Báo cáo khoa học: The hemoglobin genes of Drosophila ppt

Here we investigate the structure and evolution of the glob1 gene in other Drosophila species.. Moreover, we identified two additional putative globin genes of Drosophila, which we named

(Hemo-)globins (Hbs) are small globular proteins

usu-ally consisting of a core of about 150 amino acids,

which typically comprises eight a-helical segments

(named A–H) and displays a characteristic 3-over-3

a-helical sandwich structure [1,2] Hbs include an

iron-containing heme (Fe2+-protoporphyrin IX), by which

they reversibly bind gaseous ligands Most Hbs are

considered respiratory proteins because of their ability

to transport or to store O2, thus enhancing the

avail-ability of O2to the respiratory chain of the

mitochon-dria [3] Hbs are widespread among metazoan taxa

[4–6] and it is not surprising that Hbs are among the

best-investigated proteins in biological and biochemical

sciences [1,2] Recent studies have demonstrated that some Hbs may also detoxify NO (and possibly other noxious reactive nitrogen and oxygen species [RNS and ROS]) and or serve as O2sensing molecule [7–9] For a long time Hbs were essentially unknown in most insects Respiratory proteins had been regarded unnecessary in this taxon because of the well-devel-oped tracheal system, which enables an efficient diffu-sion of O2 from the atmosphere to the inner organs [10] Notable exceptions had been only some basal insects that possess hemocyanin as O2 transport pro-tein in the hemolymph, which they inherited from the crustacean ancestor, but has been lost in the evolution

phylogeny

Correspondence

T Burmester, Institute of Zoology,

University of Mainz, D-55099 Mainz,

Germany

Fax: +49 6131 39 24652

Tel: +49 6131 39 24477

E-mail: burmeste@uni-mainz.de

Note

The nucleotide sequences reported in this

paper have been submitted to the

GenBankTM ⁄ EMBL databases with the

accession numbers AM086021 (D virilis

glob1 gene), AM086022 (D virilis glob1

cDNA), AM086023 (D pseudoobscura glob1

cDNA) and AM086024 (D melanogaster

glob2 cDNA).

(Received 13 October 2005, revised 18

November 2005, accepted 23 November

2005)

doi:10.1111/j.1742-4658.2005.05073.x

(glob1) in the fruitfly Drosophila melanogaster Here we investigate the structure and evolution of the glob1 gene in other Drosophila species We cloned and sequenced glob1 genes and cDNA from D pseudoobscura and

D virilis, and identified the glob1 gene sequences of D simulans, D yak-uba, D erecta, D ananassae, D mojavensis and D grimshawi in the data-bases Gene structure (introns in helix positions D7.0 and G7.0), gene synteny and sequence of glob1 are highly conserved, with high ds⁄ dn ratios indicating strong purifying selection The data suggest an important role of the glob1 protein in Drosophila, which may be the control of oxygen flow from the tracheal system Furthermore, we identified two additional globin genes (glob2 and glob3) in the Drosophilidae Although the sequences are highly derived, the amino acids required for heme- and oxygen-binding are conserved In contrast to other known insect globin, the glob2 and glob3 genes harbour both globin-typical introns at positions B12.2 and G7.0 Both genes are conserved in various drosophilid species, but only expres-sion of glob2 could be demonstrated by western blotting and RT-PCR Phylogenetic analyses show that the clade leading to glob2 and glob3, which are sistergroups, diverged first in the evolution of dipteran globins glob1is closely related to the intracellular hemoglobin of the botfly Gastero-philus intestinalis, and the extracellular hemoglobins from the chironomid midges derive from this clade

Abbreviations

AIC, Akaike information criterion; EPO, erythropoietin; Hb, hemoglobin; HRE, hypoxia responsive sequence elements; PIP, percent identity plot.

