Amino acid differences between major and minor hemoglobins from lemmings (Lemmus and Dicrostonyx)

Amino acid differences between major and minor hemoglobins from lemmings (Lemmus and Dicrostonyx)

Comp. Biochem, Physiol., Vol. 6111, pp. 521 to 532 © Perclamon Pre.ss Ltd 1978. Printed in Great Britain 0305-0491/78/1115-0521502.00/0 A M I N O AC...

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Comp. Biochem, Physiol., Vol. 6111, pp. 521 to 532 © Perclamon Pre.ss Ltd 1978. Printed in Great Britain

0305-0491/78/1115-0521502.00/0

A M I N O ACID D I F F E R E N C E S B E T W E E N MAJOR AND MINOR HEMOGLOBINS FROM LEMMINGS (LEMMUS A N D DICROSTONYX)* L. K. DUFFY,~ C. T. GENAUX and P. R. MORRISON Institute of Arctic Biology, University of Alaska, Fairbanks, AK 99701, U.S.A. (Received 22 March 1978)

Abstract--1. Dual hemoglobins were isolated from both Lemmus and Discrostonyx, two genera of microtine rodents of the tribe Lemmini. 2. The dual hemoglobins result from dual or-chains and the charge difference Asp----, Ala at position ct5. 3. The fl-chains of Dicrostonyx and Lemmus hemoglobins differ in charge by Ala--,Asp at fl125. 4. Cladograms are presented for the hemoglobin chains of microtine rodents.

INTRODUCTION

The evolution of proteins and their use as measures of genetic divergence continue to be topics of active discussion (Zuckerkandl, 1976a, b; Holmquist et al., 1976). The various hypotheses a b o u t rates of protein evolution have been refined as more and varied taxa have been studied. A l t h o u g h rigorous chemical sequences are needed, the value o f alignments from tryptic peptide compositions has been d e m o n s t r a t e d (Ambler, 1974). The present study characterizes the tryptic peptides from the hemoglobins of two lemming genera to give some insight into protein evolution in this group that is of interest to b o t h theoreticians and taxonomists. Van Valen (1974) pointed out that m a n y microtine rodents have had extremely rapid rates of evolution and that such evolution could be sought in the proteins as well as in morphology. Although most authors accept the Lemmini as a natural group, Dicrostonyx shows such distinctive morphological differences from other microtines, that it has been proposed as an independent tribe within the Microtinae (Hooper & Hart, 1962). A comparison of hemoglobins provides an additional source of phyIogenetic information on b o t h Dicrostonyx and Lemmus and allows a more direct way of relating Dicrostonyx to other microtines. Little information has been reported on the hemoglobins of these two microtine genera. Both Dicrostonyx and Lemmus have two electrophoretic c o m p o n e n t s (Johnson, 1974), and solubility studies on the hemoglobins have been recently reported by Stratton et al. (1977). MATERIALS AND METHODS Animals

Hemoglobins were obtained from Lemmus sibiricus alascensis (Barrow, AK), Dicrostonyx torquatus rubricatus (Prudhoe Bay, AK) and D. t. stevensoni (Umnak Island, * A preliminary account of part of this study was presented at the Rocky Mountain Regional Meeting of the American Chemical Society, 17-19 June 1976. t Present address: Department of Chemistry, Boston University, Boston, MA 02215, U.S.A.

AK). Biogeographical and taxonomic descriptions for both genera have been presented by Rausch & Rausch (1972, 1975). With the exception of the major component from Lemmus (single animal), the hemoglobins were pooled from three to five animals. Methods Cellulose acetate strip electrophoresis was carried out using a Beckman Microzone apparatus at room temperature. Hemolysates were compared using a Tris-EDTAborate (TEB) buffer, pH9.1 (16.1g/l Tris and 1.56g/1 disodium EDTA, titrated to desired pH with boric acid). Strips were stained with amido black and scanned at 555 nm with an RFT Transidyne General scanning densitometer to determine the relative amounts of major and minor components. Separation and identification of the polypeptide chains were performed as reported by Ueda & Schneider (1969) with TEB buffer, pH 8.6, 6 M urea and 0.05 M 2-mercaptoethanol. Globin was precipitated from pooled hemolysate at - 2 0 C in acid-acetone essentially as described by Clegg et al. (1968). Separation of ct- and fl-globin chains was effected on columns of carboxymethyl-cellulose, CM52 (Whatman Microgranular). Freshly precipitated globin was dissolved in 1 ml of a solution of deionized 8 M urea, which was also 0.005 M in Na2HPO4 and 0.05 M in mercaptoethanol (starting buffer). After globin solution was loaded on the column, the starting buffer was allowed to flow for 1.5 hr at a rate of approx 14ml/hr. A gradient was established using 150 ml of the starting buffer ahead of 150ml of eluting buffer: deionized 8 M urea, 0.045 M in Na2HPO4, 0.075 M in mercaptoethanol. The fl-chain was eluted ahead of the ~t-chain. Fractions of approx 5 ml were collected, and the protein components, identified by their absorption at 280 nm, were chromatographed in 0.1~o formic acid on a Sephadex G-25 column and were recovered by lyophilization. Tryptic digestion of S-aminoethylated globin derivatives, peptide mapping and amino acid analysis have been described (Genaux & Morrison, 1973a, b). Cyanogen bromide treatment of the fl-chain of D. rubricatus was performed by adding 20mg of CNBr to 20 mg of fl-chain in 2 ml of 70~o formic acid. The reactivial (Pierce) was flushed with N 2 for 10 min and placed in the dark at 5°C for 48 hr. The mixture was diluted and lyophilized. The separated globin fragments were subsequently digested with trypsin, and the tryptic fragments were mapped. Carboxypeptidase digestion was performed as previously described (Duffy et al., 1976).

521

522

L.K. DUFFY, C. T. GENAUX and P. R. MORRISON

The general validity of the stoichiometry deduced for the tryptic peptides was established by reference to experimental data obtained by using the same methods with hemoglobins from man and from other microtine species (Genaux & Morrison, 1973a, b). Probable sequences were obtained by alignment of the data with the chemically determined sequences from Hb of white mouse (Mus) and with the Microtus sequences which have been deduced from the results accumulated to data for a number of species (Genaux et al., 1976). Where there were two or more substitutions within a peptide, assignments to probable positions followed the chemical properties, e.g. Glu to Asp, Gly to Ala. Where there were options for a substitution, assignment was made to the more labile position as seen in a range of mammalian hemoglobins (Dayhoff, 1976). Accordingly, these alignments (Figs. 3 and 5) and the cladograms based upon them (Figs. 6 and 7) are tentative and may be modified as actual sequences are determined for these species or as hemoglobins from additional species are studied.

