BIOCHIMIE, 1979, 61, 701-704.
Chemical basis of the electrophoretic variation observed at the alcohol dehydrogenase locus of Drosophila melanogaster. Anastasios D. RETZIOS and David R. THATCHER.
Department of Molecular Biology, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, U.K.
Summary.
Materials and Methods.
The amino acid substitution responsible for the different electrophoretic mobility of the ADH s alleloenzyme and the ADH ~ alleloenzyme of the alcohol dehydroqenase from a Nigerian population of Drosophila melanoqaster has been established as 1ysine (ADH s) for threonine (ADHq. This result is discussed with reference to the charge state model of electrophoretic variation, in conjunction with other know substitutions at this locus. It is concluded that electrophoretic methods should be capable of distin~tishinq m a n y alleloenzymes which have identical isoelectric points without recourse to explanations involvinq conformational variability.
ADHS and ADHf flies were obtained from the collection of Professor B. Clarke (University of Nottingham)
Introduction. The t e c h n i q u e of gel electrophoresis has revealed large a m o u n t s of allelic v a r i a t i o n in the structural genes of most species [1, 2]. These quantities of allelic v a r i a t i o n are h o w e v e r lower limits to the actual extent of genetic v a r i a b i l i t y w i t h i n a population because the electrophoretic method is incapable of resolving v a r i a t i o n w h i c h does not effect net charge. It is therefore of interest to investigate the under,lying basis for the observed electrophoretic v a r i a t i o n and define the limitations of the method e x p e r i m e n t a l l y . The alcohol d e h y d r o g e n a s e of Drosophila melanogaster is ideally suited for this purpose as the enzyme is a) expressed at a high level in both adults and larvae b) is easily purified [3] a n d has been well characterized c h e m i c a l l y [4, 5] c) the structure of the gene is b e i n g investigated both by i n t r a g e n i c r e c o m b i n a t i o n of m u t a n t s [6, 7] a n d by c l o n i n g methods [8].
having originally been isolated from the Nigerian, Kaduna population collected by Professor A. Roberfson (University of Edinburgh). The flies were cultured and grown in bulk ; extracted and the enzyme purified as described elsewhere [3]. Standard sequencing methods were used throughout and have been fully described previously [9, 10]. A detailed description of the properties and peptide evidence for the proposed sequence refered to in the results section will be summarized with publication of the complete sequence of the enzyme.
Results and Discussion. T r y p t i c , t h e r m o l y t i c and c h y m o t r y p t i c peptides derived from digests of alcohol dehydrogenases from ADHs (!Kaduna) and ADHf (Kaduna) suggested the sequence presented in figure 1. I n ADH s, lysine is substituted for file t h r e o n i n e o f ADH t p r o d u c i n g a distinctive low m o l e c u l a r weight t r y p t i c peptide ; Thr-Thr-Leu-Val-His-Lys. The c o r r e s p o n d i n g peptide in ADH~ is highly insoluble a n d r e m a i n s at the origin d u r i n g c o n v e n t i o n a l paper peptide m a p p i n g procedures. The ADH s t r y p t i c peptide is very soluble and is easily identified on peptide m a p p i n g . It has a m o b i l i t y of 0.6 on p a p e r electrophoresis at pH 6.5 a n d in a one d i m e n s i o n a l s e p a r a t i o n is only c o n t a m i n a t e d w i t h the peptide : Gln-Leu-Leu-Lys-Arg. These peptides can be separated by f u r t h e r p a p e r electrophoresis at pH 3.5. This ADH s peptide has also been identified in several isolates of a D. melanogaster p o p u l a t i o n from the Napa Valley region of California [11]. At least 90 per cent of the r e m a i n i n g sequence of ADH s alcohol d e h y d r o g e n a s e is i d e n t i c a l to that of ADHf [5] and it is possible that this substitution is responsible for the observed electrophoretic, catalytic a n d c o n f o r m a t i o n a l differences
702
A . D. R e t z i o s a n d D. R. T h a t c h e r .
Thr-Thr-Leu-Val-His-LYS
ADH s
Leu-A~a-Pr~-I~e-Thr-G~-Va~-Thr-A~a-Tyr-Thr-Va~-Asn-Pr~-G~-I~e-Thr-Arg-Thr-Thr-Leu-Val-His-THR-Phe~-'-, -'-'t "--'r
--~. - - - - r
---,,..---,
--p
---~
--~ |4
4
_~P
JD 4
(ser, Asn,)
Leu-Asp-Val-Glu-Pro-Glu-Val-Ala-Glu-Lys
C FIG. 1.