of higher neopteran insects [11] Respiratory proteins

belonging to the Hb-type were considered being

con-fined to a few insect species that are adapted to hypoxic

environments [6] According to our present knowledge,

the chironomid midges (Diptera:Nematocera) are the

only insects that possess true extracellular Hbs in their

circulatory system for O2 transport [12,13] Some

aquatic Hemiptera and the larvae of the horse botfly

Gasterophilus intestinalis harbour intracellular Hbs,

which probably carry out myoglobin (Mb)-like O2

storage-functions [6,14,15] These cellular Hbs reach

concentrations in the millimolar range and thus may

easily be identified due to the red colour of the

Hb-containing organs

Recently, however, we showed that a true Hb gene

(glob1 or Hb1) is present and expressed in the fruitfly

Drosophila melanogaster [16,17] This finding was

unprecedented because at the first glance D

melano-gasteris unlikely to face hypoxic conditions during its

life cycle The crystal structure of D melanogaster

glob1 identified a protein that displays a typical globin

fold and shows conservation of the residues important

for O2 binding [18] Although exhibiting a

hexacoordi-nated binding scheme at the Fe2+ion in the

deoxygen-ated state, the O2-binding kinetics of glob1 are similar

to those of other insect intracellular Hbs (P50¼ 0.14

Torr) [17] In larval and adult Drosophila, glob1 is

mainly expressed in tracheal cells and the fat body

These data indicate that Hbs may be in fact involved

in O2 metabolism and suggest that Hb genes may be

much more widely distributed in insects than

previ-ously thought Here we investigate the occurrence and

evolution of glob1 in other Drosophila species (Fig 1)

Moreover, we identified two additional putative globin genes of Drosophila, which we named glob2 and glob3

Results

The Drosophila glob1 genes The D melanogaster glob1 nucleotide and amino-acid sequences were used to search the databases of

genom-ic DNA sequences available from various Drosophili-dae, as they are available at the EMBL⁄ GenBank (http://www.ncbi.nlm.nih.gov) or at FlyBase (http:// flybase.net/), by the blast algorithm [19] We identi-fied the full length glob1 coding sequences from the genomes of D yakuba, D erecta, D ananassae,

D pseudoobscura, D virilis, D mojavensis and D grim-shawi, as well as a partial sequence from D simulans that covers only the first coding exon (see Supplement-ary material) The D virilis glob1 sequence was also independently obtained by screening a genomic DNA library with the D melanogaster glob1 coding sequence

as probe The D virilis glob1 sequence (Acc No AM086021), which covers the complete glob1 gene, comprises 6265 bp It differs from the genomic sequence at FlyBase (contig 462) at 20 positions and displays four short indels (1–26 bp) In addition, the cDNA sequences, including their putative 5¢ ends, from D virilis and D pseudoobscura (Acc Nos AM086022 and AM086023) were obtained by RT-PCR and subsequent RACE experiments We identified a single splice variant for each D virilis and

D pseudoobscura glob1, which contains three coding exons and a single 5¢ noncoding exon (Fig 2) The glob1 mRNAs of D pseudoobscura and D virilis cor-respond to transcript ‘A’ of the D melanogaster glob1 gene, which consists of exons 1, 4, 5 and 6 [17] In addition, the in silico analysis of the gene predicts the presence of a second splice variant in D pseudoobscura, which corresponds to transcript ‘C’ of D melanogaster glob1 (Exons 1, 3, 4, 5 and 6) However, the corres-ponding cDNA was not recovered by RT-PCR The glob1 gene plus the upstream and downstream genomic regions were scanned for the presence of puta-tive hypoxia responsive sequence elements (HREs) (Fig 2) HREs in hypoxia-regulated genes are defined

by the binding motif of the hypoxia-inducible transcrip-tion factor HIF-1 (5¢-RCGTG-3¢) [20] Typically, an HRE includes two HIF-1 motifs (in direct or inverted orientation) or one HIF-1 motif combined with an ery-thropoietin (EPO) box or a HIF ancillary sequence [21] Six putative HREs are present in the D melano-gaster and D virilis glob1 genes, while the D pseudo-obscura glob1 contains five such motifs Interestingly,

Fig 1 Evolutionary history of Drosophilidae The phylogenetic

rela-tionships of drosophilid species were taken from Russo et al [52],

divergence times are indicated as calculated by Tamura et al [24].

the HREs are located in similar positions, although

motif combinations are not always conserved

The degree of sequence conservation ± 4 kb

upstream and downstream of the glob1 genes were

evaluated with PipMaker, using the D melanogaster

glob1 gene region as reference (Fig 3A) The putative

glob1 coding exons (see below) are conserved and

clearly discernable in all Drosophila species Outside

the melanogaster subgroup (D simulans, D yakuba,

D erecta), hardly any other region displays sequence

similarities The only exceptions are the noncoding

exon 1, which is transcribed in D melanogaster,

D pseudoobscura and D virilis, and a region within

the third intron, about 2 kb 5¢ of the translation start

codon, which may harbour regulatory sequences The

D melanogaster glob1 gene is located on the right arm

of chromosome 3 at band 89A8 between the genes

CG14877, which codes for a putative natriuretic

pep-tide receptor, and CG31292, which has no putative

ortholog outside the Drosophila species (see

Supple-mentary material) In the other species of the

melano-gaster subgroup (Fig 1) for which the genome

locations are available (D simulans and D yakuba),

the glob1 gene is positioned on the same chromosome

The D pseudoobscura glob1 gene is located on

chromo-some 2, which is orthologous to chromochromo-some 3R of

the melanogaster subgroup [22,23] Gene synteny

ana-lyses of 10 neighbouring genes (five on each side)