RESULTS

Electrophoretic comparison Cellulose-acetate electrophoresis of individual hemolysates from different Dicrostonyx and Lemmus always showed the presence of two hemoglobin components. Figure l(a) compares the electrophoretic patterns of the hemolysates from Lemmus and Dicrostonyx. It can be seen that the hemoglobins of the species differ in their mobilities, with the Dicrostonyx c o m p o n e n t s moving faster at pH 8.9. In b o t h species the major c o m p o n e n t represents 60-70°,,~, of the total hemoglobin. The separation of the hemoglobins into their ~- and fl-chains by urea cellulose-acetate electrophoresis is shown in Fig. l(b). As would be expected from the presence of two hemoglobin components, three polypeptide chains were observed in approximately a 3:2:1 ratio. The two faster migrating b a n d s



U

m

m

I

m

m

~ m

, m

m

,m-Ilmm

I

I

m

m

b 8

Fig. l. Electrophoretic patterns of Dicrostonyx and Lemmus hemoglobins. (a) Hemoglobins at pH 8.9: position 1, Lemmus; position 2, Dicrostonyx. (b) Chains in urea-cellulose acetate at pH 8.9: position 3, D. r. ~f-chain; position 4, D. r. orS-chain; position 5, Lemmus chains; position 6, Dicrostonyx chains. Lemmus lemmus had an identical pattern to Lemmus sibiricus and D. stevensoni had a pattern identical to D. rubricatus.

i

Amino acid differences between major and minor hemoglobins Structure of fl-chain

,0

©

Figure 2 compares the "map" locations of fl-chain peptides from Dicrostonyx with those from Lemmus. The only significant difference in electrophoretic and chromatographic mobility was seen in peptide flTl3 which moved farther toward the anode in Dicrostonyx than in Lemmus. An additional negative charge in flTl3 in Dicrostonyx accounts for the greater mobility of its hemoglobin components as compared to those from Lemmus. By means of the compositions presented in Table l, the tryptic fl-peptides of Dicrostonyx and Lemmus hemoglobins have been aligned (Fig. 3) with the homologous peptides of Microtus hemoglobin and Mus hemoglobin (Popp, 1973) and are compared with the recent results of Garrick et al. (1977) for rat. There were no differences observed between Lemmus and Dicrostonyx in peptides fiT1, fiT2, fiT3, fiT7, fiT8, flTll and fiT15. Peptide fiT4. Dicrostonyx showed a glycine vs valine difference in fiT4. This peptide contacts the or-chain and the two valines (fl33, fl34) have been found to be invariant in mammals until recently Runkel et al. (1974) reported a V a l - * Ile substitution in Canids, and Morrison et al. (1977) a similar substitution in Calomys callosus (Rodentia, Cricetinae, Hesperomyini).

/9- chain

0 ~5

90

14/ 8-90

III

/-,, 9b

0

15

~)

c5 ©_°°,o I f, L.,c2:P +

,,

523

7 %.3,8_

Joblrigin--pH6.4

Fig. 2. Composite peptide map of Lemmus and Dicrostonyx fl-chains. Position of fiT13 should be noted; clear spots indicate that the peptides appear in similar locations on both Lemmus and Dicrostonyx maps. of both genera were identified as or-chains after CMC chromatography and peptide mapping. The slow migrating third band, accounting for 50% of the chains, was identified as the ~6-chain. The difference in mobilities of the fl-chains between the two species accounts for the electrophoretic differences in Fig. l(a).

Species

BTI MUS D.T. L.S. I.._.CC M RAT

N

D.T. L.S. MIC RAT

F. IF.

MUS D.T. LS. MIC RAT

~T4

A. A.A. D

P

P W T Q R' (40)

A. A.

]~"1"5 BT6 /3T7 /~T8 /3T9 FG D L S S A S A I M G N A K ' V K ' A HG K ' K ' V I T A F S DG L N H L (50) (60) (70) Z .H. A__. V. Q. H. G. K. A.H.L. T.G S V __M

'Y F D S

D.T. L.S. MIC RAT

/~T3

I. I.

MUS

MUS

/~T2

IVH L T D A E K ' A A V S G L W G K ' V N A D E V G G EA LG R'L L V V Y (I 0 ) (20) (30) I. A. A. G.

F.Z.H

~

~' L

Q. Q

]

H N

P

G.

A. N

DN L K ' (80)

K. K K

/~TJ0 /3TII /~TI2 'GT F A S L S E L H C D K ' L H V D P EN F R ' L L G N M I V I V LG HH L G K' (90) (100) (llOI (120)

S.

K.

[IIIIIIIIIIIIIIIIIIIIIIIIII

]

K. [/////////////////////////////////////] S.

8.

H /~TI3 ,~TI4 ~TP5 'D F T PA AQ A A F Q K ' V V A G V A A A L A H K'YH' (130) (140) D. S. G.S. S. G.T. S. S. E S_-=X. S

Fig. 3. Amino acid sequence alignment of fl-peptides. Inferred amino acid replacements are indicated relative to the sequence for fl-chain of hemoglobin from white mouse (Mus: Popp, 1973) and from rat (Garrick et al., 1977). A minor, probably allelic, component with A, N, T, T at the underlined positions in the rat sequence was also identified by Garrick et al. (1977). Blank spaces indicate identity with Mus. The single letter amino acid code is given as in Dayhoff (1972). The fir5 alignment for D.T. represents only D. t. rubricatus and is based on analysis of CNBr fragments (see text).

No of residues: Posit ions :

AE-CYS

lrp

]yr

Phe

Leu

lie

Mel

Val

Ala

Gly

Pro

GIx

Set

Thr

Asx

Atg

His

I.ys

9

~ 17

8

[1] + Ill

129 072 1.00 1(14 tl.86 1.00

2.33 2.56 [2] 1.85 ] 147 [2] ] 142 [2] 1.64 [2]

i '~

1.20 105 I>06

I 33 [I] 135 [I] 091 0.76 0.60 [ I ] 67 [ ]

I./1t/ 0.88 119

1.10 0.94 11.76

1.10 100 100

1.00 130 0.86 0.73 0.60 [ 1] 0.8(1

LI6 1.12 1.17 073 I II 1 15

fiT2

flTl

13 18 ~0

095 I 03 0 94

10 31 40

0.18 [1] 0.2711] 0.4l [1] + fil NA"

1.63 [21 1184 [ 1 65 ]2]

070 I1) 1 235 [21

1.50 [11 107 105 0.86 0.83 1.(X) 093 073 (0 27)