~4
)C
The aminoaeid sequence around the ADFI/ADHs substitution between [lies isolated [rom a Nigerian population o[ D. melanogaster ( ~ l ~ l ~ ) represents tryptie peptides, ( r l) ehymotryptie peptides -
-
and ( ( - - ) )
b e t w e e n ADHf elsewhere).
thermolytie peptides. (
and
A.DHs
(to
be
7 ) denotes residues identified by the dansyl-Edman procedure.
discussed
C u r r e n t t h e o r i e s a c c o u n t i n g for the origin and d i s t r i b u t i o n of e l e c t r o p h o r e t i e allelic v a r i a t i o n by r a n d o m genetic drift [12, 13, 141 assume that elect r o p h o r e t i c m e t h o d s only detect c h a n g e s in unit net charge a n d that the e l e c t r o p h o r e t i c allele, in a p o p u l a t i o n sense, r e p r e s e n t s an e x t r e m e l y diverse class of sequences w h i c h h a p p e n to have the same net charge a n d are b e t t e r d e s c r i b e d as e l e c t r o m o r p h s . E l e c t r o m o r p h s , it is claimed, could consist of a c o m p l e x v a r i e t y of e l e c t r o p h o r e t i c a l l y i n d i s t i n g u i s h a b l e alleles w h i c h w o u l d all m i g r a t e at the same v e l o c i t y in a dilute gel system. Different charge classes w o u l d m i g r a t e at p r o p o r t i o n a tely different rates d e p e n d i n g on t h e i r average net charge. As these e l e c t r o m o r p h s differ by a single unit charge mutatio,ns w h i c h r e s u l t in a change in charge of a p a r t i c u l a r surface r e s i d u e w o u l d result in the n e w allele j o i n i n g an a d j a c e n t e l e c t r o m o r p h class d u r i n g e l e c t r o p h o r e s i s . The t h e o r y p r e d i c t s a s y m m e t r i c a l d i s t r i b u t i o n of frequencies for each charge class a r o u n d a p r e d o m i n a n t e l e c t r o m o r p h frequency, i.e. charge state 0 is f o u n d in the h i g h e s t f r e q u e n c y , state + 1 and--1 lower, + 2 a n d - - 2 l o w e r f r e q u e n c i e s still. By assuming v a r i o u s levels of d e t e r m i n i s t i c selection against c h a r g e changes from this p r e d o m i n a n t e l e c t r o m o r p h ( c h a r g e state 0), the m o d e l can a c c o u n t for the observed, but not p r e d i c t e d i n d e p e n d e n c e of e l e c t r o p h o r e t i c allele n u m b e r over effect p o p u l a t i o n size [15]. BIOCHIMIE, 1979, 61, n ° 5-6.
J o h n s o n [16] has c r i t i c i s e d this t h e o r y and argued that it is u n l i k e l y that a n y two c h a r g e changes w i l l result in i d e n t i c a l c h a n g e s in net charge. This is because the p K a of the i o n i s i n g side c h a i n s of a n u m b e r of p r o t e i n s have been f o u n d to be e x t r e m e l y v a r i a b l e a n d also that a large prop o r t i o n of the charge substitutions o c c u r r i n g in p r o t e i n s w i l l not result in unit charge changes at the p H values c o m m o n l y e m p l o y e d in e l e c t r o p h o rests. F i g u r e 2 s u m m a r i s e s the sequence i n f o r m a t i o n available on 6 e l e c t r o p h o r e t i c alleles of D. melanogaster alcohol d e h y d r o g e n a s e . Neutral amino a c i d residues, p r e s u m a b l y on the surface of the enzyme of ADHf have m u t a t e d to a c i d i c r e s i d u e s in ADH uf, and ADH n-ll a n d ADH0. The m e m b e r s of this new e l e c t r o m o r p h class are e l e c t r o p h o r e t i c a l l y i n d i s t i n g u i s h a b l e although the substitutions o c c u r at different sites in the enzyme molecule and i n c l u d e the a c q u i s i t i o n of both a s p a r t i c and glutamic a c i d residues. In c o n t r a s t ADH s differs from ADHf b y the substitution of a lysine r e s i d u e for a t h r e o n i n e causing a d e c r e a s e in net c h a r g e w i t h a c o n c o m i t a n t r e d u c t i o n in e l e c t r o p h o r e t i c m o b i l i t y at p H 8.5. At this pH, h o w e v e r , the n e w lysine r e s i d u e is o n l y p a r t i a l l y i o n i s e d w h i c h results in a f r a c t i o nal r e d u c t i o n in e l e c t r o p h o r e t i c mobility. In other w o r d s ADHf does not differ f r o m ADH s b y a unit c h a r g e c h a n g e at p H 8.5. I n a n o t h e r new v a r i a n t a unit m o b i l i t y change is o b s e r v e d : in ADH n
Alcohol dehydrogenase alleloenzymes.
