The Cytochrome b and ATPase Genes of Honeybee Mitochondrial DNA ’ R. H. CrozieP
and Y. C. Croziert
*Department of Genetics and Human Variation, La Trobe University; and $School of Biological Science, University of New South Wales
The gene sequences for honeybee cytochrome b, ATPase 6, and ATPase 8 are presented, along with the inferred amino acid sequences of the proteins. These mitochondrial genes are in the same relative positions as are their counterparts in Drosophilamitochondrial mtDNA and have evolved at a significantly greater overall rate than have those of Drosophila. Comparisons using both amino acid identity and the proportion of conservative replacements between the inferred Apis and vertebrate cytochrome b sequences shows the two highly conserved sections reported by Howell, but his recognition of five conserved regions is not well supported. A very high AT bias is reflected in very high codon biases. The best predictors of the number of occurrences of an amino acid in honeybee cytochrome b are the T and G contents of its codon family-unlike the case for vertebrate cytochrome b, in which the codon family size and AT bias are the strongest predictors; protein function, at least as judged by hydrophilicity characteristics, appears to be unaffected by these differing influences on amino acid composition. Introduction
The sequences and order for the mitochondrial genes for cytochrome c oxidase subunits I and II (CO-I and CO-II, respectively), for four tRNAs, (Crozier et al. 1989), and for most of the large subunit of rRNA (Vlasak et al. 1987) have been published previously for the honeybee Apis mellifera. The DNA sequences for the two protein-encoding genes are extremely AT rich, with the CO-II gene lacking guanine altogether at the third codon position (Crozier 1990)) a bias stronger than that seen in Drosophila (Clary and Wolstenholme 1985; also see Crozier 1990). Apart from some shifts of tRNA genes, the Apis protein-encoding and rRNA genes identified elsewhere (Vlasak et al. 1987; Crozier et al. 1989) are in the same relative positions as are their Drosophila counterparts (Clary and Wolstenholme 1985 ). Phylogenetic analysis shows that, during their separation of -280 Myr (Carpenter and Burnham 1985), the CO-I and CO-II genes of the honeybee have evolved significantly more than have the corresponding Drosophila genes (Crozier et al. 1989). Despite this long period of separate development, the similarities between Drosophila and Apis mitochondrial DNA (mtDNA) at the third codon position for the CO-II (Crozier 1990) and CO-I genes are very close, whereas those at the first and second codon positions differ markedly (Crozier, accepted). Drosophila and Apis are more similar than expected by chance, for all three codon positions for both of these genes. We here report and analyze the honeybee gene sequences for cytochrome b and 1. Key words: cytochrome b, ATPase genes, honeybee. Address for correspondence and reprints: R. H. Crozier, Department of Genetics and Human Variation, La Trobe University, Bundoora, Victoria 3083, Australia. Mol. Bid. Ed. 9(3):474-482. 1992. 0 1992 by The University of Chicago. All rights reserved. 0737-4038/92/0903-0008$02.00
474
Cytochrome b/ATPase Genes of Honeybee Mitochondrial DNA
475
the ATPase subunits 6 and 8. The cytochrome b gene is of special interest both because of its increasing use as a tracer of phylogeny (Kocher et al. 1989; Irwin et al. 199 1) and because of evolutionary deductions about its structure and function (Howell 1989; Kocher et al. 1989; Irwin et al. 199 1) . Various authors (e.g., see Almagor 1985) have suggested that there could be an interaction between DNA-level and protein-level forces; for example, Dover ( 1989) suggests that DNA-level processes such as slippage replication have altered the per locus of Drosophila and have affected courtship behavior. The different associations between DNA parameters and cytochrome b amino acid content in Apis and vertebrates provide an opportunity to see whether, at least at a gross level, there is any sign of such an interaction for this mitochondrial gene. Methods
The source of the material and the methods of mtDNA preparation, cloning, and sequencing have been presented elsewhere (Crozier et al. 1989). Sequence analyses were carried out using the Nucleic Acids Analysis System package (Genesearch, Broadbeach, Queensland) run on an Apple II microcomputer, the MacVector (IBI) and Statview packages on a Macintosh, and Macintosh programs written by R.H.C. Inferred amino acid replacements in a four-species Wagner network were restricted to those whose change of state could be unambiguously assigned to a specific branch under parsimony. For example, given the grouping of taxa (Apis, Drosophila) (MS, Xenopus), the observation ABBB (Apis having one amino acid and the three other species having another) implies a single replacement with a single most-parsimonious placement in the terminal branch leading to Apis. The observation ABBC implies two changes most parsimoniously placed in the terminal branches leading to Apis and Xenopus. The observation ABCC (Apis and Drosophila each having unique amino acids, with the vertebrates possessing a third) implies two changes which can be placed in three equally parsimonious ways. The observation AABB implies a single change in the internal branch between the insect and vertebrate pairs. Hence, ABBB and ABBC each contribute to inferred changes in terminal branches, and AABB contributes to inferred changes in the internal branch, but ABCC leads to the inference of two replacements of unknown position. Because the mitochondrial genetic code differs for these organisms, it is not. practical to infer the number of replacements by using code information. We used multiple stepwise regression (Sokal and Rohlf 198 1, pp. 66 l-67 1) to determine which of various variables predict significantly the number of times an amino acid occurs in cytochrome b molecules. In brief, although many independent variables may each give a good prediction of the values of a dependent variable, many of these may be correlated with each other. In such cases, one might infer that some of these variables are in fact dependent on others for their effects on the dependent variable, and the task is then to determine which of the examined variables underlie(s) the patterns observed. Under multiple stepwise regression, the correlations between variables are calculated, and those with the largest independent effects are selected. As variables are added, their contribution to the prediction is then tested following a partial correlation, and any shown to be making an insignificant contribution to the predictive power of the multiple-regression equation are removed. The Kyte and Doolittle ( 1982) method of determining hydrophilicity curves was used to provide a broad picture of protein functional composition. In brief, the hydrophilicity values of the amino acids in a moving window are averaged to provide a
476 Crozier and Crozier
mean value for each amino acid position. These values provide an indication of the tendency that that portion of the protein is on the interior (negative values) or exterior (positive values) of the molecule. The comparison of hydrophilicity curves therefore gives one indication of the functional similarity of the molecules; because all amino acid positions are considered, the curves are liable to reflect changes at many weakly selected positions in addition to the relatively few strongly selected positions. Results and Discussion Genome Organization The ATPase 6 and 8 genes, and that for cytochrome b, are in the same relative positions in both Apis mtDNA and Drosophila mtDNA (Clary and Wolstenholme 1985 ) . The sequence of the region inferred to contain the genes responsible for encoding mitochondrial ATPase subunits 6 and 8 is shown in figure 1, and the sequence for the inferred cy-tochrome b gene is shown in figure 2. The amino acid sequences inferred from the Drosophila genetic code are also shown. The inferred amino acid sequences for the two ATPase subunits and for cytochrome b are shown in figures 3 and 4, along with those of representative other animals from the literature. We infer the Apis ATPase 6 gene to be slightly larger than that for Drosophila. The amino acid sequence begins with the ATG methionine codon at position 14 1 in figure 2-rather than with the ATA codon at position 156-because ATG is a stronger promoter than is ATA (Jacobs et al. 1988 ) . Similarly, Jacobs et al.