116 1.30 0.86

1.00 100 0.84

[fl-4

acid compositions

1.87 1.70 2 18 365 [4] 3.62 [4] 3.99 1.40 [2] 1.54 [2] 2.11/

1.08 1 /)2 111

1.00 0.84 0.85 1.98 2.23 2.28

fiT3

1. A m i n o

21 41 61

] 1.22 [2],"

3 00

031)

177

4.33

202

(0.39)

1.1 ~

1.34

0.68 [1]

2,14

1.70

0.92

fiT5 6~.

of tryptic

4 62 65

1 (14 11.93 I 33 0.71 0.73 1.00

0.93 0.95 11.96 1.00

1.00 1.00

fiT7

fl-peptides

10 67 76

028 [1] /).4311 / o.61 I l l 106 0.97 11)4 103 078 0 82

207 2 24 238[2] 124 102 1.22 0.33 ]1] 0.40 [1] (.48 [ ]

087

0,93 0,91 092

II 66 76

085 107

120 I 13

051 [1] /I.71

068 [I] 061 I l l

1.08 1.18

2 34 2 14

091 1.04

1130 1/.96

11,811 i184

fiTS 9a

6 77 82

227 I 711 2 12

1.74 2.10 2,34 [2]

1.00 0.80 0.85 0.40 [I] 0.25 [ ]

1.00

flTgb

13 83 95

031 [ I j 025 [ t ] (141 [I]

1.81 226 232 0.70 113 I 12

0.93 116 116511] 090 151 [1] 1.48 [11

1.24

1.10 3.12 3.07 2.08 0.93 088

1.15 0.97 1.16

1.00 1.00 0.93 1.08 1.16

1.00

flTI0

9 96 1 0 4

0.84 1.20 0.76 /I.91 090 0.88

(075) 1077) 105 1.20 1116

0.95 1.27 1.03 (0.16) (038) (0.47i

I 20

(0.33) 10.231 105 11.91

1013t 1.74 1.85 1.70 10.16) 10331

1.10 0.90 0.82 0.94 0.94

1.00

flTII

Dicrostonyx and Lemmus hemoglobins*

0.61 [1] 0.68 [1] 0.54 [1]

1.00 1,00

fiTga

from

12 121 132

01631 206 I 79 I 78

(038) 223 213 ~11

1.79 1.55 [2] .0( 0.83 1.14 100 1.04 1.14 1.06 1.86 185 2 04 143 [ I ] 1.19 1.22

(0.20i

1.10 1130

1.00

flTI3

12 133 144

III 109 125

2.70 296 304 2.10 [3] 225 [31 298

1.92

2.06 2.29

0.97 141 [1] 0.62 [1] (0.261

10.601

1.25 1.311 0.96 1.00

1.(30 1.00

1/T14

2 145 146

052 LU 051 [I] 5 [ l

1.00 1.00 1,17

flTI5

++T h e c o m p o s i t i o n o f t h i s p e p t i d e f r o m D. t. rubricatus, b a s e d o n w e r e n o t o b t a i n e d f o r e i t h e r D. t. rubricatus o r D. t. sterensoni. Poor yield resulted from the terminal Phe-Phe (Duffv & Genaux.

19771.

CNBr

peptides

(flT5a

and

f l T 5 b + 6J is g i v e n

in the

text.

Satisfactory

analyses

for fiT5

* An integral number o f r e s i d u e s is a s s u m e d when the data shown are within + 0 . 3 r e s i d u e s o f 1, 2, 3, e t c . I n o t h e r c a s e s t h e i n t e g r a l n u m b e r inferred is s h o w n i n b r a c k e t s [ ] f o l l o w i n g t h e d a t a . D a t a i n p a r e n t h e s i s () a r e a t t r i b u t e d t o s o u r c e s o u t s i d e t h e g i v e n p e p t i d e a n d a r e e x c l u d e d f r o m t h e s t o i c h i o m e t r y . Tryptophan was detected in intact peptides by dipping the paper in a fresh solution o f 1°0 p - d i m e t h y l a m i n o b e n z a l d e h y d e in acetone cone. HCI, 9:1 by volume. Cysteine was determined as S-(aminoethyl)-cysteine. M---missing p e p t i d e . A b a r , I, t o left o f d a t a i n d i c a t e s N - t e r m i n a l residues, ffI'8 = K. f Tryptophan not analyzed.

Dll Dts b, DIr Dis Ls

Dr) Dis Ls Dtr [)is

Ls

Dtr Dis Ls Dtr Dt s Ls Dtr Dis Ls Dir Dts Ls Dtr Dis Ls Dlr Dis Ls Dtr Dts Ls Dtr Dis ks Dtr Dts Ls Dir Dts Ls Dtr Dis Ls Dtr Dts Ls Dtr Dts ks Dtr Dis

Table

(,

V~

Y

1

k

1

M

V

A

G

P

Z

S

T

B

R

It

K

© Z

©

e~

z

7:

t-"

No. of residues: Positions:

TRP

PH E

TYR

LEU

ILE

MET

CYS

VAL

ALA

GLY

PRO

GLX

SER

THR

ASX

ARG

HIS

LYS

Peptide

7 1 7

1.04 0.79 0.84

/ 0.88

/0.77

| 0.78

1.12 1.04 1.17

1.04 0.93 1.05

4 8 11

0.94 0.81 1.00

1.17 1.20 1.00 0.88 0.89 0.76

1.00 1.00 1.02

0tT2

5 12 16

+ [1] + [11 + [1]

0.93 0.72 1.18 0.94 0.72 1.26

0.78 1.02 0.74

1.07 1.15 0.94

~tT3

15 17 31

0.62 0.64 0.46 1.12 1.15 0,86 0.69 0.40 0,12

(0.31)

[1] [1] [1]

[ 1] [1] [ ]

2.54 [3] 2.70 2.71 4.21 3.60 [41 4.40 [4]

2.03 2.60 2.43

(0.34)

(0.21)

0.83 0.91 0.56 [1] 1.00 0.84 1.05 0.40 0.83 0.16

(0.28)

aT4

9 32 40

0.76 0.40 [11 0.59 [1] 0.87 0.90 0.93

0.20 [1] 0.41 [ I ] 0.26 [ ]

1.17 1.01 2.17

1.22 t.02

0.98 0.95 0.94

1.75 1.87 1.70

1.00 1.00 0.96

~T5

16 41 56

0,78 0.40 [11 0.67 [-1] 2.12 1.89 1.99

1.23 0.98 1.14 0.75 0.99 0.85 2.22 2,34 1.55 [2] 1.08 0.81 1.12 1.10 0.72 1.00 1.28 0.99 1.08 1.40 [1] 1.02 1.06 1.79 1.70 2.06

1.00 1.00 1.07 1.99 2.05 2.24

~tT6

* As given for Table 1. t Data have been corrected to remove interfering proportions of ~T11.