703 ADH
ADH
s
ADH
~
uf
%'~x',,,A~/o ' ~
ADHd
ADHfl Charge State
41
-I
0
Fro. 2. - - K n o w n amino acid s u b s t i l u t i o n s at the alcohol dehydrogenase locus. ADHuf (La Mancha) (R. G. Camfield and D. R. Thatcher (1977) Biochem. Sac. Trans., 5, 271-272. ADHd (Schwartz and H. J6rnvall (1976) Eur. J. Bioehem., 68, 159-168. ADHn-n and ADHfl (David) (Retzios, Thatcher, Clarke and David, unpublished). ( D a v i d , [17]), g l u t a n f i c a c i d r e s i d u e r e p l a c e s a n e u t r a l a l a n i n e r e s i d u e f o u n d in ADHs. T h u s A D H f a n d A D H n c a n be r e s o l v e d b y e l e c t r o p h o r e _ s i s at h i g h p H , A D H fl b e i n g s l i g h t l y m o r e e l e c t r o negative.
It a p p e a r s f r o m t h e s e p r e l i m i n a r y data, t h e r e fore, t h a t e l e c t r o p h o r e s i s is c a p a b l e of d i s t i n g u i s h i n g at l e a s t t w o m o d e s of c h a r g e s u b s t i t u t i o n . S i m i l a r c h a r g e s u b s t i t u t i o n s on t h e s u r f a c e of an e n z y m e (i.e. n e u t r a l ~- b a s i c OR n e u t r a l ~ actl
-nr&+l
Chemical basis of the electrophoretic variation observed at the alcohol dehydrogenase locus of Drosophila melanogaster. Anastasios D. RETZIOS and David R. THATCHER.
Department of Molecular Biology, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, U.K.
Summary.
Materials and Methods.
The amino acid substitution responsible for the different electrophoretic mobility of the ADH s alleloenzyme and the ADH ~ alleloenzyme of the alcohol dehydroqenase from a Nigerian population of Drosophila melanoqaster has been established as 1ysine (ADH s) for threonine (ADHq. This result is discussed with reference to the charge state model of electrophoretic variation, in conjunction with other know substitutions at this locus. It is concluded that electrophoretic methods should be capable of distin~tishinq m a n y alleloenzymes which have identical isoelectric points without recourse to explanations involvinq conformational variability.
ADHS and ADHf flies were obtained from the collection of Professor B. Clarke (University of Nottingham)
Introduction. The t e c h n i q u e of gel electrophoresis has revealed large a m o u n t s of allelic v a r i a t i o n in the structural genes of most species [1, 2]. These quantities of allelic v a r i a t i o n are h o w e v e r lower limits to the actual extent of genetic v a r i a b i l i t y w i t h i n a population because the electrophoretic method is incapable of resolving v a r i a t i o n w h i c h does not effect net charge. It is therefore of interest to investigate the under,lying basis for the observed electrophoretic v a r i a t i o n and define the limitations of the method e x p e r i m e n t a l l y . The alcohol d e h y d r o g e n a s e of Drosophila melanogaster is ideally suited for this purpose as the enzyme is a) expressed at a high level in both adults and larvae b) is easily purified [3] a n d has been well characterized c h e m i c a l l y [4, 5] c) the structure of the gene is b e i n g investigated both by i n t r a g e n i c r e c o m b i n a t i o n of m u t a n t s [6, 7] a n d by c l o n i n g methods [8].
having originally been isolated from the Nigerian, Kaduna population collected by Professor A. Roberfson (University of Edinburgh). The flies were cultured and grown in bulk ; extracted and the enzyme purified as described elsewhere [3]. Standard sequencing methods were used throughout and have been fully described previously [9, 10]. A detailed description of the properties and peptide evidence for the proposed sequence refered to in the results section will be summarized with publication of the complete sequence of the enzyme.
