATA-box occurs at nt -32. In the 3’-flanking sequence, a polyadenylation site is found at nt 50 downstream from the stop codon. Comparison of the flanking sequences of Ctp

this

that

allelic

Ctp

genes.

(approx. 70%) can also be

HbVZZB gene family. Such multiple gene copies have already been reported for the HbVZZB gene family (Trewitt et al., 1987) and also for the monomeric HbZZZ and HbZV genes of Ctt (Antoine and Niessing, 1984; Antoine et al., 1987). They are probably the result of duplication events followed by divergent evolution. This interpretation is supported by the

member

imply

represent

HbZZZ and HbZV genes (Antoine

a further

over a dis-

100 nt (see Fig. 2). However,

found between the 3’-untranslated

represents

In high

nucleotide sequence homology of about 76% of the 3’-flanking region of Ctp HbVZZB-5 with the 3’-un-

of the

HbVZZB-5

50% divergence.

there is a surprisingly

regions of the Ctt et al., 1987).

(d) In situ localization By in situ hybridization of the whole recombinant phage to the salivary gland polytene chromosomes of Ctp the globin gene cluster could be localized to chromosome II, position F2b2 (according to the map of Haegele, 1970; see Fig. 4A). This locus is different from the one determined for the globin gene cluster containing the ‘monomeric’ HbZZZ and HbZV genes. The HbZZZ and HbZV genes most likely originate from the end of chromosome III, position Alcl, which has been determined by in situ hybridization (Fig. 4B) using the isolated 0.9-kb Hind111 fragment of LGl containing Ctt HbZV gene (Antoine and Niessing, 1984). No cross-hybridization can be detected between the Ctp HbVZZB-5 containing gene cluster and region Alcl on chromosome III, containing the HbZZZ and HbZV genes (Fig. 4A). In contrast, there is some cross-hybridization of the isolated HbZV gene to region F2b2, containing the Ctp HbVZZB-5 gene (Fig. 4B). The reason why crosshybridization occurs only in one direction is because we used the entire cluster including intergenic regions in one case and the isolated HbZV gene in the other. Control hybridizations using the entire Ctt HbZZZ/ZV gene cluster (iG1) confirmed this interpretation; cross-hybridization to the HbVZZB locus was not further detectable. However, with ilG1 as probe several other weak hybridizations occurred, which can either be the result of the presence of a hidden repetitive element within the Ctt HbZZZ/ZV gene cluster or a cross-hybridization to other so far not identified gene loci. The possibility of more than two Hb loci is suggested by the results of in situ hybridizations using uncloned cDNAs which gave a num-

302

Fig. 4. Chromosome

localization

111situ hybridization

with C/p HhVIIB-5

chromosomes

to chromosome

ofthc

Crp HhVIfB-5 containing

and Crr Hhl V genes. Fluorescence gene cluster

I. II and III). The large white arrowheads II, region F2b2 (A) and of Ctr Hhf V gene

indicate

micrograph

(A) and with Cl/ HbIV the prominent

to chromosome

hybridization

of CQ polytenc

gene (B) (magnification of Cfp HbVIIB-5

chromosomes 450 x

gene containing

III region AlcI (B). The small arrowheads

after

; I, II, 111, ApiHhl

indicate the position

303

ber of different strong hybridization signals along chromosomes II and III (Laufer et al., 1982).

REFERENCES Antoine,

M. and Niessing, Chjro~~rn~

insect

(e) Conclusions

Antoine,

of the Chironomid

has been isolated

and sequenced. demonstrated

HbVIIB

gene

from the genome of Ctp

Although

its activity

gous

gene

is

probably

non-allelic

organization

W.D. and

Braun,

V., Chrichton,

und dimere Physiol. Feinberg,

could be expected

with biotinylated

DNA probes, from IGl

Frischauf,

G.: Uber monomere

(Chironomus thummi).

B.: A technique

endonuclease

A.-M.,

Lambda

2.

for radiolabeling

fragments

to high specific

132 (1983) 6-13.

Lehrach,

replacement

H., Poustka,

vectors

carrying

A. and

Murray,

polylinker

N.:

sequences,

J. Mol. Biol. 170 (1983) 827-842. Goodman,

M., Braunitzer,

H.: The analysis monomeric rim)

and

G., Kleinschmidt,

T. and Aschauer.

of a protein-polymorphism. homodimeric

Evolution

haemoglobins

of

(erythrocruo-

of Chironomus fhummi thummi (Insecta,

Diptera).

Z.

Chem. 364 (1983) 205-217.

K.:

DNS-Repiikationsmuster

der

van Chironomiden.

Speicheldrtisen(Berl.) 3 1

Chromosoma

P.R., Levine, M. and Ward,

ethanol-dehydrated (ENZO

(A) or occurred

DNA was detected (Langer-Safer

Laufer.

I-I.. VafopouloLi-Matldnios.

G. and Ramircz, hemoglobin Weher,

X., Kuliawat.

F.: Tissue-specific

synthesis

immunologically et al., 1982).

in 2

by rabbit

x

R.. Gundling,

and gcnc-specific

in C~~r~~i7~~~?7z~.s. In Burger.