ATPase 8 I P Q MMPMKWFL IYFIYLLIFYLFIM ATTCCTCAAATAATACCTATAAAATGATTTTTAATTTTT~TTTATTTTATTTATTTATT~TTTTTTATTTATTCATTATA ATPase M K L INSMLIKTKINKETLKIKLKKWNW TTAATTAATTCAATACTAATCAAAACTAAAATCAATAAAATT
AAAAZUATGAAATTGA
I LMMNLFEMFDP STSNNLSMNWLFM F w x TTTTGATAATAAATTTATTTGAAATATTTGATCCATCAAC ML P I I I F P S IFWLIQSRIMF TATTACCAATTATTATTTTTCCAAGAATTTTTTTGATT~TTC~TCACG~TTATATTTATTAT
75
6 L 150
225 I
M
K T L AAAAACACTAA
MNFMYNEFKVVSKSKYQSNII I F TAAATTTTATATATAATGAATTTAAAGTTGTTGTTTC~TCT~TATC~TCT~TATTATTATTTTTATTAGAT
I
300
S 375
LMLYIMITNIFSLIPYVFTLTSHLL TAATACTTTATATTATMTTACTAACATTTTTAGATTTTTAGATT~TTCCTTATGTTTTTACATT~C~GACATCTACTTT
450
L N M I LSLTLWFSFLIYLIYNNYIMF TAAATATAATTTTATCATTAATTATGATTTAGATTTCT~TTTATTT~TTTAT~T~TTATATTATATTCC
525
L s H L V P L N S PVFLMNFMVI TTAGTCATTTAGTTCCATTRTTCACCAGTATTTTTAAT
600
IELISL
I R L S AN L I SGHLILTLL I IRPWTLS TTATTCGACCTTGAACATTATCAATTCGATTCGATTATCAGC~TTT~TTTCTGGACATTT~TTTT~CATTATTAG
675
ILPINLMIQNMLLTLE GIFISNFIS GAATTTTTATTAGAAACTTTATTTCAATTTTACCAATTAA
750
IFMSMIQSYVFS TTTTTATATCAATAATTCATTATGTATTTTCAATTCT
ILLILYFSESNX
FIG. 1.--Sequence of genes for honeybee ATPase subunits 6 and 8, together with amino acid sequences inferred from Drosophila mitochondrial genetic code.
Cytochrome b/ATPase Genes of Honeybee Mitochondrial DNA SNEFLKMIMST MKKFMNFFS I ATGAAAAAATTTATAAACTTTTTTAGTTCCAATGAATTTCT~T~TTATATCTAC~TTTATTTACC~CT
Y
L
P
477 T 75
LGIFLMIQIISG PVNINYMWNFGSI CCAGTAAATATTAATTATATATGAAATTTTT~ATTCTCATTTCTGGA
150
F ILSMHYCPNID I AF W S ITNIMKDM TTTATTTTATCAATACATTATTGTCCAAATATTGATATTGATATT~TTTCTGATC~TTACT~TATTAT~GATATA
225
NSGWLFRLIHMNGASFYFLMMYIHI AATTCAGGATGATTATTTCGTTTAATTCACACATGGAGATATTCATATT
300
SRNLFYCSYKLNNVWGIGIMI AGTCGAAATTTATTTTATTGTTCATATAAATTAAATAATGTATCA
L
L
M
S 375
MAAAFMGYVLPWGQMS YWGATVITN ATAGCAGCTGCATTTATAGGATATGTACTACCATGAGGAC~TATCATATTGAGGTGC~CAGTTATTACT~T LLSAIPYIGDTIVLWIWGGFS CTTTTATCAGCAATTCCTTATATTGGTGATACAATTGTATTGCT
I
450 N
N
A 525
T L N RF F SLHFILPLLI L F M V I L H ACATTAAATCGATTTTTTTCTTTACATTTTATTTTACCATTATT~TTTTATTTATAGTTATTCTTCATTTATTT SNPLGSNFNNYKISFHPY ALHLTGS GCCTTACATTTAACTGGATCATCTAATCCTCCTCAATTTCATTTCATCCATAT
L
F 600
675
F S IKDLLGFYIILFIFMFINFQFPY TTTTCAATTAAAGATCTTTTAGGATTTTATATCATCTTATTTATCTTTATATTCATT~TTTTC~TTTCCATAT
750
HLGDPDNFKIANPMNTP CATTTAGGAGATCCAGACAATTTCAAAATTGCAAATCCAATGATAT
825
THIKPEWY
FLFAYSI L RA IPNKLGGVIGLVMSI TTCCTATTTGCATATTCAATTTTACGAGCAATTCCTAATATT
9ao
L I L Y I M I FYNNKMMNNKFNMLNKIY CTTATTCTTTATATTATAATTTTTTATAATAATAAAATAATTTAT
975
Y WM F I N N F ILLTWLGKQLIEYPFTN TATTGAATATTTATTAATATTCATTTTATTAACATGATGATTA~T~C~TT~TTG~TATCCATTTACT~T
LO50
INMLFTTTYFLYFFLNFYLSKLWDN ATTAATATATTATTTACAACAACATATATTTTTTTTT
L125
LIWNSP L N X TTAATTTGAAATTCACCATTAAATTAA
FIG. 2.-Sequence of gene for honeybee cytochrome b, together with amino acid sequences inferred from Drosophila mitochondrial genetic code.
( 1988) inferred that the cytochrome b gene in the echinoderm Strongylocentrotus is longer than that seen in various other organisms.
Evolutionary
Rates
Appropriate comparisons using the inferred amino acid sequences from figures 3 and 4 can be used to test the hypothesis that the evolutionary rates of honeybee and fly mtDNA are the same (table 1). x: Tests reject the possibility that the number of amino acid replacements that occurred in the line leading to Apis from the common ancestor with Drosophila has been the same as that leading to Drosophila, both for the total number of changes reported in the present paper and for those found in cytochrome b alone (94 for Apis vs. 32 for Drosophila). The replacement-rate difference is also reflected in the number of times each insect has one amino acid and all the other taxa another one (for cytochrome b, 7 1 times for Apis vs. 17 times for Drosophila). The three proteins differ markedly in the proportions of unvaried amino acids-and hence in the likelihood of multiple changes per site-and this factor explains
478 BEE DRO XEN MUS
Crazier and Oozier
MKLILMM-NLFEMFDPSTS~LS~F~PIIIFPSIFWLI--QSRI~I~T~FMYNEFKWSK-SKYQS ..T...Sv....aIF...1...STF.gllmI...y..m--P ..YNIFWNsilLTlHK...TlLGP.GHnG .NLSF.dQ.MSPVILGiP1IAiA.1D.FT1ISWPIQSNGFnN.liTlQSWFlHNFTTI.YQ1tSP-GHKW nN.lIiSFQhW.vKLiIKqMMliHTPKGRTW .NE...AS.ITP.MMGFPiWAIi.F.S.l...SKR..--
BEE NIIIFISLMLYIMITNIFSLIPWFTLTSHLLLNMILSLTLWFSFLIYLIYNNYIMFLSHLVPLNSPVFLMNFMV DRO STF.....FSL.lFN.FMG.F..i..S . . ..T.TlS.a.P..LC.ml.GWI.HTQHMFa....QGt.AI..P... XEN Alll-T . . ..Ll.SL.lLG.l..T..P.tQ.S...G.avP ..LaTv.MASKPTNYA-.G..l.EGt.TP.iPVli MIJS Tim.-v..imf.GS ..lLG.l.HT..P.tQ.Sm.lSmaiP ..AGAv.TGFRHKLKSS.a.Fl.QGt.iS.iPMli BEE DRO XEN MUS
IIELISLIIRPWTLSIRLSANLISGHLILTLLGIFISNFISILPIN---~IQ~LLT-LEIFMSMIQSYVFSIL C..T..N . . ..G..av..t..m.a...l.....NTGPS-m.Y.L-VTFL.vA.IA..V-..SAvt.......av. . ..T...F...La.Gv..t...Ta . ..liQ.iATARFVLl..m.TVAILTS.VLF...L...Ava...a...Vl. . ..T . ..F.Q.Ma.av..t..iTa... lmH.i.GATLVLmN.S.PTATITF.ILl...I..FAval..a...tl.