Results and Discussion. T r y p t i c , t h e r m o l y t i c and c h y m o t r y p t i c peptides derived from digests of alcohol dehydrogenases from ADHs (!Kaduna) and ADHf (Kaduna) suggested the sequence presented in figure 1. I n ADH s, lysine is substituted for file t h r e o n i n e o f ADH t p r o d u c i n g a distinctive low m o l e c u l a r weight t r y p t i c peptide ; Thr-Thr-Leu-Val-His-Lys. The c o r r e s p o n d i n g peptide in ADH~ is highly insoluble a n d r e m a i n s at the origin d u r i n g c o n v e n t i o n a l paper peptide m a p p i n g procedures. The ADH s t r y p t i c peptide is very soluble and is easily identified on peptide m a p p i n g . It has a m o b i l i t y of 0.6 on p a p e r electrophoresis at pH 6.5 a n d in a one d i m e n s i o n a l s e p a r a t i o n is only c o n t a m i n a t e d w i t h the peptide : Gln-Leu-Leu-Lys-Arg. These peptides can be separated by f u r t h e r p a p e r electrophoresis at pH 3.5. This ADH s peptide has also been identified in several isolates of a D. melanogaster p o p u l a t i o n from the Napa Valley region of California [11]. At least 90 per cent of the r e m a i n i n g sequence of ADH s alcohol d e h y d r o g e n a s e is i d e n t i c a l to that of ADHf [5] and it is possible that this substitution is responsible for the observed electrophoretic, catalytic a n d c o n f o r m a t i o n a l differences
702
A . D. R e t z i o s a n d D. R. T h a t c h e r .
Thr-Thr-Leu-Val-His-LYS
ADH s
Leu-A~a-Pr~-I~e-Thr-G~-Va~-Thr-A~a-Tyr-Thr-Va~-Asn-Pr~-G~-I~e-Thr-Arg-Thr-Thr-Leu-Val-His-THR-Phe~-'-, -'-'t "--'r
--~. - - - - r
---,,..---,
--p
---~
--~ |4
4
_~P
JD 4
(ser, Asn,)
Leu-Asp-Val-Glu-Pro-Glu-Val-Ala-Glu-Lys
C FIG. 1.
~4
)C
The aminoaeid sequence around the ADFI/ADHs substitution between [lies isolated [rom a Nigerian population o[ D. melanogaster ( ~ l ~ l ~ ) represents tryptie peptides, ( r l) ehymotryptie peptides -
-
and ( ( - - ) )
b e t w e e n ADHf elsewhere).
thermolytie peptides. (
and
A.DHs
(to
be
7 ) denotes residues identified by the dansyl-Edman procedure.
discussed
C u r r e n t t h e o r i e s a c c o u n t i n g for the origin and d i s t r i b u t i o n of e l e c t r o p h o r e t i e allelic v a r i a t i o n by r a n d o m genetic drift [12, 13, 141 assume that elect r o p h o r e t i c m e t h o d s only detect c h a n g e s in unit net charge a n d that the e l e c t r o p h o r e t i c allele, in a p o p u l a t i o n sense, r e p r e s e n t s an e x t r e m e l y diverse class of sequences w h i c h h a p p e n to have the same net charge a n d are b e t t e r d e s c r i b e d as e l e c t r o m o r p h s . E l e c t r o m o r p h s , it is claimed, could consist of a c o m p l e x v a r i e t y of e l e c t r o p h o r e t i c a l l y i n d i s t i n g u i s h a b l e alleles w h i c h w o u l d all m i g r a t e at the same v e l o c i t y in a dilute gel system. Different charge classes w o u l d m i g r a t e at p r o p o r t i o n a tely different rates d e p e n d i n g on t h e i r average net charge. As these e l e c t r o m o r p h s differ by a single unit charge mutatio,ns w h i c h r e s u l t in a change in charge of a p a r t i c u l a r surface r e s i d u e w o u l d result in the n e w allele j o i n i n g an a d j a c e n t e l e c t r o m o r p h class d u r i n g e l e c t r o p h o r e s i s . The t h e o r y p r e d i c t s a s y m m e t r i c a l d i s t r i b u t i o n of frequencies for each charge class a r o u n d a p r e d o m i n a n t e l e c t r o m o r p h frequency, i.e. charge state 0 is f o u n d in the h i g h e s t f r e q u e n c y , state + 1 and--1 lower, + 2 a n d - - 2 l o w e r f r e q u e n c i e s still. By assuming v a r i o u s levels of d e t e r m i n i s t i c selection against c h a r g e changes from this p r e d o m i n a n t e l e c t r o m o r p h ( c h a r g e state 0), the m o d e l can a c c o u n t for the observed, but not p r e d i c t e d i n d e p e n d e n c e of e l e c t r o p h o r e t i c allele n u m b e r over effect p o p u l a t i o n size [15]. BIOCHIMIE, 1979, 61, n ° 5-6.