reveals the conservation of gene locations and

orienta-tions in this region between D melanogaster and

D pseudoobscura, which diverged about 55 million

years ago (Fig 1) Because of the absence of data from

other genomes, gene synteny analyses was restricted to

the two neighbouring gene in these species This shows

the presence of both CG14877 and CG31292 adjacent

to the glob1 in all Drosophila species (see

Supplement-ary material) Gene orientations were found to be

con-served in all Drosophila genomes except D ananassae,

in which the putative CG31292 ortholog was found in reversed orientation However, this may also reflect an assembly error

The Drosophila glob1 proteins and gene evolution

Gene predictions and comparisons with the available cDNA sequences show that Drosophila glob1 genes consistently harbour two introns, which are located at positions D7.0 (i.e between codons 6 and 7 of globin helix D) and G7.0 (Fig 4) The introns are short, vary-ing from 64 to 73 bp for the first intron and between

59 and 198 bp for the second intron Except of

D pseudoobscura glob1, the coding sequences of all Drosophila glob1 genes measures 462 bp (including the stop codon) and translate into polypeptides of 153 amino acids with estimated molecular masses of 17.1– 17.5 kDa The D pseudoobscura glob1 sequence has an insertion of three bp close to the 3¢ end, resulting in a protein of 154 amino acids Otherwise no indel was observed in the coding region The interspecies amino-acid differences sum up from two to 36 positions (75.2–98.7% identity and 91.5–100% similarity, consid-ering isofunctional replacements) The heme- and ligand-associated residues, i.e the proximal and distal histidines in helix positions E7 and F8, LeuB10, PheCD1, ArgE10, and IleE11 [18], are invariant in the glob1 proteins of all Drosophila species (Fig 4; Table 1) Only about a third of the amino-acid posi-tions in the glob1 proteins were found variable Most

of the observed variations are located in the loop regions, in particular in the CD, EF and FG corners, while the helices are well conserved

Rates of molecular evolution were estimated on the basis of a PAM substitution matrix assuming the Dro-sophiladivergence times suggested by Tamura et al [24]

We found that the glob1 proteins have evolved with an

Fig 2 Structure of Drosophila glob1 genes The genomic sequences of glob1 genes of D melanogaster, D pseudoobscura and D virilis are displayed Exonic sequences are boxed and numbered 1–6 on top, the coding regions are hatched Putative TATA boxes are indicated and the positions of potentially relevant hypoxia-responsive sequence elements in the gene region are shown as open squares.

average rate of 1.61 ± 0.56· 10)9amino-acid

replace-ments per site and per year (Table 2) The lowest rate

was calculated from the D melanogaster – D yakuba

comparison (0.50· 10)9), which is likely due to a

mutational slow-down in the D yakuba lineage, whereas

on the other hand a faster average rate of evolution

was observed in the Drosophila subgenus clade,

partic-ularly in the Hawaiian species D grimshawi On the

nucleotide level, 0.90 ± 0.31· 10)9 nonsynonymous

(dn) and 10.97 ± 5.61· 10)9synonymous (ds) replace-ments per site per year were inferred Levels of selective constraints were measured by ds⁄ dn ratios [25] Strong selective pressure on a coding region favours synonymous substitutions (ds) over nonsynonymous substitutions that cause amino-acid replacements (dn) The high average ds⁄ dn ratio of 14.7 indicates that significant purifying selection has been imposed on the glob1gene

Identification of two novel globin genes

of Drosophila

By searching the D melanogaster genome with the tblastn algorithm and by employing various inverteb-rate globin sequences as templates, we identified two novel putative globin genes, which we tentatively called glob2and glob3 (Figs 5 and 6) The glob2 gene is anno-tated as orphan D melanogaster gene CG15180, glob3 is known as CG14675 Reverse blastp searches were then performed with exclusion of the Drosophila sequences Assuming the BLOSUM45 model we obtained the high-est sequence similarity of D melanogaster glob2 with the Hb of the bivalve Barbatia virescens (Acc No BAA09587; e-value: 0.003), and of D melanogaster glob3 with the globin D of the sea cucumber Caudina arenicola (Acc No AAB19247; e-value: 5· 10)5) The relatedness of glob2 and glob3 to other globins was cor-roborated by a random shuffling approach, as it is implemented in the prss program [26] The analyses yield an e-value of 6.96· 10)10for the D melanogaster glob2–B virescens Hb comparison and an e-value 1.02· 10)9 for D melanogaster glob3 vs globin D