BEE DRO XEN MUS
LILYFSESN ST..S..V. .S..LQ.-.V vS..LHd-.T
BEE DRO XEN MUS
IPQMMPMKWFLIYFIYLLI-FYLFIMLINSMLIKTKINKET-LK-IKLKK-WNWFW . ..-.A.iS.L.i- ..VFS.T.I..CS-..YYSYMPTSP.SNE..N.N.NS-M..K. SI:.lN. GP . . ..LIFSW.vLLTFIPPKvLKHKAFNEPTTq.TE.-S.PNP-...P.T m..lDTST..iTIISSmiTL.I..QlKvS.QTFP~SPKsLTT-~.v.TP.eLK.TKIYLPHSLPQQ
FIG. 3.-Inferred amino acid sequencesfor subunits 6 (upper) and 8 (lower) of ATPase. for Apis melliferu (present paper), Drosophila yukuba (Clary and Wolstenholme 1985), Xenopus laevis (Roe et al. 1985), and Mus species ( Bibb et al. 198 1), aligned by eye. Amino acids identical to those in the honeybee are indicated by dots; conservative replacements are indicated by lowercase letters; and replacements nonconservative relative to the honeybee are indicated by capital letters. Replacements were treated as conservative if, according to the protocol of French and Robson ( 1983), they involved amino acids of the same group (KHR, DENQ, GP, AST, ILMV, or FWY). Hyphens denote gaps inserted to improve the alignment.
the notable variation between them in the relative evolutionary
rates of Apis and
Drosophila.
For slowly evolving genes such as COI, COIL and cytochrome b, if the much larger number of replacements in the Apis than in the Drosophila lineage resulted from differences in average evolutionary rates, such rate differences have a number of possible causes (Crozier et al. 1989 ) . When population-biology factors are considered, generation-time ( Wu and Li 1985) differences would lead to a trend opposite to that observed, because honeybee queens live for many Drosophila generations, whereas honeybees have much smaller effective population sizes than do fruit flies (Crozier 1979), which would favor the trend observed. However, it is uncertain that honeybees and fruit flies have had their current life-pattern characteristics for more than about -40% of the time since their separation, so invocation of population characteristics is not firmly based. It is also possible that differences either in DNA polymerases or in other cellular constituents could lead to a higher mutation rate in honeybees. The actual magnitude of the difference in numbers of replacements is also liable to be increased by the difference in the stationary nucleotide proportions (Holmquist 1983). It is not clear to what extent the instantaneous evolutionary rates for fruit fly mtDNA and for honeybee mtDNA differ: to some extent the greater evolutionary divergence of the honeybee genes than of the fruit fly genes from their common ancestor could result from a shift in nucleotide composition in Apis.
Cytochrome b/ATPase Genes of Honeybee Mitochondrial DNA
479
BEE ~KFMNFFSSNEFLKMIMSTIYLPTPVNINYMWNFGSILGIF~IQIISGFILSMHYCPNIDIAFWSITNIMKDM .h.PlRN-.HPL.KIANNaLvD..a.i..SSW.....l..lC.i...lt.LF.a...TAdvnl..y.vNH.Cr.v DRO .APNiRK-.HPLIKIiNN.F.D....S..SSl.....l..vC.iA...t.LF.a...TAdTSm..S.vaH.CF.v XEN M'JS .T-N.RK-tHPL.KIiNH.F.D..a.S..SSW.....l..vC..v...t.LF.a...TSdTMT..S.v.H.Cr.v BEE DRO XEN MUS
NSGWLFRLIHMNGASFYFLMMYIHISRNLFYCSYKLNNVWGIGIMILLMSMAAAFMGYVLPWGQMSYWGATVITN .Y...L.Tl.A..... f.iCi.l..G.Giy.G..LFTPT.Lv.vi..FlV.Gt.............f........ .Y.L.I.Nl.A..L..f.iCi.l..G.G.y.G.fLYKeT.N..vil.FlV..t..v..........f........ .Y...I.Ym.A.... Mf.iClfl.vG.G.y.G..TFMeT.N..vll.FAV..t.............f........
BEE DRO XEN MUS
LLSAIPYIGDTIVLWIWGGFSI,~ATLNRFFSLHFILPLLILFMVILHLFALHLTGSSNPLGSNFNNYKISFHPY . . . . . ..l.MDl.Q.l....avd....T...tF.....Fiv.A.Rni..LF..Q...N..i.L.S.ID..P.... . . ..K.. ..nVl.Q.SL....vd....T...aF..l..Fi.AGAS....LF..E...t..T.L.SdPD.vP.... . . . . . . . ..T.l.E.......vdK...T...aF.....Fi.AAlA.v..LF..E...N..T.L.SdAD..P....
BEE DRO XEN MUS
FSIKDLLGFYIILFIFMFINFQFPYHLGDPDNFKI~P~TPTHIKPE~FLFAYSIL~IPNKLGGVIGL~SI .tF..iv..Ivmi..LiSlVLIS.NL.......IP...lV..a..Q.........a...s.........A..l.. ..Y......L.m.TALTLlAMFS.NL.......TP...lI..P............a...sm-......lA..l.. yt... i..IL.mFL.L.TlVLF..DM......yMP...l...P............a...s........lA.il..
BEE DRO XEN MUS
LILYIMIFYNN-KMMNNKFNMLNKIYYWMFINNFILLTWLGKQLIEYPFTNINMLFTTTYFLYFFLNFYLSKLWD i.ARPv.E.yVL.GQiL.II....yLi.PLvt.W.. A..M.lP . ..LS.FRGIQ.YPi.Q.Lf.SMlVTV..... i.G.Pv.D.y.M.GQ.AsVI..SI.IiM.P.MGWVe . ..Al.PLLHTS.QRSLM.RPFTQ.Mf.ALvAdTli... . ..A1.P.LHTS.QRSLM.RPiTQ.L..iLvA.Lli...i.G.Pv.H..II.GQ.AsIs..SIILiLMPi.GiIe
BEE DRO XEN MUS
NLIWNSPLN ..-----.. .KlL.W dKmLKLYP
FIG.