R. (Eds.), Embryonic

Development,

sites of

MM.

IX Congress Biologists;

Research,

Vol. 85. Liss, New York, 1982, pp. 327-335.

D.A.. Trewitt.

Bergtrom,

of the International Progress

Society of Dcvel-

in Clinical

P.M.. Castro,

G.: Deoxynucleotide

and

M.. Wejksnora.

of an

sequence

and

part .4, Genctic

Aspects:

Saffarini.

Biological P.J. and

insect cDNA

codes for an unreported

member

of the C’iliro~ro~r~u.r rirwmri

globin tamily. Biochem.

Biophys.

Res. Commun.

133

(1985)

641-637.

(B). Squash

preparations

of salivary

several Ctp Hb genes including

The DNA probes

Inc.). ffybridization

chromo-

opmental

only the C!t HblV gene (Antoine

the slides were washed

D.C.: Immunological

genes on Drosophila polytene

and Niessing,

gland

chromosomes

were biotinylated

was carried

of Crp were

C’tr,HbVIIB-5 (see Fig. 1) or a 0.9-kb 1984). The chromosomes

A, 250 u/ml RNase Tl, in 2 x SSC for 30 min at room temperature)

and air-dried. Biochem.

for mapping

somes. Proc. Natl. Acad. Sci. USA 79 (1982) 4381-4385.

either ApiHbl containing

containing

with RNase (5 pg!ml RNase

antibody

dgt recombinant ‘in situ’. Science 196

R.R. and Braunitzer,

Anal. Biochem.

Langer-Safer,

per slide. After hybridization as second

globins

(1970) 91-138.

where cross-hybridization

hybridized

Screening

Insektenhlmoglobine

restriction

activity.

We are grateful to Drs. M. Antoine and J. Niessing who kindly provided us with the cloned HblV and HbHI genes from Ctt and who communicated their sequence data as a preprint to us. We wish to thank Prof. Dr. H.-G. Key1 for the excellent working facilities, his cytological advice and stimulating discussions. The expert technical asistance of B. Weich and R. Ross as well as the help of I-I. Sommerfeld in typing the manuscript is greatly acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft (Schm 523/3-5).

1983), using biotin-dCTP

R.W.:

to single plaques

A.P. and Vogelstein,

method

in 0.07 N NaOH.

secretory

Chem. 349 (1968) 197-210.

DNA

ACKNOWLEDGEMENTS

isolated

J.:

of five homolo-

(1977) 180-182.

to

Chromosomen

fragment

(1984)

S. and Niessing,

structure

genes encoding

Davis,

clones by hybridization

Hagele,

HitldIII

310

41-51.

Physiol.

were digested

E., Schnell,

and primary

pairs of intron-less

Benton,

Chironomus thummi HbVIIB-3 and HbVIIB-4 genes, which can be concluded from the comparison of the DNA flanking the genes. The Ctp HbVfIB-5 containing gene cluster is located at position F2b2 in chromosome II, a site different from the locus for Ctt HbZZZ/IV at chromosome III, position Alcl. Thus at least two different chromosome loci of Hb genes exist in Ctt and Ctp, which has important implications on the discussion of the evolution of the Chironomid Hb gene family.

hybridized

genes in the

from the insect Chirotzamus thummi ~hummi. Gene 56 (1987)

has not been

we assume that it is an active member

This

M., Erbil, C., Munch,

Genomic

of the HbVIIB gene subfamily, because it displays the typical regulatory sequences in its 5’- and 3’4lanking regions.

globin

795-198.

A new member subfamily

J.: Intron-less

fh~rn~~~ zhummj. Nature

on the slides

denatured

by ‘oligo labelling’ (Feinberg

for

out in 5 x SSC at 60°C for 6-8 h with G-10 ng of DNA

SSC (30 min. 5O’C) and then in PBS (5 min, room temperature).

anti-biotin

I min

and ~~ogelstciil,

IgG as primary

and fluorescein-conjugated

goat anti-rabbit

The IgG

304

Sladic-Simic, Sequenz

D., Kleinschmidt, eines dimeren

T. and

Hamoglobins

Braunitzer,

G.:

(Komponente

Chironomus thummi fhummi, Diptera).

Die

VII B,

Z. Physiol. Chem. 358

(1977) 591-594. Smith,

site

Trewitt,

P.M.,

sequence

H.O. and Birnstiel,

restriction

different ligand states refined at 1.4 A resolution.

M.L.: A simple method

mapping.

Nucl.

Acids

Res.

Saffarini,

D.A. and Bergtrom,

of the intronless

VIIB subfamily

W. and Weber,

E.: Structure

3

Nucl. Acids Res. 15 (1987) 5494.

of erythrocruorin

in

Communicated

G.: Nucleotide a member

of the

from Chironomus thummi (Diptera).

globin

2387-2398. Steigemann,

gene expressing

for DNA (1976)

J. Mol. Biol.

(1979) 309-338.

by S.T. Case.