C arenicola These comparisons further confirm the identity of glob2 and glob3 as true globins

Analyses of Drosophila glob2 genes and proteins The predicted D melanogaster glob2 coding sequence covers 669 bp (including stop codon), which was con-firmed by sequencing a cDNA fragment that had been obtained by RT-PCR experiments on adult D melano-gaster total RNA (Acc No AM086024) In addition, six entries in the database of D melanogaster expressed sequence tags (ESTs [27]); (BE976463, BE978352, BE978841, BE976596, CB305306, CO331008) demon-strate that glob2 is actually expressed Moreover, an antiserum that had been raised against synthetic glob2 peptides detects a protein band of about 26 kDa in extracts from total flies (Fig 7), which corresponds to the expected size of glob2 (see below)

Comparison of cDNA and genomic sequences show that the glob2 gene contains two introns, which are

A

B

C

Fig 3 Conservation of Drosophila globin gene regions Percent

identity plot (PIP) showing the comparisons of the Drosophila

glo-bin 1 (Gb1; A), gloglo-bin 2 (Gb2; B) and gloglo-bin 3 (Gb3; C) genes and

their ± 4 kb flanking regions The D melanogaster genes were

used as reference sequences, in the upper row the exons are

boxed, with the black boxes representing the coding sequences.

Gene extensions and transcriptional orientations are indicated by

arrows Interspecies sequence identities are shown as horizontal

bars on a 50–100% scale GC-rich regions are indicted as shown

on the right hand side The boxes in dark grey indicate missing

data Species abbreviations are: Dsi, D simulans; Dya, D yakuba;

Der, D erecta; Dan, D ananassae; Dps, D pseudoobscura; Dvi,

D virilis; Dmo, D mojavensis; Dgr, D grimshawi.

positioned in B12.2 and G7.0 (Fig 5) The introns are

short, measuring 52 and 127 bp, respectively The

pre-dicted D melanogaster glob2 protein covers 222

amino-acid positions (25.3 kDa), which is longer than glob1 and most other globins (typically
140–150 amino acids) This is mainly due to the 50 and 30 amino-acid extensions at the N- and C-termini, respectively Nevertheless, except of an indel of six amino acids in the DF corner (positions D6 to E4), the globin core is well conserved This also comprises the residues crucial for heme- and O2- binding, e.g the PheCD1 and the proximal and distal histidines E7 and F8 (Table 1) The calculated pI of glob2 is 9.18, indi-cating a highly basic protein This coincides with the analyses according to the Reinhardt’s method [28], which predicts glob2 to be nuclear protein (reliability 76.7%)

Putative glob2 orthologs were identified from the genomic sequences of D simulans, D yakuba,

D erecta, D ananassae and D pseudoobscura (see Supplementary material; Fig 5) They all display a similar length, varying from 218 to 223 amino acids, and all have short introns in positions B12.2 and G7.0 Except of an insertion of five amino acids in the CD loop in D ananassae glob2 the different lengths of these proteins result from variations of the N- and C-terminal extensions All glob2 proteins were predicted to reside in the nucleus The rate cal-culations, as estimated as described above (Table 2), show that the Drosophila glob2 proteins evolve with 7.62 ± 3.89· 10)9 replacements per site and per year This is about six times faster than in the glob1 proteins The relaxed selective constraint is also indi-cated by a ds⁄ dn ratio of 6.5 The PipMaker analy-sis showed discernable sequence similarities within

Fig 4 Comparison of Drosophila glob1 amino-acid sequences The secondary structure elements of Drosophila glob1 (PDB entry 2BK9 [18])

is shown in the upper row The alpha-helices are designated A through H and the globin consensus helix numbering is given in the lower row Conserved amino acids are shaded in grey The abbreviations are: DmeGb1, D melanogaster globin 1; DyaGb1, D yakuba globin 1; DerGb1, D erecta globin 1; DanGb1, D ananassae globin 1; DpsGb1, D pseudoobscura globin 1; DviGb1, D virilis globin 1; DmoGb1,

D mojavensis globin 1, and DgrGb1, D grimshawi globin 1.

Table 1 Functionally important residues in selected globins Amino

acids at key positions in human Hb, myoglobin, Chironomus Hb,

G intestinalis Hb, and Drosophila glob1–3 are given.