4.-Inferred
cytochrome b amino acid sequences, according to conventions of fig. 3
Functional Structure of Cytochrome
b
Howell ( 1989) reviewed models for the functional attributes for cytochrome b and, from comparisons between seven phylogenetically diverse species of prokaryotes and eukaryotes, concluded that there are five regions, of -20 amino acids each, that are relatively conserved during evolution and that two of these show very high conservation. From a survey of mammal cytochrome b protein sequences, Irwin et al. ( 199 1) extended Howell’s ( 1989 ) analysis and concluded that eight conserved regions can be discerned and that the outer surface of the molecule (in relation to the portions penetrating the mitochondrial membrane) is evolving slowly, compared with the transmembrane or inner-surface portions. A comparison between the inferred cytochrome b sequences of honeybee and Xenopus (fig. 5 ) clearly shows the two highly conserved regions identified by Howell ( 1989) and lends more support to the occurrence of eight relatively conserved regions (Irwin et al. 199 1) than to the occurrence of five (Howell 1989). Apparent Effects of Sequence Composition on Amino Acid Composition The protein-encoding mitochondrial genes known so far from the Apis mitochondrion show overall AT proportions of 0.76-0.90 (Crozier 1990, and accepted), and the results conform to this trend. The proportion for third-codon positions is uniformly >0.90, whereas the second-codon positions show lower and variable AT proportions. There is a particularly strong bias against guanine, since both the gene for ATPase 8 and that for cytochrome b lack any in the third-codon position. Further data are needed to test the apparent negative correlation between AT proportions and constraint on evolutionary rate (as measured by similarity to the homologous Drosophila gene products). The extreme AT bias of honeybee mtDNA is associated with a skewed usage of
480
Crozier and Crozier
Table 1 Numbers of Amino Acid Replacements
in Proteins
No. OF AMINO ACID REPLACEMENTS
Terminal PROPORTION
Apis
ATPase 6 ATPase 8 Cytochrome b Total
Drosophila
Xenopus
Mus
23 7
22 6
94
32
128
62
32 2
Internal
Other
Total
UNVARIED
25 8
17 4
205 70
324 97
0.21 0.08
32
28
16
126
328
0.40
60
61
37
401
749
NOTE.-sequences are inferredfor the terminalbranchesleadingto Apis, Drosophila, Xenopus, and Mus and for the internalbranchbetweenthe insectsand vertebrates.The number of replacements inferred to have occurred but which
cannot be placed in the phylogeny is also shown (as “Other”), as are both the total number of replacements inferred for each protein and the proportion of those positions with amino acids present for all four animals that is unvaried. Ten positions supported the other two possible trees.
codons. For example, in the inferred Apis cytochrome b amino acid sequence, phenylalanine is specified by TTC seven times and is specified by TTT 34 times. In MUS cytochrome b, by contrast, TTT occurs 12 times and TTC occurs 17 times. Despite being less AT biased than that of Apis, the Drosophila mitochondrial genome (Clary and Wolstenholme 1985) shows a bias similar to that of Apis (28 TTT codons and three TTC codons for the cytochrome b gene). Is the strong nucleotide-composition bias of its gene reflected in the amino acid makeup of cytochrome b? We examined this possibility by using forward stepwise regression analysis, using as independent variables the number of codons per family, the proportion of AT per family, and the proportions of each of the four bases in a family. Analysis of the inferred Mus cytochrome b sequence shows that the number of occurrences of an amino acid is significantly associated with both the number of codons in the family and the proportion of AT in the codon family but not with the separate proportions of the four nucleotides in the codon family. The same conclusion results from analysis of Xenopus cytochrome b. For these same variables, by contrast, only the proportions of T and G are significant predictors of the number of occurrences of an amino acidin Apis cytochrome b. An association between size of an amino acid’s codon-family size and its abundance has long been known for nuclear genes (e.g., see King and Jukes 1969) ; it is surprising that codon-family size appears to be relatively unimportant for honeybee cytochrome b although not for that of vertebrates. It is reasonable to ascribe this effect to the very high AT bias of honeybee mtDNA and to regard it as an example of a DNA-level process (AT bias) affecting a protein-level process (amino acid composition) (Almagor 1985 ). The possibility is then raised that DNA-level processes ( nucleotidecompositional bias) affect protein function and, possibly, the biology of the organism (e.g., see Dover 1986 ) . It is hard to evaluate this possibility directly, but hydrophilicity (plots available from the authors) and surface-probability analyses of MUS and Apis cytochrome b yield plots that appear very similar despite the different evolutionary dynamics of these genomes (comparisons involving other animals yield similar results). If the biases observed in codon usage have not altered the hydrophilic characteristics of most amino acid positions, then it is quite unlikely that the relatively few strongly constrained residues have been affected. This suggestion concurs with the general
Cytochrome b/ATPase Genes of Honeybee Mitochondrial DNA
481
Amino Acid Position FIG. 5.--Identity values between inferred Apis and Xenopus cytochrome b amino acid sequences, according to numbering sequence derived from fig. 4. Comparisons were made using a moving window of 11 amino acids. The solid-line curve denotes exact identity, whereas the dotted-line curve denotes functional identity according to the criteria used for fig. 3. The horizontal lines give the random expectations for exact and functional identity.
impression that selection at the organism level (or higher) overwhelms contrary DNAlevel selective processes (Crozier, accepted). Sequence Availability
The GenBank accession numbers are M87065 for the ATPase genes and M87052 for the cytochrome b gene. Acknowledgments We thank B. Crespi, S. Edwards, W. M. Fitch, T. Kocher, S. Koulianos, M. Kreitman, R. Kusmierski, and two anonymous reviewers for constructive comments on various drafts of the manuscript, and we thank the Australian Research Council, the University of New South Wales, and La Trobe University for grants supporting this work. LITERATURE
CITED
ALMAGOR, H. 1985. Nucleotide
distribution and the recognition of coding regions in DNA theory approach. J. Theoret. Biol. 117:127-136. BIBB, M. J., R. A. VAN ETTEN, C. T. WRIGHT, M. W. WALBERG, and D. A. CLAYTON. 1981. Sequence and gene organization of mouse mitochondrial DNA. Cell 26: 167- 180. CARPENTER,F. M., and L. BURNHAM. 1985. The geological record of insects. Annu. Rev. Earth Planetary Sci. 13:297-3 14. CLARY, D. O., and D. R. WOLSTENHOLME. 1985. The mitochondrial DNA molecule of Drosophila yakuba: nucleotide sequence, gene organization, and genetic code. J. Mol. Evol. 22: 252-27 1. CROZIER, R. H. 1979. Genetics of sociality. Pp. 223-286 in H. R. HERMANN, ed. Social insects. Vol. 1. Academic Press, New York. sequences:
an information
482 Crazier and Oozier
-.