Ls

0.84 1.06 1.00

Dtr Dts Ls Dtr Dts Ls Dtr Dts Ls Dtr Dis Ls Dtr Dts Ls Dtr Dts Ls Dtr Dts Ls Dtr Dts Ls Dtr Dts Ls Dtr Dts Ls Dtr Dis Ls Dtr Dts Ls Dtr Dts Ls Dtr Dts Ls Dlr Dts Ls Dtr Dts Ls Dtr Dts Ls Dtr Dis

2.26 1.73 1.82

:tTI

let')

4 57 60

1.05 1.24 1.70 0.95 0.97

1.00 1.00 0.97 0.89 1.06 1.07

~tT7

29 62 90

(0.27) (0.431

5.00 5.25 M

10.24t

1.50 [1] 0.82 M 2.18 2,85 M 4.65 [5] 6.46 M I 1.00 I 1.92 M

3.95 2.27 M 2.84 2.12 M 3.47 2.66 [3] M 0.64

(0.20)

1.33 1.00 M 2.87 2.40 [31 M

ctT9#

2 91 92

0.70 0.69 [1] 0.80

1.00 1.00 1.06

~tTl0

7 93 99

1.00 0.92 0.81

2.11 1.93 . 0 [-2]

0.60 [1] 0.80 0.76

1.85 2.49 [2] 2.26

1.00 0.91 1.05

~tTl I

5 100 104

[ 1.77 [ 1.72 1.80

0.78 0.60 [1] 0.62 [1]

1.05 1.16 0.73

1.00 1.00 0.96

¢tT12a

23 105 127

1.02 1.23 1.00

0.86 0.78 0.91 [ 3.16 [ 3.72 [31 3.20

2.41 [2] 1.68 [_2] 2.93 1.61 [21 1.20 [2] 1.92 2.03 1.45 [2] 1.34 [_11 (0.78) (0.59) (0.27) 1.16 [2] 1.76 2.18 1.37 [1] 1.08 0.91 3.97 3.36 [4] 4.22 1.86 2.01 1.86

1.00 1.36111 0.77 1.70 1.64 [2] 1.41 [2]

~tTl2b

Table 2. Amino acid composition of tryptic hemoglobin ct-peptides from Lemmus and Dicrostonyx (fast component-err) *

2 14(~141

0,42 [1] 0.65 [1] 0.56 [1]

1.00 1.00 1.00

ctTl4

(6)

1.14

1.05

0.75

2.09

1.00

X

W

F

Y

L

1

M

C

V

A

G

p

Z

S

T

B

R

H

K

O

O

o

O

>.

No. of residues: Positions

TRP

PHE

TYR

LEU

ILE

MET

CYS

VAL

ALA

GLY

PRO

GLX

SER

THR

ASX

ARG

HIS

LYS

Peptide

7 1 7

(0.151 (0.06) (0,25)

0.50 [ 1] 1.22 0.61 [1]

0.15 [1] 0.43 [ I ] 0.62 [ I ] 1.6011] 1.53 [1] 0.78 0.3011] 0.49 L1] 0.50 [ ]

10.28)

4 8 11

0.87 0.66 [1] 0.56 [1]

1,20 1.07 1,04 0.80 0.84 0.83

1,11 1.10 1.22

'~T2

5 12 16

+[i]

M M 1.06 M M 1.03

(0.44t

M M 11.07

M M 1.00

~T3

15 17 31

10,78 0.98 1.18 1.23 0.50 [1] 0.67 [ I ] 0,80

0.23 [1] 0.45 [1] [0.10)

{0.421

2.87 3.25 2.60 [3] 3.07 [4] 355 [4] 374

2.27 2.16 2.60 [3]

(0.311 [0.72)

0.93 0.99 1.00 0.85 1.00 1.00 1,43 [ I ] 0.78

:~T4

9 32-40

[0.60J 0.47 [ I ] 0.60 [1] 0.30 [1] 0.84 1.00 0.60 [1]

0.21 [1] 0.24 [1] 0.20 [ ]

40.60) 0.89 0.88 10.60~ 1.01 0.94 1.40 [2]

0.79 0.88 0.60 [1]

(1.14)

2,18 1.94 1.70

0.73 1,00 1.00

~tT5

16 41 56

0.46 [1] 0.61 [1] 0.88 2.12 2.07 2 18

1.40 [1] 1.11 1.04 1.29 0.98 0.78 2.43 [2] 2.22 2.10 1.00 1.04 0.88 1.25 1.29 0,94 1.04 1.13 1.27 1.08 1.07 1.2 I 1.87 2.16 2.10

0.75 1.00 0.92 1.70 2.07 1.85

:cT6

4 57 60

1.13 0.88 1.70 0.95 0.74

1.00 1.06 I. 18 1.08 0.93 1.01

aT7

29 62 90

4.61 [5] M 6.23

0.82 1.32 M 1.03 2.12 M 130 5.12 M 5,70 11.69 [2] M 11.77

5.15 M 3.04 [4] 2.13 M 1.70 3.58 M 2.13

1.48 [2] M 1.08 2.26 M 2.82

:~T9{

2 91 92

0.6011] 0.66 [1] 0.6 [ ]

1,00 1.00 1.00

:~TI0

7 93 99

0.73 0.56 [1] 0.91

1.67 [2] 1.77 .90

0.71 1.13 0.73

2.30 2.16 1.73

0.70 0.93 1.00

~T11

5 1130 104

1.33 [2] 1.71 .63 [2]

1.14 0.76

050 [1]

1.25 1.19 0.96

0.79 1.00 1.00

:(T 12a

2 140 141

0.45 [ I ] 0.62 [1] 0.46 [ ]

1.00 1,00 1.OO

:(TI4

161

1.13 0.83 0.94

1.38 0,91 0.94 1.04

1.22

1.45

1.67 1.42 1.46

1.00 1.00

1.00

X

* As given for Table 1. t Data for ~tT1 complicated by contamination from ctT9; chemical sequence obtained for Dtr-ct ~ through residue 9, given in Fig. 5, shows ~4 Gly and ct5 Ala. Data for Dtr pertain to ctT8-9 t~et~tide.