Chironomus Hb Trp ⁄ Phe ⁄

Tyr

Leu Phe His Arg Ile ⁄ Val His

G intestinalis Hb Trp Leu Phe His Arg Ile His

Drosophila glob3 Trp Phe ⁄ Tyr Phe His Arg ⁄ Ala ⁄

Thr

Table 2 Substitution rates in Drosophila globins Amino-acid

evolu-tion was modeled according to the PAM matrix [49] Corrected

nonsynonymous (dn) and synonymous (ds) nucleotide substitutions

per site were calculated by the method of Ota and Nei [51]

Evolu-tion rates are given as estimated replacements per site and per

year.

Substitution rates (x 10)9)

ds ⁄ dn

Globin 1 1.61 ± 0.56 0.90 ± 0.31 10.97 ± 5.61 14.7

Globin 2 7.62 ± 3.89 4.21 ± 2.02 14.59 ± 6.88 6.5

Globin 3 7.28 ± 1.53 3.86 ± 0.78 17.64 ± 7.27 4.6

the melanogaster subgroup (Fig 3B), while in

D ananassae and D pseudoobscura only the coding

exons are conserved

The gene of D melanogaster glob2 was found localized on the right arm of chromosome 3 at band 83F4 The flanking genes are CG31482 on the proxi-mal side, which encodes for chain A of the Golgi-associated Pr-1 protein, and CG15184 on the distal side, for which no putative ortholog is available and which is poorly conserved even within the Drosophili-dae We therefore used the next neighbouring gene CG15178 for synteny analyses (see below), which encodes a highly conserved EF hand calcium binding protein In all species of the melanogaster subgroup, glob2 is located on chromosome 3R, and on the orthologous chromosome 2 of D pseudoobscura Gene synteny analyses of the neighbouring genes showed conservation of gene order within the melanogaster subgroup However, there is no syntenic conservation of the glob2 regions between D melano-gaster and D pseudoobscura or D ananassae The

D melanogaster orthologs of the genes adjacent to

D pseudoobscura glob2 are located on chromosome 3R in regions 83C, which is the glob3 locus (see below), and 100D

Fig 5 Comparison of Drosophila glob2 amino-acid sequences The secondary structure of Drosophila glob1 is shown in the upper row, alpha-helices designations and shading was performed as described in Fig 4 Abbreviations: DmeGb2, D melanogaster globin 2; DsiGb2,

D simulans globin 2; DerGb2, D erecta globin 2; DyaGb2, D yakuba globin 2; DpsGb2, D pseudoobscura globin 2; DmoGb2, D mojavensis globin 2.

Fig 6 Comparison of Drosophila glob3 amino-acid sequences The secondary structure in the upper row derives from Drosophila glob1, other decorations were performed as described in the legends to Figs 4 and 5 Abbreviations: DmeGb3, D melanogaster globin 3; DsiGb3,

D simulans globin 3; DerGb3, D erecta globin 3; DyaGb3, D yakuba globin 3; DanGb3, D ananassae globin 3; DpsGb3, D pseudoobscura globin 3; DviGb3, D virilis globin 3.

Fig 7 Western blot analyses About 50 lg of total adult protein

were applied per lane and the glob1 (lane 1) and glob2 (lane 2)

pro-teins were detected using specific antibodies.

Analyses of Drosophila glob3 genes and proteins

The putative coding sequence of the D melanogaster

glob3 gene measures 588 bp, which translates into a

protein of 195 amino acids However, we could not

obtain any glob3 cDNA from larval or adult RNA by

RT-PCR Moreover, no entry in the Drosophila EST

database was recovered that corresponds to glob3

Because at that stage we could not exclude that glob3 were a pseudogene, we searched the genomic sequences

of the other Drosophila species In fact, we obtained apparently conserved orthologs genes of glob3 from

D simulans, D yakuba, D erecta, D pseudoobscura and D virilis (see Supplementary material; Fig 6) Gene prediction and comparison with glob2 suggest the presence of the two introns in B12.2 and G7.0 also

Fig 8 Phylogeny of Drosophila and other dipteran globins An alignment of the Drosophila globins, G intestinalis Hb and selected Hbs from chironomid midges was analysed by MrBayes 3.1, assuming a WAG model of evolution with gamma-distribution of rates The Daphnia Hb domains represent the outgroup Bayesian posterior probabilities are given at the nodes The bar corresponds to 0.1 PAM distance The abbreviations are: DmaHb1.1 and DmaHb1.2, first and second domain of the hemoglobin (U67067) of D magna; DpuHb1.1 and DpuHb1.2, first and second domain of the hemoglobin (Q9U8H0) of D pulex; Chironomus thummi thummi hemoglobins CttHbI (P02221), CttHbIIb (AF001292), CttHbIII (M17691), CttHbIV (P02230), CttHbVIIB6 (A30477), CttHbVIII (P02227), CttHbIX (P02223), CttHbX (P02228), CttHbE (P11582), CttHbZ (P29245); C thummi piger hemoglobins: CtpHbV (X56271), CtpHbW (X56271), CtpHbY (X56271); GinHb, Gasterophilus intestinalis hemoglobin (AF063938); for abbreviations of the Drosophila globins refer to Figs 4, 5 and 6.