1990. From population genetics to phylogeny: uses and limits of mitochondrial DNA. Aust. Syst. Bot. 3:111-124. -. Molecular methods for insect systematics. In WHITTEN, M. J., and J. G. OAKESHOTT, eds. Molecular approaches to pure and applied entomology. Springer, New York. (accepted). CROZIER, R. H., Y. C. CROZIER, and A. G. MACKINLAY. 1989. The CO-I and CO-II region of honeybee mitochondrial DNA: evidence for variation in insect mitochondrial evolutionary rates. Mol. Biol. Evol. 6:399-4 11. DOVER, G. A. 1986. Molecular drive in multigene families: how biological novelties arise, spread and are assimilated. Trends Genet. 2: 159-165. -. 1989. Slips, strings and species. Trends Genet. 5: 1OO- 102. FRENCH, S., and B. ROBSON. 1983. What is a conservative substitution? J. Mol. Evol. 19: 17 l175. HOLMQUIST, R. 1983. Transitions and transversions in evolutionary descent: an approach to understanding. J. Mol. Evol. 19: 134- 144. HOWELL, N. 1989. Evolutionary conservation of protein regions in the protonomotive cytochrome b and their possible roles in redox catalysis. J. Mol. Evol. 29: 157- 169. IRWIN, D. M., T. D. KOCHER, and A. C. WILSON. 1991. Evolution of the cytochrome b gene of mammals. J. Mol. Evol. 32:128-144. JACOBS,H. T., D. J. ELLIOTT, V. B. MATH, and A. FARQUARSON.1988. Nucleotide sequence and gene organization of sea urchin mitochondrial DNA. J. Mol. Biol. 202: 185-2 17. KING, J. L., and T. H. JUKES. 1969. Non-Darwinian evolution. Science 164:788-798. KOCHER, T. D., W. K. THOMAS, A. MEYER, S. V. EDWARDS,S. PUBO, F. X. VILLEBLANCA, and A. C. WILSON. 1989. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc. Natl. Acad. Sci. USA 86:6 196-6200. KYTE, J., and R. F. DOOLITTLE. 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157: 105- 132. ROE, B. A., D. P. MA, R. K. WILSON, and J. F. H. WANG. 1985. The complete nucleotide sequence of the Xenopus laevis mitochondrial genome. J. Biol. Chem. 260:9759-9774. SOKAL, R. R., and F. J. ROHLF. 198 1. Biometry. 2d ed. W. H. Freeman, San Francisco. VLASAK,I., S. BURGSCHWAIGER, and G. KREIL. 1987. Nucleotide sequence ofthe large ribosomal RNA of honeybee mitochondria. Nucleic Acids Res. 15:2388. Wu, C.-I., and Ll, W.-H. 1985. Evidence for higher rates of nucleotide substitution in rodents than in man. Proc. Natl. Acad. Sci. USA 82:1741-1745. MARTIN KREITMAN, reviewing
editor
Received March 12, 199 1; revision received October 24, 199 1 Accepted November 22, 199 1
and Y. C. Croziert
*Department of Genetics and Human Variation, La Trobe University; and $School of Biological Science, University of New South Wales
The gene sequences for honeybee cytochrome b, ATPase 6, and ATPase 8 are presented, along with the inferred amino acid sequences of the proteins. These mitochondrial genes are in the same relative positions as are their counterparts in Drosophilamitochondrial mtDNA and have evolved at a significantly greater overall rate than have those of Drosophila. Comparisons using both amino acid identity and the proportion of conservative replacements between the inferred Apis and vertebrate cytochrome b sequences shows the two highly conserved sections reported by Howell, but his recognition of five conserved regions is not well supported. A very high AT bias is reflected in very high codon biases. The best predictors of the number of occurrences of an amino acid in honeybee cytochrome b are the T and G contents of its codon family-unlike the case for vertebrate cytochrome b, in which the codon family size and AT bias are the strongest predictors; protein function, at least as judged by hydrophilicity characteristics, appears to be unaffected by these differing influences on amino acid composition. Introduction
The sequences and order for the mitochondrial genes for cytochrome c oxidase subunits I and II (CO-I and CO-II, respectively), for four tRNAs, (Crozier et al. 1989), and for most of the large subunit of rRNA (Vlasak et al. 1987) have been published previously for the honeybee Apis mellifera. The DNA sequences for the two protein-encoding genes are extremely AT rich, with the CO-II gene lacking guanine altogether at the third codon position (Crozier 1990)) a bias stronger than that seen in Drosophila (Clary and Wolstenholme 1985; also see Crozier 1990). Apart from some shifts of tRNA genes, the Apis protein-encoding and rRNA genes identified elsewhere (Vlasak et al. 1987; Crozier et al. 1989) are in the same relative positions as are their Drosophila counterparts (Clary and Wolstenholme 1985 ). Phylogenetic analysis shows that, during their separation of -280 Myr (Carpenter and Burnham 1985), the CO-I and CO-II genes of the honeybee have evolved significantly more than have the corresponding Drosophila genes (Crozier et al. 1989). Despite this long period of separate development, the similarities between Drosophila and Apis mitochondrial DNA (mtDNA) at the third codon position for the CO-II (Crozier 1990) and CO-I genes are very close, whereas those at the first and second codon positions differ markedly (Crozier, accepted). Drosophila and Apis are more similar than expected by chance, for all three codon positions for both of these genes. We here report and analyze the honeybee gene sequences for cytochrome b and 1. Key words: cytochrome b, ATPase genes, honeybee. Address for correspondence and reprints: R. H. Crozier, Department of Genetics and Human Variation, La Trobe University, Bundoora, Victoria 3083, Australia. Mol. Bid. Ed. 9(3):474-482. 1992. 0 1992 by The University of Chicago. All rights reserved. 0737-4038/92/0903-0008$02.00
474
Cytochrome b/ATPase Genes of Honeybee Mitochondrial DNA
475
the ATPase subunits 6 and 8. The cytochrome b gene is of special interest both because of its increasing use as a tracer of phylogeny (Kocher et al. 1989; Irwin et al. 199 1) and because of evolutionary deductions about its structure and function (Howell 1989; Kocher et al. 1989; Irwin et al. 199 1) . Various authors (e.g., see Almagor 1985) have suggested that there could be an interaction between DNA-level and protein-level forces; for example, Dover ( 1989) suggests that DNA-level processes such as slippage replication have altered the per locus of Drosophila and have affected courtship behavior. The different associations between DNA parameters and cytochrome b amino acid content in Apis and vertebrates provide an opportunity to see whether, at least at a gross level, there is any sign of such an interaction for this mitochondrial gene. Methods
The source of the material and the methods of mtDNA preparation, cloning, and sequencing have been presented elsewhere (Crozier et al. 1989). Sequence analyses were carried out using the Nucleic Acids Analysis System package (Genesearch, Broadbeach, Queensland) run on an Apple II microcomputer, the MacVector (IBI) and Statview packages on a Macintosh, and Macintosh programs written by R.H.C. Inferred amino acid replacements in a four-species Wagner network were restricted to those whose change of state could be unambiguously assigned to a specific branch under parsimony. For example, given the grouping of taxa (Apis, Drosophila) (MS, Xenopus), the observation ABBB (Apis having one amino acid and the three other species having another) implies a single replacement with a single most-parsimonious placement in the terminal branch leading to Apis. The observation ABBC implies two changes most parsimoniously placed in the terminal branches leading to Apis and Xenopus. The observation ABCC (Apis and Drosophila each having unique amino acids, with the vertebrates possessing a third) implies two changes which can be placed in three equally parsimonious ways. The observation AABB implies a single change in the internal branch between the insect and vertebrate pairs. Hence, ABBB and ABBC each contribute to inferred changes in terminal branches, and AABB contributes to inferred changes in the internal branch, but ABCC leads to the inference of two replacements of unknown position. Because the mitochondrial genetic code differs for these organisms, it is not. practical to infer the number of replacements by using code information. We used multiple stepwise regression (Sokal and Rohlf 198 1, pp. 66 l-67 1) to determine which of various variables predict significantly the number of times an amino acid occurs in cytochrome b molecules. In brief, although many independent variables may each give a good prediction of the values of a dependent variable, many of these may be correlated with each other. In such cases, one might infer that some of these variables are in fact dependent on others for their effects on the dependent variable, and the task is then to determine which of the examined variables underlie(s) the patterns observed. Under multiple stepwise regression, the correlations between variables are calculated, and those with the largest independent effects are selected. As variables are added, their contribution to the prediction is then tested following a partial correlation, and any shown to be making an insignificant contribution to the predictive power of the multiple-regression equation are removed. The Kyte and Doolittle ( 1982) method of determining hydrophilicity curves was used to provide a broad picture of protein functional composition. In brief, the hydrophilicity values of the amino acids in a moving window are averaged to provide a
476 Crozier and Crozier
mean value for each amino acid position. These values provide an indication of the tendency that that portion of the protein is on the interior (negative values) or exterior (positive values) of the molecule. The comparison of hydrophilicity curves therefore gives one indication of the functional similarity of the molecules; because all amino acid positions are considered, the curves are liable to reflect changes at many weakly selected positions in addition to the relatively few strongly selected positions. Results and Discussion Genome Organization The ATPase 6 and 8 genes, and that for cytochrome b, are in the same relative positions in both Apis mtDNA and Drosophila mtDNA (Clary and Wolstenholme 1985 ) . The sequence of the region inferred to contain the genes responsible for encoding mitochondrial ATPase subunits 6 and 8 is shown in figure 1, and the sequence for the inferred cy-tochrome b gene is shown in figure 2. The amino acid sequences inferred from the Drosophila genetic code are also shown. The inferred amino acid sequences for the two ATPase subunits and for cytochrome b are shown in figures 3 and 4, along with those of representative other animals from the literature. We infer the Apis ATPase 6 gene to be slightly larger than that for Drosophila. The amino acid sequence begins with the ATG methionine codon at position 14 1 in figure 2-rather than with the ATA codon at position 156-because ATG is a stronger promoter than is ATA (Jacobs et al. 1988 ) . Similarly, Jacobs et al.