Dts Ls Dlr Dts Ls Dtr Dis Ls Dtr Dts Ls Dtr Dis Ls Dtr DIs Ls Dtr Dis Ls Dlr Dts Ls Dtr Dts Ls

Dtr

Ls Dtr Dis Ls Dtr Dts Ls Dtr Dts Ls

Dis

1,00 0.55 [1] 1.00 10.25) {0.55} (0.7H

Dlr Dts Ls Dtr Dts Ls Dtr Dts Ls Dtr Dts Ls Dtr Dts Ls Dtr

1.30 1.30 0.40 [ 11 (0.15) 10.34) {0,28) 0.40 [ 1] 0.50 [1] 0.48 [ I ]

:~T 1"i"

(:~')

Table 3. Amino acid composition of tryptic hemoglobin ~-Peptides from Lemmus and Dicrostonyx (slow component-~) *

W

L

I

M

C

V

A

G

P

Z

Z

N ©

g

X

Z >

[-"

527

Amino acid differences between major and minor hemoglobins Peptide fiT5 6. A discrete peptide fiT6 (Val-Lys), as obtained from Mus or Ondatra (muskrat), was not found in lemmings thus confirming the absence of lysine at fl59. Poor stoichiometry was obtained for the intact peptide fiT5-6 from both D. t. stevensoni and D. t. rubricatus, and their data are omitted from Table 1. Cyanogen bromide cleavage of the fl-chain from D. t. rubricatus yielded, subsequently, a tryptic peptide (flT5b-6, residues 56-61) in the map location expected for the six C-terminal residues. Its analysis was found to be: 0.89 Gly, 0.60 Asx, 1.33 Ala, 0.86 Glx, 1.00 Val, 1.00 Lys, which is the same as that found in Hb from Microtus and Clethrionomys (Genaux et al., 1976). This confirmed the presence of methionine in fiT5 of Dicrostonyx and supported the Lys--~ Glx substitution at position fl59. A peptide flT5a, residues 41-55, was found with good stoichiometry provided one accepts a Gly/Ala ambiguity at f151 as well as loss of Phe due to incomplete hydrolysis of the terminal Phe.Phe: 1.83 Phe [3]; 0.97 Glx: 1.12 His: 1.48 Gly: 1.07 Asx: 1.05 Leu; 2.40 [3] Ser: 1.66 Ala; 0.80 Val; 0.30 Homoserine (= Met [1]); and no threonine. The present data do not support the concept of deletions at fl52 and fl54, as suggested for several Microtus (Genaux & Morrison, 1973a, b), but do support the alignment proposed for M. xanthognathus (Duffy & Genaux, 1977). The proposed alignment for Lemmus (Fig. 3) suggests ambiguities S/H and M/L in view of the proportions of histidine and leucine. Peptides flT8-9a, flT9a and flT9b. These three peptide fragments from fiT9 indicate a lysine substitution at fl76 as found in other microtines, in the fl-chain of rat (Garrick et al., 1977), and in the minor fl-chain of Mus (Gilman, 1976; Popp, 1973). The two lemming genera differ by a two-base change in the codon for fl69 residue (His vs Thr). Glycine at position fl72 has also been reported in Clethrionomys (Genaux et al., 1976). The map positions of tryptic peptides from the badger fl-chain (Hombrados et al., 1976) support the identification of peptides fl9a, f18-9a and fl9b, as do the sequence studies on M. xanthognathus (Duffy & Genaux, 1977). Peptide flTlO. The composition of this Dicrostonyx peptide differs by one amino acid from that of Lemmus (Thr vs Ser) as shown at position fl84 in Fig. 3. Peptide fiT12. Cyanogen bromide treatment of the fl-chain from Dicrostonyx yielded an N-terminal fragment (flT12a) with the composition 1.10 Leu [2], 1.18 Gly, 0.60 Asx [1], and 0.35 Homoserine ( = Met [1]) corresponding to positions fl105-109. The expected two leucine residues are not observed presumably due to incomplete hydrolysis of the Leu-Leu bond. The C-terminal fragment of this peptide (fl110-120) was not recovered. Peptide flTl3. The replacement of an alanine residue by an aspartic acid residue has been located by homology with hemoglobins from other mammals at position fl125. Carboxypeptidase digestion gave the following C-terminal composition for this peptide (fl128-132) from D. t. stevensoni: 0.51 [1] Ala; 0,33 [1] Ser; 0.71 Phe; 0.49 [1] Glx; 1.00 Lys. It may be noted that position fl125 has a functional role in making subunit contact with residue at34 (Goodman et aL, 1975). (',F,,P 61/4B

D

Peptide fiT14. This peptide differs between the two genera of lemmings: serine in Dicrostonyx vs threonine in Lemmus. Both peptides showed a glycine residue at position fl138. Glycine was also found in the Ondatra (Genaux et al., 1976) but not in Microtus. Additional support for this alignment comes from carboxypeptidase digestion of the fl-chain from D. t. rubricatus: (ffI'14b-15, fl137-146) 0.95 Val; 1.01 Gly; 1.13 Ser; 1.48 [2] Ala; 0.95 Leu; 1.78 His; 1.19 Lys; 1.07 Tyr. Structure of or-chains

The similarity between or-chains from the major and minor components and also between the hemoglobins from the two lemming genera was apparent upon peptide mapping. Several differences, however, were noted (Fig. 4): 1. Peptide ~T1 was located below peptide ctT11 in the map of the slow component a-chain (~) of both Lemmus and Dicrostonyx. This is the same location that had been observed for the peptide ~tT1 from the minor o-chain from Microtus hemoglobin (Duffy et al., 1976). 2. A peptide (designated ctT4') was located below peptide ctT4 in the peptide maps of Dicrostonyx ~. This peptide was not observed in the map of the cdchain (fast component) of the D. t. stevensoni but was observed in the ~tf-chain peptide map of D. t. rubriC~Ius.

3. A peptide not obtained from other rodent hemoglobins appeared to the left (anodic side) of ctT14 in peptide maps from both Dicrostonyx and Lemmus. This peptide could not be assigned and was designated "X". Tables 2 and 3 present the compositions for the tryptic peptides isolated from the or-chains, fast and slow, respectively, of Dicrostonyx and Lemmus hemoglobin. Data from the two species are consistent with the similar mobilities observed for the respective homologous peptides. Apart from several ambiguities in the data which can be attributed to poor stoichiometry, it has not been possible to specify any amino acid differences between the homologous ct- and

"-'~

a - chain

,~P2b Io

4

7

+

=

I

Origin

- -

pH 6.4

8

-

Fig. 4. Composite peptide map of Lemmus and Dicrostonyx ~t-chains. Peptide T4' was not observed in either Lemmus or-chains or D. t. stevensoni 0if-chain. Peptide T3 was not identified for Dicrostonyx ~S-chains. Peptide ls and 9 overlap each other.