in the glob3 genes As for glob2, the glob3 amino-acid

sequences include extensions at the N- and C-terminal

ends, and are longer than the typical globin proteins

Another similarity of glob2 and glob3 is the six-amino

acids indel in the DF corner The residues important

for heme-contact and O2-binding globins are conserved

(Fig 6; Table 1) Reinhardt’s method [28] predicts the

glob3 proteins to reside in the cytoplasm The

evolu-tion rate of the glob3 proteins was calculated to be

7.28 ± 1.53· 10)9 replacements per site and per year,

which is in the same range as that of glob2 (Table 2)

The glob3 genes also show relaxed selective constraints,

as indicated by the ds⁄ dn ratio of 4.6 pipmaker

com-parisons clearly identify the glob3 coding exons

(Fig 3C), while otherwise there is little conservation

except of closely related species The 5¢ noncoding

region was found GC-rich The D virilis sequence was

too diverged to allow PipMaker analyses

Database searches show that the glob3 gene of

D melanogaster is located on chromosome 3R at band

83C4 On the proximal side of glob3, the gene

CG10981encodes a putative RING zinc finger protein,

on the distal side resides the gene CG1208, which

codes for a sugar-transporter (see Supplementary

material) Gene orders and orientations in this region

are identical in the species of the melanogaster group

(D melanogaster, D simulans, D yakuba, D erecta,

D ananassae) Gene synteny is only partially conserved

outside this taxon: in D pseudoobscura, D mojavensis

and D virilis, the genes located proximal to D

melano-gaster glob3are not on the same contig as glob3 Gene

synteny is only conserved on the distal side While in

D melanogaster glob2 and glob3 are separated on

chromosome 3R by 800 kb, they are neighbouring

genes in D pseudoobscura on chromosome 2 in a head

to tail orientation separated by 388 bp

Drosophila hemoglobin phylogeny

Because preliminary phylogenetic studies had

demon-strated the monophyly of arthropod globins (data

not shown), we constructed an alignment that covers

a total of 33 insect globin amino-acid sequences and

includes all Drosophila globins The globin domains

of the extracellular Hbs of the branchiopod

crusta-ceans Daphnia magna and D pulex were used as

outgroup As expected, each of the Drosophila

glob1–3 clades is monophyletic (Fig 8) The closest

relative of Drosophila glob1 is the intracellular Hb of

the horse botfly G intestinalis [15] This clade is

associ-ated with the extracellular Hbs of the Chironomidae

Although such grouping is supported by only 0.84

Bayesian posterior probability, it was consistently

recovered in all types of analyses Glob2 and glob3 are highly supported sistergroups Within the Dro-sophila clades, the globins generally follow the accep-ted phylogeny of the species, with the exception of

D pseudoobscura glob1, which was found to be asso-ciated with D erecta glob1 However, the support value was low and additional Bayesian analyses with other evolution models or Neighbor joining trees did not resolve this clade (data not shown)

Discussion

Conservation of Drosophila glob1 and functional implications

Until recently, it has been assumed that Drosophila has

no Hb genes and even after the determination of the full D melanogaster genome [29] their presence in this species was explicitly denied [30] Nevertheless, we identified a true Hb gene in D melanogaster [16], which is closely related to the intracellular Hb of the horse botfly G intestinalis and to the extracellular Hbs

of the Chironomidae (Fig 8) While there is no doubt that the G intestinalis and Chironomus Hbs carry out respiratory functions in transporting and storing O2 [12,14], the actual physiological role of the Drosophila Hbs remains to be determined Similarities in the O2 affinities and tissue distributions of G intestinalis Hb and D melanogaster glob1 may be considered as sup-port for a respiratory function of glob1 in Drosophila, possibly facilitating the flow of O2 from the trachea and tracheoles into the tissues [17] This view may sup-ported by the high expression rate of glob1 mRNA, as reflected by
130 D melanogaster ESTs (which corres-ponds to
0.03% of all D melanogaster ESTs) More-over, a rough estimate of glob1 protein content by comparing the western blot signal intensities (Fig 7) with known concentrations of recombinant glob1 sug-gests that glob1 corresponds to approximately 0.1% of total adult proteins Taking into account that the expression of glob1 is largely restricted to the larval and adult tracheal system, as well as some regions of the fat body, this appears to be a significant amount, which is compatible with the idea that glob1 enhances local oxygen diffusion rates or acts as oxygen store Alternatively, glob1 may function as a buffer system that protects the inner organs from an excess of O2 [31,32] Moreover, other, nonrespiratory functions of glob1, such as the detoxification of reactive oxygen species or NO, are also conceivable