ATPase 8 I P Q MMPMKWFL IYFIYLLIFYLFIM ATTCCTCAAATAATACCTATAAAATGATTTTTAATTTTT~TTTATTTTATTTATTTATT~TTTTTTATTTATTCATTATA ATPase M K L INSMLIKTKINKETLKIKLKKWNW TTAATTAATTCAATACTAATCAAAACTAAAATCAATAAAATT
AAAAZUATGAAATTGA
I LMMNLFEMFDP STSNNLSMNWLFM F w x TTTTGATAATAAATTTATTTGAAATATTTGATCCATCAAC ML P I I I F P S IFWLIQSRIMF TATTACCAATTATTATTTTTCCAAGAATTTTTTTGATT~TTC~TCACG~TTATATTTATTAT
75
6 L 150
225 I
M
K T L AAAAACACTAA
MNFMYNEFKVVSKSKYQSNII I F TAAATTTTATATATAATGAATTTAAAGTTGTTGTTTC~TCT~TATC~TCT~TATTATTATTTTTATTAGAT
I
300
S 375
LMLYIMITNIFSLIPYVFTLTSHLL TAATACTTTATATTATMTTACTAACATTTTTAGATTTTTAGATT~TTCCTTATGTTTTTACATT~C~GACATCTACTTT
450
L N M I LSLTLWFSFLIYLIYNNYIMF TAAATATAATTTTATCATTAATTATGATTTAGATTTCT~TTTATTT~TTTAT~T~TTATATTATATTCC
525
L s H L V P L N S PVFLMNFMVI TTAGTCATTTAGTTCCATTRTTCACCAGTATTTTTAAT
600
IELISL
I R L S AN L I SGHLILTLL I IRPWTLS TTATTCGACCTTGAACATTATCAATTCGATTCGATTATCAGC~TTT~TTTCTGGACATTT~TTTT~CATTATTAG
675
ILPINLMIQNMLLTLE GIFISNFIS GAATTTTTATTAGAAACTTTATTTCAATTTTACCAATTAA
750
IFMSMIQSYVFS TTTTTATATCAATAATTCATTATGTATTTTCAATTCT
ILLILYFSESNX
FIG. 1.--Sequence of genes for honeybee ATPase subunits 6 and 8, together with amino acid sequences inferred from Drosophila mitochondrial genetic code.
Cytochrome b/ATPase Genes of Honeybee Mitochondrial DNA SNEFLKMIMST MKKFMNFFS I ATGAAAAAATTTATAAACTTTTTTAGTTCCAATGAATTTCT~T~TTATATCTAC~TTTATTTACC~CT
Y
L
P
477 T 75
LGIFLMIQIISG PVNINYMWNFGSI CCAGTAAATATTAATTATATATGAAATTTTT~ATTCTCATTTCTGGA
150
F ILSMHYCPNID I AF W S ITNIMKDM TTTATTTTATCAATACATTATTGTCCAAATATTGATATTGATATT~TTTCTGATC~TTACT~TATTAT~GATATA
225
NSGWLFRLIHMNGASFYFLMMYIHI AATTCAGGATGATTATTTCGTTTAATTCACACATGGAGATATTCATATT
300
SRNLFYCSYKLNNVWGIGIMI AGTCGAAATTTATTTTATTGTTCATATAAATTAAATAATGTATCA
L
L
M
S 375
MAAAFMGYVLPWGQMS YWGATVITN ATAGCAGCTGCATTTATAGGATATGTACTACCATGAGGAC~TATCATATTGAGGTGC~CAGTTATTACT~T LLSAIPYIGDTIVLWIWGGFS CTTTTATCAGCAATTCCTTATATTGGTGATACAATTGTATTGCT
I
450 N
N
A 525
T L N RF F SLHFILPLLI L F M V I L H ACATTAAATCGATTTTTTTCTTTACATTTTATTTTACCATTATT~TTTTATTTATAGTTATTCTTCATTTATTT SNPLGSNFNNYKISFHPY ALHLTGS GCCTTACATTTAACTGGATCATCTAATCCTCCTCAATTTCATTTCATCCATAT
L
F 600
675
F S IKDLLGFYIILFIFMFINFQFPY TTTTCAATTAAAGATCTTTTAGGATTTTATATCATCTTATTTATCTTTATATTCATT~TTTTC~TTTCCATAT
750
HLGDPDNFKIANPMNTP CATTTAGGAGATCCAGACAATTTCAAAATTGCAAATCCAATGATAT
825
THIKPEWY
FLFAYSI L RA IPNKLGGVIGLVMSI TTCCTATTTGCATATTCAATTTTACGAGCAATTCCTAATATT
9ao
L I L Y I M I FYNNKMMNNKFNMLNKIY CTTATTCTTTATATTATAATTTTTTATAATAATAAAATAATTTAT
975
Y WM F I N N F ILLTWLGKQLIEYPFTN TATTGAATATTTATTAATATTCATTTTATTAACATGATGATTA~T~C~TT~TTG~TATCCATTTACT~T
LO50
INMLFTTTYFLYFFLNFYLSKLWDN ATTAATATATTATTTACAACAACATATATTTTTTTTT
L125
LIWNSP L N X TTAATTTGAAATTCACCATTAAATTAA
FIG. 2.-Sequence of gene for honeybee cytochrome b, together with amino acid sequences inferred from Drosophila mitochondrial genetic code.
( 1988) inferred that the cytochrome b gene in the echinoderm Strongylocentrotus is longer than that seen in various other organisms.