528

L. K. DUFFY, C. T. GENAUXand P. R. MORRISON (Garrick et al., 1975; Chua et al., 1975). Positions 4 and 5 seem to be more variable than the other positions in this peptide. As obtained from Lernmus and D. t. stevensoni this =~'-peptide contained a large amount of histidine, indicating an overlap with other peptides. (It was noted in other studies that the ~T1 peptide from Lemrnus lemmus (c~f) had a composition identical with that of Lemrnus sibiricus.) Peptide ctT3. The three lemming c~-peptides as well as the Lemmus c¢-peptide are characteristic microtine peptides. The peptide was not obtained from maps of the Dicrostonyx ~'-chain suggesting a change in composition. Peptides ~ T 4 and ~T4', Each of the peptides ~T4 obtained from the c~-chains of Dicrostonyx as well as those from the ~ - and c~-chains of Lemmus hemoglobin has an isoleucine residue whereas Dicrostonyx cC-peptides have instead a methionine residue. The l l e - - , M e t change involves a single base change between the corresponding codons.

fl-chains of the two subspecies D. t. rubricatus and D. t. stevensoni. Figure 5 gives the alignment of amino acids inferred from the compositions of the respective ~'- and ~-peptides by homology with the sequences for or-chain of hemoglobins from white mouse (Popp, 1965; Dayhoff, 1972), from Microtus, and from rat (Garrick et al., 1975; Chua et al., 1975). When peptides of the major (of) chains of Lemmus and Dicrost o n y x are compared (Fig. 5), no differences are observed in peptides ctT1, ~tT2, ~¢T3, ~tT4, ~tT6, ~T8(K), ~T10, ctTll, ~Tl2a and ctTl4. No data were obtained for ctT13. Peptide ~ T l . All ~tf-peptides contained two aspartyl residues and a single glycine residue. The difference in electrophoretic mobility between the cCTl-peptides and the ~rTl-peptides can be accounted for by the absence of a second aspartyl residue in the former, a situation observed for the corresponding peptides from M . xanthognathus (Duffy et al., 1976) and rat Species MUS,

'VLS

aTI aT2 aT3 G ED K'SN I K'AAWGK'I

aT4 G G H(AG) E Y G A

(io) D .T.f

D.

T.

D.T. s

A.

T.

L. s.f

D.

T.

A.

] T.

D. A D

T. T

L. s.s

[

MIC RAT

(20) A.

N. [/I////1111] M . A . N. A, N. A.

(3Ol _.E. D.

E. D

N. N[C]

G

aT5

E A L E R'

E

Q

aT6

MUS

'M F A S F P T T

D.T. f D.T. s L.S f L.S s MIC RAT

V.Y. V.Y, V.V.Y. V.V. Y V.Y. A

QT7 aT8

K'T Y F P H F D V S H G S A Q V K'G H G K'K' (40) (50) (60) A. A.

N [S--]

I

A. A

P

aT9 MUS

'VADA

LANAG

D.T.S f D'T" R'f D'T" R'S

[ B. [ B. A [ B.

T.S. T.S. T.S.

L'SS MIC

[

T. T.

X

RAT

AHLDD (70l

L.

HK'LR'V (90l

T. T. T.

] ] ]

SVv" " V.

Z.

T. T.

]

[7.] IV]

LVTLA

FK'

aTI3

S HH P A D FT P A V H A S

(110) G.

a TI I DPVN

T.

aTI2b

'L L S H C ' L

(120)

LDK'FLASVS (130)

aTI4

T V L T S KY R" (140)

[1//////1/Ill/Ill/////////H/] [////I////////////////////////1/////////////1////1///////////1/////////1///////] G.B. I. [///////////////////////////] [i///11//i//111//i/I/I//i/I///I//////I/11111/I////I//////////11111//I//////111] G. B. Z. [///////////////////////////]

O. T. f

D.T.s L.S. f L.S. s

MI__..£C RAT

LS D LHA

G. Z. S

K.

(100)

LSA (SOl

V. V. HT --

aTI2a MUS

aTlO

LPGA

r.F]

It]

I.

[G]

M.

Fig. 5. Amino acid sequence alignment of c~-peptides. Inferred amino acid replacements are indicated relative to the sequences for a-chain of hemoglobin from white mouse (Popp, 1965; Dayhoff, 1972) and from rat ~c~-chain (Garrick et al., 1975). Bracketed residues in the rat sequence resulted from the work of Chua et al. (1975). Blank spaces indicate identity with Mus. No differences were found between D. t. rubricatus and D. t. stevensoni for both fast and slow components except in ~T9 in which are indicated possible differences at two of the 29 positions. Sequenced residues for D.T? refer to D. t. rubrieatus (D.t.r.-c~s). Bracketed sequences refer to imprecise stoichiometry. The symbol X stands for a missing residue.

Amino acid differences between major and minor hemoglobins The peptide ctT4' indicated in Fig. 4 has the composition expected for =T4 and resulted presumably from the oxidation of methionine in the 0t~-peptides. The presence of a 4'-peptide also in conjunction with the D. t. rubricatus cr-4-peptide (isoleucine) suggests the possibility of a polymorphism in this species, a situation postulated to exist in rat fl-chains (L. M. Garrick, personal communication). Peptide otT5. Both of the Leramus ctf- and ct'-peptides are distinguished from those of Dicrostonyx by the presence of valine in place of alanine at position 34. Peptide otT7. The Lemmus ctf- and ~tS-peptides are distinguished from those of Dicrostonyx by glycine in place of alanine at position 57. Peptides ctT9 and ctT8-9. The single lysine residue, otherwise designated otT8, remains associated at the N-terminal of some of the peptides ;iT9 and produces the diagonal map distribution seen in Fig. 4. Imprecision in the overall stoichiometry for each of these very large peptides is indicated by the bracketed alignments in Fig. 5 and by the several ambiguous

positions given in terms of alternative residues B/A, etc. Microtine-like characteristics are evident in the common residues T, V, T at positions 67, 73 and 82, respectively. The Dicrostonyx hemoglobin is distinguished by the residue S at position 68. Except for the possible differences at two of the 29 positions of ~T9 f, no other fl + ~r + ~, differences were observed with which D. t. rubricatus could be distinguished in this study from D. t. stevensoni. Peptide ~tl2b. This hemoglobin peptide has been identified in two species of Microtus and in Ondatra and Clethrionomys (Genaux et al., 1976). Dicrostonyx and Lemmus 0tf-peptides (Table 2) are found in the same area of the map as those from Ondatra and Clethrionomys and are similar in composition, both containing isoleucine. Figure 5 shows the alignments proposed for the lemming ~r-peptides and compares then to the homologous peptides from Mus and Rat hemoglobins. The alignment for such a large peptide must be considered tentative. Peptide otT12b was not identified in hydrolysates of the slow hemoglobins from any of the lemmings.