An important role of glob1 in the flies’ metabolism

is supported by the strong conservation of the gene and protein Its evolution rate of 1.61 ± 0.56· 10)9

mean ds⁄ dn ratio (14.7), these data indicate that glob1

is a slowly evolving gene that is under significant

selective pressure

Recent studies employing the D melanogaster SL2

cell line have demonstrated that glob1 expression levels

decreased under low oxygen conditions [33] As in

other animals, hypoxia-response in Drosophila is

medi-ated by binding of HIF-1, which consists of an

a- (Similar in Drosophila) and a b-subunit (tango in

Drosophila), to HREs [34] Unexpectedly, HIF-1 was

demonstrated to cause a hypoxia-dependent

down-regulation of glob1 in the SL2 cells [33] Here we have

shown that the glob1 genes of D melanogaster, D

viri-lis and D pseudoobscura harbour several putative

HREs Although without experimental evidence we

cannot conclude which HRE is functional, the

posi-tional conservation of the HREs in distantly related

Drosophila species suggest that these elements may

actually mediate a hypoxia-response of glob1

Two novel globin genes in Drosophila with

uncertain function

While the function of glob1 may be related to oxygen

supply, the physiological roles of glob2 and glob3 are

presently unknown Moreover, we failed to amplify

any glob3 mRNA from D melanogaster Although we

cannot exclude technical problems, we must consider

the possibility that this gene is not expressed under

normal physiological conditions Latter assumption is

also supported by the fact that no glob3 cDNA could

be found in the > 300.000 EST sequences of D

mel-anogaster Nevertheless, the conservation of glob3 in

Drosophila evolution (Fig 6) suggests that this gene is

not a pseudogene, but is functional It is conceivable

that glob3 is only expressed under particular

physio-logical conditions or only during a short period of

development

glob2 is expressed in Drosophila, as confirmed by

RT-PCR experiments, EST database entries and

western blotting The expression level is much lower

than that of glob1, as reflected by only six EST

sequences in the database and own estimations based

ESTs are also available (CV790354, CV790570, CV784980), which were obtained from testis cDNA, too This might suggest a testis-specific function of glob2 as transcriptional regulator, which, however, requires further investigations

Insect globin phylogeny Globins are widespread in all kingdoms of life and display a complex evolutionary pattern [4,6,36] The last common ancestor of all insects certainly har-boured a globin gene, although is must remain uncertain whether it had respiratory functions Our analyses indicate that the insect Hbs are monophyletic, and that a clade comprising of glob2 and glob3 is the earliest offshoot of the insect globins Therefore, the separation of the glob2⁄ glob3 clade and other insect globins likely occurred early in the evolution

of this taxon On the other hand we cannot exclude the possibility that the high evolution rate of glob2 and glob3 has masked its actual relatedness and have led to a long-branch attraction effect The rela-tionship of glob2 and glob3 is not only confirmed by the phylogenetic analysis, but also by the fact that they are neighbouring genes in D pseudoobscura The most parsimonious explanation is therefore that glob2 and glob3 arose by gene duplication in the Drosophila stemline During the evolution of the melanogaster group the glob2 gene was moved to a different region of the 3R chromosome This is com-patible with the notion that within the Drosophila genus there is a strong tendency for genes to remain

on the same chromosome arm [22,23]