Evolutionary
Rates
Appropriate comparisons using the inferred amino acid sequences from figures 3 and 4 can be used to test the hypothesis that the evolutionary rates of honeybee and fly mtDNA are the same (table 1). x: Tests reject the possibility that the number of amino acid replacements that occurred in the line leading to Apis from the common ancestor with Drosophila has been the same as that leading to Drosophila, both for the total number of changes reported in the present paper and for those found in cytochrome b alone (94 for Apis vs. 32 for Drosophila). The replacement-rate difference is also reflected in the number of times each insect has one amino acid and all the other taxa another one (for cytochrome b, 7 1 times for Apis vs. 17 times for Drosophila). The three proteins differ markedly in the proportions of unvaried amino acids-and hence in the likelihood of multiple changes per site-and this factor explains
478 BEE DRO XEN MUS
Crazier and Oozier
MKLILMM-NLFEMFDPSTS~LS~F~PIIIFPSIFWLI--QSRI~I~T~FMYNEFKWSK-SKYQS ..T...Sv....aIF...1...STF.gllmI...y..m--P ..YNIFWNsilLTlHK...TlLGP.GHnG .NLSF.dQ.MSPVILGiP1IAiA.1D.FT1ISWPIQSNGFnN.liTlQSWFlHNFTTI.YQ1tSP-GHKW nN.lIiSFQhW.vKLiIKqMMliHTPKGRTW .NE...AS.ITP.MMGFPiWAIi.F.S.l...SKR..--
BEE NIIIFISLMLYIMITNIFSLIPWFTLTSHLLLNMILSLTLWFSFLIYLIYNNYIMFLSHLVPLNSPVFLMNFMV DRO STF.....FSL.lFN.FMG.F..i..S . . ..T.TlS.a.P..LC.ml.GWI.HTQHMFa....QGt.AI..P... XEN Alll-T . . ..Ll.SL.lLG.l..T..P.tQ.S...G.avP ..LaTv.MASKPTNYA-.G..l.EGt.TP.iPVli MIJS Tim.-v..imf.GS ..lLG.l.HT..P.tQ.Sm.lSmaiP ..AGAv.TGFRHKLKSS.a.Fl.QGt.iS.iPMli BEE DRO XEN MUS
IIELISLIIRPWTLSIRLSANLISGHLILTLLGIFISNFISILPIN---~IQ~LLT-LEIFMSMIQSYVFSIL C..T..N . . ..G..av..t..m.a...l.....NTGPS-m.Y.L-VTFL.vA.IA..V-..SAvt.......av. . ..T...F...La.Gv..t...Ta . ..liQ.iATARFVLl..m.TVAILTS.VLF...L...Ava...a...Vl. . ..T . ..F.Q.Ma.av..t..iTa... lmH.i.GATLVLmN.S.PTATITF.ILl...I..FAval..a...tl.
BEE DRO XEN MUS
LILYFSESN ST..S..V. .S..LQ.-.V vS..LHd-.T
BEE DRO XEN MUS
IPQMMPMKWFLIYFIYLLI-FYLFIMLINSMLIKTKINKET-LK-IKLKK-WNWFW . ..-.A.iS.L.i- ..VFS.T.I..CS-..YYSYMPTSP.SNE..N.N.NS-M..K. SI:.lN. GP . . ..LIFSW.vLLTFIPPKvLKHKAFNEPTTq.TE.-S.PNP-...P.T m..lDTST..iTIISSmiTL.I..QlKvS.QTFP~SPKsLTT-~.v.TP.eLK.TKIYLPHSLPQQ
FIG. 3.-Inferred amino acid sequencesfor subunits 6 (upper) and 8 (lower) of ATPase. for Apis melliferu (present paper), Drosophila yukuba (Clary and Wolstenholme 1985), Xenopus laevis (Roe et al. 1985), and Mus species ( Bibb et al. 198 1), aligned by eye. Amino acids identical to those in the honeybee are indicated by dots; conservative replacements are indicated by lowercase letters; and replacements nonconservative relative to the honeybee are indicated by capital letters. Replacements were treated as conservative if, according to the protocol of French and Robson ( 1983), they involved amino acids of the same group (KHR, DENQ, GP, AST, ILMV, or FWY). Hyphens denote gaps inserted to improve the alignment.
the notable variation between them in the relative evolutionary
rates of Apis and
Drosophila.
For slowly evolving genes such as COI, COIL and cytochrome b, if the much larger number of replacements in the Apis than in the Drosophila lineage resulted from differences in average evolutionary rates, such rate differences have a number of possible causes (Crozier et al. 1989 ) . When population-biology factors are considered, generation-time ( Wu and Li 1985) differences would lead to a trend opposite to that observed, because honeybee queens live for many Drosophila generations, whereas honeybees have much smaller effective population sizes than do fruit flies (Crozier 1979), which would favor the trend observed. However, it is uncertain that honeybees and fruit flies have had their current life-pattern characteristics for more than about -40% of the time since their separation, so invocation of population characteristics is not firmly based. It is also possible that differences either in DNA polymerases or in other cellular constituents could lead to a higher mutation rate in honeybees. The actual magnitude of the difference in numbers of replacements is also liable to be increased by the difference in the stationary nucleotide proportions (Holmquist 1983). It is not clear to what extent the instantaneous evolutionary rates for fruit fly mtDNA and for honeybee mtDNA differ: to some extent the greater evolutionary divergence of the honeybee genes than of the fruit fly genes from their common ancestor could result from a shift in nucleotide composition in Apis.
Cytochrome b/ATPase Genes of Honeybee Mitochondrial DNA
479
BEE ~KFMNFFSSNEFLKMIMSTIYLPTPVNINYMWNFGSILGIF~IQIISGFILSMHYCPNIDIAFWSITNIMKDM .h.PlRN-.HPL.KIANNaLvD..a.i..SSW.....l..lC.i...lt.LF.a...TAdvnl..y.vNH.Cr.v DRO .APNiRK-.HPLIKIiNN.F.D....S..SSl.....l..vC.iA...t.LF.a...TAdTSm..S.vaH.CF.v XEN M'JS .T-N.RK-tHPL.KIiNH.F.D..a.S..SSW.....l..vC..v...t.LF.a...TSdTMT..S.v.H.Cr.v BEE DRO XEN MUS
NSGWLFRLIHMNGASFYFLMMYIHISRNLFYCSYKLNNVWGIGIMILLMSMAAAFMGYVLPWGQMSYWGATVITN .Y...L.Tl.A..... f.iCi.l..G.Giy.G..LFTPT.Lv.vi..FlV.Gt.............f........ .Y.L.I.Nl.A..L..f.iCi.l..G.G.y.G.fLYKeT.N..vil.FlV..t..v..........f........ .Y...I.Ym.A.... Mf.iClfl.vG.G.y.G..TFMeT.N..vll.FAV..t.............f........
BEE DRO XEN MUS
LLSAIPYIGDTIVLWIWGGFSI,~ATLNRFFSLHFILPLLILFMVILHLFALHLTGSSNPLGSNFNNYKISFHPY . . . . . ..l.MDl.Q.l....avd....T...tF.....Fiv.A.Rni..LF..Q...N..i.L.S.ID..P.... . . ..K.. ..nVl.Q.SL....vd....T...aF..l..Fi.AGAS....LF..E...t..T.L.SdPD.vP.... . . . . . . . ..T.l.E.......vdK...T...aF.....Fi.AAlA.v..LF..E...N..T.L.SdAD..P....