= [ Dicr°st°nyxf

I

D.T.r. t 6 3 : A-'~B t 75 : D--='Z t

B-~S~

- I II1:

D.T.S f 75: I

I

68

_[

5 : D --~A t 17 : I'-='M 2 3 : E --="D

1 Lemmini

D-~G

Dicrostonyx s

j~

N-,-S

-I

1 1

~

,09: G----L~I

~lj

A " ~ B "r L'S" i

5: -{ 70: 75:

A_.,.G ~

t

D.T.r. s

6:3:

I

Lemmus 1 34 : A--~V ~ 57 : A"~G t

J

D-=~A t

G-~L O-~Z t

M.X. 23 : E--"D.

M.

Mierotinae

4 : 12: 19 : 35: 36: 73: II I: 113:

Ondotra

A-~G A-~T G-z~A S-~V F"~Y L-'-V S-~B H'~I

t Cricetidoe 109 : L--~G

1

8: 34 : 53: 68 :

I I

~

T~N ~ A'-"V t A"~G N--~S

=JHe$peromylnl

J

_l -I

[

1

M.P. 8 : T "~'S 12: N'e"S

J

t Muroldeo (rodent)

J ]

529

Murinoe

Fig. 6. Cladogram relating ctHb chains, t, parallel mutations in different lines. §, back mutation.

L. K. DUFFY,C. T. GENAUXand P. R. MORRISON

530

DISCUSSION Based on the proposed alignments, cladograms relating the ct- and fl-hemoglobin chains of Lemmus and Dicrostonyx are presented in Figs 6 and 7. Classification follows Simpson (1945) who designates tribes Microtini and Lemmini. The biochemical criteria show that the genera Lemmus and Dicrostonyx are characteristic microtines, a group whose hemoglobins show a homogeneity not seen in either cricetid or murid hemoglobins. Despite the unique morphological features of Dicrostonyx which distinguish it from other microtines, the hemoglobins of Dicrostonyx and Lemmus each show the same number of amino acid differences from hemoglobin of Microtus (12-14). However, these two lemmings also show about the same number of amino acid differences between each other and thus support Hooper & HaWs (1962) conclusion from anatomical data that the two genera need not be combined in a single group such as Simpson's (1945) tribe Lemmini or Ognev's (1948) supergenus Lemmi. Clethrionomys also shows a difference of 10 from Microtus.

Dicrostonyx 33: V-~G 69: T - - ~ H ¢ 8 4 : T --,.- S 125: A--~D

I

_l

Lemmini 72: 138:

25:

A--~-G t A-~G t

Lemmus 43:

Z--~A

45:

F --~-Lt

-1

50 : S --~T t 1 3 9 : S --D-T

1

Microtus

G --'-A

23 :

59:

69:

I

104:

K-~Q

86 :

Microtinae 22:

V--~I E -,-- A S-~-A I -,=S

17: 29:

H-~B A -~S

30:

Y -" F

A--~$t

~ l

_[

MR. ll2:

S--~I'

M.O./M. P. M.A./M.M. 45: F --,-L t 50: S --~T t 86: A --"S t

Ondatra

S-,~H

44: 72: 12:

V --~A T --,=H t K --~R §

Clefhrionornys 7'2: A -,-=G t

t

If:

The cladograms also show several parallel mutations to have occurred in different rodents at positions fl45, 50, 69, 72, 86, 138 and ct5, 34, 57, 63 and 75. Position ~t34, which is considered to be functional for cqfll-subunit contact (Goodman et al., 1975) may exemplify Zuckerkandl's concept of "evolutionary noise". Lowered solubility in phosphate buffers is observed for the hemoglobin which has ~t34 Val as compared with the hemoglobin which has ct34 Ala. Either valine or alanine appears in three microtine lineages (supergenera) and in the cricetines (two species). This may indicate a fluctuation around an optimal hydrophobicity for this area of the hemoglobin molecule in this family of rodents. Alignments based on compositions of tryptic peptides generally indicate the minimum number of amino acid differences between species, so that given a time of ancestral divergence we can estimate a minimum rate of amino acid fixation. Earlier broad comparisons of hemoglobins suggested that the rate of change was fairly constant, sufficiently so indeed to characterize the process as an "evolutionary clock", with a mean rate of 0.4 x l0 -6 mutations/yr (Day-

51: 62: 125: 138:

A-~S A--~S A --,.- D A---G t

135

A----S

f J

Cricetidoe 43: 04:

O-"Z R-~K

Muroideo (rodent)

'I

:1

]

Fig. 7. Cladogram relating fl-Hb chains, t, parallel mutations in different lines. §, back mutation. Ondatra 135:A ~ S, revision, Morrison et al. (1977).

Amino acid differences between major and minor hemoglobins hoff, 1972, p. 50). Later, more detailed comparisons, however, show striking differences in various lineages and tend to refute the "clock" concept (Holmquist et al., 1976). Z u c k e r k a n d l (1976) has proposed that the "clock" concept may still be valid under special circumstances: " t h e p r o p o r t i o n of the randomly occurring m u t a t i o n s that are fixed in relation to functional adjustments at constant e n v i r o n m e n t should remain constant, within limits of course, as long as the functional density of the protein is not significantly altered". O u r analyses show a m i n i m u m of 12 differences between Dicrostonyx and Lemmus hemoglobins, and, since the earliest fossils of these two genera are estimated to be 2 × 106 yr old (Guthrie & Matthews, 1971), we may compute a rate of change of at least 3 x 10 -6 mutations/yr. This faster rate of change is at variance with the conclusions of Sarich (1972) in his study of albumins of rodents and suggests to us that the short generation time of microtines may be a factor in accelerating their evolution. In the case of Dicrostonyx, its rapid directional evolution, as illustrated by dentition (Guthrie & Matthews, 1971), may have carried along m u t a t i o n s in its proteins (Morrison et al., 1977). Duplication of the or-chain loci seems to have occurred independently in these two lemming genera. Since b o t h c o m p o n e n t s are always present, the two ~t-chains appear to be non-allelic, and the unequal hemoglobin ratios (2:1) may result from an unequal synthesis from the two genes as described in rat and guinea pig (Weiser et al., 1976). The significance of gene duplications in general is still an open question (Maclntyre, 1976). Acknowledgements--We are grateful to Andr6e Porchet for her technical assistance, and to Christine Grilling who conducted much of the analysis of the major hemoglobin component from Lemmus. We are indebted to Dr. F. R. N. Gurd, Professor of Biochemistry, Indiana University for his hospitality, to L. K. Duffy, and to Dr. G. C. West, Director of the Institute of Arctic Biology, for his continuing support. We thank Dr. R. T. Jones, Professor of Biochemistry, University of Oregon, for unpublished amino acid sequences for carnivore hemoglobins* and Dr. U M. Garrick, State University of New York at Buffalo for the sequences of the rat hemoglobin fl-chains. This study was supported in part by PHS grant GM-10402 and from a special federal appropriation through the Center for Disease Control and represents a part of a thesis submitted by L. K. Duffy (1977) in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