The Drosophila glob1 genes share a recent common ancestry with the G intestinalis Hb, and it may be assumed that the genes are orthologs that diverged upon the separation of the species (about 80–100 mil-lion years ago [37,38]) As mentioned above, this gene orthology might be considered as support for a role in respiration also of Drosophila glob1 The respiratory function of the Chironomus Hbs has convincingly been proven (for a review, see [5]) The phylogenetic tree show that the Chironomus Hb genes most likely derive

from intracellular Hbs, as represented today by

G intestinalis Hb and Drosophila glob1 Therefore,

with the evolution of the chironomid midges there was

a shift in function from an intracellular O2-binding

protein with possible O2 supply function to an

extra-cellular O2-transport protein This event must have

occurred after the separation of the Chironomidae

(Nematocera) and the brachyceran flies An ancient

origin of the Chironomus Hbs, as originally proposed,

e.g by Goodman et al [39,40], is not supported

Introns and evolution

It is well established that intron positions may be

phy-logenetically informative, particularly in case of globin

genes In fact, the relatedness and the basal position of

glob2 and glob3within the insect globins are supported

by the presence of introns B12.2 and G7.0 In fact,

these two intron positions are conserved in all

verte-brate and most inverteverte-brate globin genes, and almost

certainly reflect the ancient gene structure of the

eukaryote globins [4] Therefore, the loss and gain of

introns occurred in the clade of glob1 and chironomid

globin genes only after they diverged from a common

ancestor The G7.0 intron has been retained in the

glob1 and G intestinalis Hb, while the B12.2 intron

was lost in this clade Instead, a novel intron had

emerged in the common ancestor of glob1 and

G intestinalis Hb at position D7.0 None of the known

chironomid globin genes have introns in the ‘classical’

positions B12.2 or G7.0 Taking into account the

phylogenetic tree (Fig 8), the most parsimonious

explanation for this pattern is independent intron

losses While the B12.2 intron was lost in the clade

leading to the last common ancestor of Drosophila

glob1, G intestinalis Hb and the chironomid Hbs, the

G7.0 intron was deleted only before the radiation of

the chironomid globins While most present-day

chironomid globin genes do not have any introns,

some have acquired during evolution central introns at

positions E9.1 and E15.0 [41] The complex intron

pattern in dipteran globin genes can only be

inter-preted in terms of a dynamic evolution of gene

struc-ture by intron loss and insertion [42,43]

Experimental procedures

Animals

D melanogaster Oregon R, D pseudoobscura and D virilis

were maintained at 18
or 25
C on standard

yeast-corn-meal-agar-sucrose medium sprinkled with active dry yeast

Propionic acid and 4-hydroxymethylbenzoate were added

as mould inhibitors D virilis were a kind gift of C Kra-emer (Institute of Molecular Genetics, Mainz)

Database searches and sequence analyses The blast algorithm [19] was employed to search the databases of genomic DNA sequences available at GenBank (http://www.ncbi.nlm.nih.gov) and FlyBase (http://flybase net) The significance of the observed amino-acid sequence similarities were evaluated using a Monte-Carlo shuffling approach applying the prss3 program (fasta package [26]); Probability scores were estimated using the blosum50 matrix assuming a gap creation penalty of )12 and a gap length penalty of )2 with 1000 shuffles The relevant nucleotide sequences of the genes were extracted from the databases and assembled by the aid of genedoc 2.6 [44] DNA translation and analyses of primary structures were performed with the programs of the ExPASy Molecular Biology Server (http:// www.expasy.org) Interspecific comparison of gene sequences was performed with multipipmaker (http://bio.cse.psu.edu/ pipmaker/), which computes and visualizes local alignments

in two or more sequences based on the blastz algorithm [45,46] The D melanogaster genes were used as templates The multiple sequence alignments from pipmaker were visualized as ‘percent identity plots’ (PIP) Promoter predic-tions were carried out using the server at BDGP (http:// www.fruitfly.org/seq_tools/promoter.html) [47]

Molecular biology RNA from Drosophila species were extracted either with the GITC method [48] or by the ENZA total RNA extrac-tion kit (Peqlab) Various specific or degenerate oligonucleo-tide primers were used to amplify cDNA fragments by reverse transcription-PCR experiments, employing the Qiagen OneStep kit system or the Superscript II RNase H–

(Invitrogen) according to the manufacturer’s instructions The primer sequences are available from the authors upon request The sequences of the PCR products were either obtained by direct sequencing or after cloning of the frag-ments into the pCR4-TOPO vector (Invitrogen) Sequences were obtained from both strands using a commercial sequencing service (GENterprise) Missing 5¢- and 3¢ ends

of the cDNAs were obtained using the RACE system by Invitrogen

A kFIX II-library of D virilis genomic DNA was kindly provided by H Kress Screening was performed with a

D melanogaster glob1 probe that had been labelled with

32

P-ATP employing the random labelling kit by Roche Positive phage clones were grown on E coli p2392 in standard l-medium until lysis [48] Phage were then precipi-tated over night with 7% polyethylene glycol, 6% NaCl and suspended in 10 mm MgCl2 Contaminations by bacterial nucleic acids were removed by 30 min digestion with RNase A and DNase I Phage DNA was obtained by