BEE DRO XEN MUS
FSIKDLLGFYIILFIFMFINFQFPYHLGDPDNFKI~P~TPTHIKPE~FLFAYSIL~IPNKLGGVIGL~SI .tF..iv..Ivmi..LiSlVLIS.NL.......IP...lV..a..Q.........a...s.........A..l.. ..Y......L.m.TALTLlAMFS.NL.......TP...lI..P............a...sm-......lA..l.. yt... i..IL.mFL.L.TlVLF..DM......yMP...l...P............a...s........lA.il..
BEE DRO XEN MUS
LILYIMIFYNN-KMMNNKFNMLNKIYYWMFINNFILLTWLGKQLIEYPFTNINMLFTTTYFLYFFLNFYLSKLWD i.ARPv.E.yVL.GQiL.II....yLi.PLvt.W.. A..M.lP . ..LS.FRGIQ.YPi.Q.Lf.SMlVTV..... i.G.Pv.D.y.M.GQ.AsVI..SI.IiM.P.MGWVe . ..Al.PLLHTS.QRSLM.RPFTQ.Mf.ALvAdTli... . ..A1.P.LHTS.QRSLM.RPiTQ.L..iLvA.Lli...i.G.Pv.H..II.GQ.AsIs..SIILiLMPi.GiIe
BEE DRO XEN MUS
NLIWNSPLN ..-----.. .KlL.W dKmLKLYP
FIG.
4.-Inferred
cytochrome b amino acid sequences, according to conventions of fig. 3
Functional Structure of Cytochrome
b
Howell ( 1989) reviewed models for the functional attributes for cytochrome b and, from comparisons between seven phylogenetically diverse species of prokaryotes and eukaryotes, concluded that there are five regions, of -20 amino acids each, that are relatively conserved during evolution and that two of these show very high conservation. From a survey of mammal cytochrome b protein sequences, Irwin et al. ( 199 1) extended Howell’s ( 1989 ) analysis and concluded that eight conserved regions can be discerned and that the outer surface of the molecule (in relation to the portions penetrating the mitochondrial membrane) is evolving slowly, compared with the transmembrane or inner-surface portions. A comparison between the inferred cytochrome b sequences of honeybee and Xenopus (fig. 5 ) clearly shows the two highly conserved regions identified by Howell ( 1989) and lends more support to the occurrence of eight relatively conserved regions (Irwin et al. 199 1) than to the occurrence of five (Howell 1989). Apparent Effects of Sequence Composition on Amino Acid Composition The protein-encoding mitochondrial genes known so far from the Apis mitochondrion show overall AT proportions of 0.76-0.90 (Crozier 1990, and accepted), and the results conform to this trend. The proportion for third-codon positions is uniformly >0.90, whereas the second-codon positions show lower and variable AT proportions. There is a particularly strong bias against guanine, since both the gene for ATPase 8 and that for cytochrome b lack any in the third-codon position. Further data are needed to test the apparent negative correlation between AT proportions and constraint on evolutionary rate (as measured by similarity to the homologous Drosophila gene products). The extreme AT bias of honeybee mtDNA is associated with a skewed usage of
480
Crozier and Crozier
Table 1 Numbers of Amino Acid Replacements
in Proteins
No. OF AMINO ACID REPLACEMENTS
Terminal PROPORTION
Apis
ATPase 6 ATPase 8 Cytochrome b Total
Drosophila
Xenopus
Mus
23 7
22 6
94
32
128
62
32 2
Internal
Other
Total
UNVARIED
25 8
17 4
205 70
324 97
0.21 0.08
32
28
16
126
328
0.40
60
61
37
401
749
NOTE.-sequences are inferredfor the terminalbranchesleadingto Apis, Drosophila, Xenopus, and Mus and for the internalbranchbetweenthe insectsand vertebrates.The number of replacements inferred to have occurred but which
cannot be placed in the phylogeny is also shown (as “Other”), as are both the total number of replacements inferred for each protein and the proportion of those positions with amino acids present for all four animals that is unvaried. Ten positions supported the other two possible trees.
codons. For example, in the inferred Apis cytochrome b amino acid sequence, phenylalanine is specified by TTC seven times and is specified by TTT 34 times. In MUS cytochrome b, by contrast, TTT occurs 12 times and TTC occurs 17 times. Despite being less AT biased than that of Apis, the Drosophila mitochondrial genome (Clary and Wolstenholme 1985) shows a bias similar to that of Apis (28 TTT codons and three TTC codons for the cytochrome b gene). Is the strong nucleotide-composition bias of its gene reflected in the amino acid makeup of cytochrome b? We examined this possibility by using forward stepwise regression analysis, using as independent variables the number of codons per family, the proportion of AT per family, and the proportions of each of the four bases in a family. Analysis of the inferred Mus cytochrome b sequence shows that the number of occurrences of an amino acid is significantly associated with both the number of codons in the family and the proportion of AT in the codon family but not with the separate proportions of the four nucleotides in the codon family. The same conclusion results from analysis of Xenopus cytochrome b. For these same variables, by contrast, only the proportions of T and G are significant predictors of the number of occurrences of an amino acidin Apis cytochrome b. An association between size of an amino acid’s codon-family size and its abundance has long been known for nuclear genes (e.g., see King and Jukes 1969) ; it is surprising that codon-family size appears to be relatively unimportant for honeybee cytochrome b although not for that of vertebrates. It is reasonable to ascribe this effect to the very high AT bias of honeybee mtDNA and to regard it as an example of a DNA-level process (AT bias) affecting a protein-level process (amino acid composition) (Almagor 1985 ). The possibility is then raised that DNA-level processes ( nucleotidecompositional bias) affect protein function and, possibly, the biology of the organism (e.g., see Dover 1986 ) . It is hard to evaluate this possibility directly, but hydrophilicity (plots available from the authors) and surface-probability analyses of MUS and Apis cytochrome b yield plots that appear very similar despite the different evolutionary dynamics of these genomes (comparisons involving other animals yield similar results). If the biases observed in codon usage have not altered the hydrophilic characteristics of most amino acid positions, then it is quite unlikely that the relatively few strongly constrained residues have been affected. This suggestion concurs with the general
Cytochrome b/ATPase Genes of Honeybee Mitochondrial DNA
481
Amino Acid Position FIG. 5.--Identity values between inferred Apis and Xenopus cytochrome b amino acid sequences, according to numbering sequence derived from fig. 4. Comparisons were made using a moving window of 11 amino acids. The solid-line curve denotes exact identity, whereas the dotted-line curve denotes functional identity according to the criteria used for fig. 3. The horizontal lines give the random expectations for exact and functional identity.
impression that selection at the organism level (or higher) overwhelms contrary DNAlevel selective processes (Crozier, accepted). Sequence Availability
The GenBank accession numbers are M87065 for the ATPase genes and M87052 for the cytochrome b gene. Acknowledgments We thank B. Crespi, S. Edwards, W. M. Fitch, T. Kocher, S. Koulianos, M. Kreitman, R. Kusmierski, and two anonymous reviewers for constructive comments on various drafts of the manuscript, and we thank the Australian Research Council, the University of New South Wales, and La Trobe University for grants supporting this work. LITERATURE
CITED
ALMAGOR, H. 1985. Nucleotide
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editor
Received March 12, 199 1; revision received October 24, 199 1 Accepted November 22, 199 1