J o h n s o n [16] has c r i t i c i s e d this t h e o r y and argued that it is u n l i k e l y that a n y two c h a r g e changes w i l l result in i d e n t i c a l c h a n g e s in net charge. This is because the p K a of the i o n i s i n g side c h a i n s of a n u m b e r of p r o t e i n s have been f o u n d to be e x t r e m e l y v a r i a b l e a n d also that a large prop o r t i o n of the charge substitutions o c c u r r i n g in p r o t e i n s w i l l not result in unit charge changes at the p H values c o m m o n l y e m p l o y e d in e l e c t r o p h o rests. F i g u r e 2 s u m m a r i s e s the sequence i n f o r m a t i o n available on 6 e l e c t r o p h o r e t i c alleles of D. melanogaster alcohol d e h y d r o g e n a s e . Neutral amino a c i d residues, p r e s u m a b l y on the surface of the enzyme of ADHf have m u t a t e d to a c i d i c r e s i d u e s in ADH uf, and ADH n-ll a n d ADH0. The m e m b e r s of this new e l e c t r o m o r p h class are e l e c t r o p h o r e t i c a l l y i n d i s t i n g u i s h a b l e although the substitutions o c c u r at different sites in the enzyme molecule and i n c l u d e the a c q u i s i t i o n of both a s p a r t i c and glutamic a c i d residues. In c o n t r a s t ADH s differs from ADHf b y the substitution of a lysine r e s i d u e for a t h r e o n i n e causing a d e c r e a s e in net c h a r g e w i t h a c o n c o m i t a n t r e d u c t i o n in e l e c t r o p h o r e t i c m o b i l i t y at p H 8.5. At this pH, h o w e v e r , the n e w lysine r e s i d u e is o n l y p a r t i a l l y i o n i s e d w h i c h results in a f r a c t i o nal r e d u c t i o n in e l e c t r o p h o r e t i c mobility. In other w o r d s ADHf does not differ f r o m ADH s b y a unit c h a r g e c h a n g e at p H 8.5. I n a n o t h e r new v a r i a n t a unit m o b i l i t y change is o b s e r v e d : in ADH n
Alcohol dehydrogenase alleloenzymes.
703 ADH
ADH
s
ADH
~
uf
%'~x',,,A~/o ' ~
ADHd
ADHfl Charge State
41
-I
0
Fro. 2. - - K n o w n amino acid s u b s t i l u t i o n s at the alcohol dehydrogenase locus. ADHuf (La Mancha) (R. G. Camfield and D. R. Thatcher (1977) Biochem. Sac. Trans., 5, 271-272. ADHd (Schwartz and H. J6rnvall (1976) Eur. J. Bioehem., 68, 159-168. ADHn-n and ADHfl (David) (Retzios, Thatcher, Clarke and David, unpublished). ( D a v i d , [17]), g l u t a n f i c a c i d r e s i d u e r e p l a c e s a n e u t r a l a l a n i n e r e s i d u e f o u n d in ADHs. T h u s A D H f a n d A D H n c a n be r e s o l v e d b y e l e c t r o p h o r e _ s i s at h i g h p H , A D H fl b e i n g s l i g h t l y m o r e e l e c t r o negative.
It a p p e a r s f r o m t h e s e p r e l i m i n a r y data, t h e r e fore, t h a t e l e c t r o p h o r e s i s is c a p a b l e of d i s t i n g u i s h i n g at l e a s t t w o m o d e s of c h a r g e s u b s t i t u t i o n . S i m i l a r c h a r g e s u b s t i t u t i o n s on t h e s u r f a c e of an e n z y m e (i.e. n e u t r a l ~- b a s i c OR n e u t r a l ~ actl
-nr&+l