REFERENCES AMBLER R. P. (1974) The evolutionary stability of cytochrome c-551 in Pseudomonas aeruginosa and Pseudomonas fluorescens biotype C. Biochem. J. 137, 3-14. CHUA C. G., CARRELL R. W. & HOWARD B. H. (1975) The amino acid sequence of the or-chain of the major haemoglobin of the rat (Rattus norvegicus). Biochem. J. 149, 259-269. CLEGO J. B., NAUGHTON M. A. & WEATHERALL D. J. (1968) Separation of the ct- and fl-chains of human hemoglobin. Nature, Lond. 219, 69-70. * A preliminary survey of the primary structures of the carnivore hemoglobins has now been published: BRIMnALL B. et al. (1977). J. Molec. Evol. 9, 231-278.

531

DAVrtOVF M. O. (1972) Atlas of Protein Sequence and Structure, Vol. 5. The National Biochemical Research Foundation, Silver Spring. DAYHOFF M. O. (1976) Atlas of Protein Sequence and Structure, Supplement 2. The National Biochemical Research Foundation, Silver Spring. DUFFV L. K. (1977) A comparison of the primary structure of hemoglobins from two microtine tribes: Microtini and Lemmini. Ph.D. Thesis, University of Alaska. DUEFY L. K. & GENAUXC. T. (1977) The primary structure of the hemoglobin fl-chains of Microtus xanthognathus. Comp. Biochem. Physiol. 56B, 143-146. DUFFY L. K., GENAUX C. T. & STRATTON L. P. (1976) Amino acid differences between the or-chains from two hemoglobins of the yellowcheeked vole (Family Cricetidae). Biochem. Genet. 14, 809-821. GARRICK L, M., KLONOWSKI T. J., SLOAN R. L., RYAN T. W. & GARRICK M. D. (1977) Primary structure of the major fl-chain of rat hemoglobin. Fedn. Proc. Fedn Am. Socs exp. Biol. 36, (3), Abstract 2560. GARRICK L. M., SHARMAV. S., McDONALD M. J. & RANNEY H. M. (1975) Rat haemoglobin heterogeneity: two structurally distinct ct chains. Biochem. J. 149, 245-258. GENAUX C. T., ERNST K. U. & MORRISON P. R. (1976) A comparison of the tryptic peptides of hemoglobin from two microtine genera: Clethrionomys and Ondatra. Biochem. Syst. Ecol. 4, 295-301. GENAUX C, T. & MORRISON P. R. (1973a) A comparison of the tryptic peptides of hemoglobin from Microtus pennsylvanicus tananaensis, Mus musculus and man. BiDchem. Syst. 1, 211 219. GENAUX C. T. & MORRISON P. R. (1973b) A comparison of hemoglobins in five species of Microtus. Biochem. Syst. 1, 221-230. GILMAN J. (1976) Mouse hemoglobin fl-chains: sequence data on embryonic y-chains. Biochem. J. 155, 231 241. GOODMAN M., MOORE G. W. & MATSUDA G. (1975) Darwinian evolution in the geneology of hemoglobin. Nature, Lond. 253, 603-608. GUTHRIE R. D. & MATTHEWSJ. V. (1971) The Cape Deceit fauna---early Pleistocene mammalian assemblage from the Alaskan Arctic. Quaternary Res. 1, 474--510. HOLMQUIST R., JUKES T. H., MORSE H., GOODMAN M. & MOORE G. W. (1976) The evolution of the globin family genes: concordance of stochastic and augmented maximum parsimony genetic distances for ct- and fl-hemoglobin and myoglobin. J. molec. Biol. 105, 39-74. HOMBRADOS I., DUCASTAING S., IRON A., NEUZIL E., DEBUmE D. & HAN K. (1976) Primary sequence of the fl-chain of badger hemoglobin. Biochim. biophys. Acta 427, 107-118. HOOVER E. T. & HART B. S. (1962) A synopsis of recent North American microtine rodents. Misc'. Publ. Mus. Zool., Unit,. Michigan 120, 68 pp. JOHNSON M. (1974) Mammals. In Biochemical and Immunological Taxonomy of Animals (Edited by WRIGHT C.), pp. l 76. Academic Press, New York. MACINTYRE R. J. (1976) Evolution and ecological value of duplicate genes. A. Rer. Ecol. Syst. 7, 421-468. MORRISON P. R., RAMAKRISHNAN P., DUEFY L. K. & GENAUX C. T. (1977) A comparison of the tryptic peptides of hemoglobin from two cricetine genera: Peromyscus and Calomys. Biochem. Syst. Ecol. 5, 309-316. OGNEV S. I. (1963) Mammals of the USSR and Adjacent Countries, Vol. 6. Rodents (Transl, from Russian), 508 pp. Israel Program for Scientific Translations, Jerusalem. PoPP R. A. (1965) The separation and amino acid composition of the tryptic peptides of the or-chain of hemoglobin from C57BL mice. J. biol. Chem. 240, 2863--2867. PoPP R. A. (1973) Sequence of amino acids in the fl-chain of single hemoglobins from C57BL, SWR and NB mice. Biochim. biophys. Acta 303, 52-60.

532

L.K. DUFFY, C. T. GENAUX and P. R. MORRISON

RAUSCH R. L. & RAtlscH V. R. (1972) Observations on chromosomes of Dicrostonyx torquatus stevensoni Nelson and chromosomal diversity in varying lemmings. Z. SaiJ#etier. 37, 372-384. RAuscH, R. L, & R~.USCH V. R. (1975) Taxonomy and zoogeography of Leramus spp. (Rodentia: Arvicolinae), with notes on laboratory-reared lemmings. Z. SaiJoetier. 40, 8-34. RUNKEL D., DRESLER S. L., BRIMHALL B. & JONES R. T. (1974) The tryptic peptides of coyote (Canis latrans) hemoglobin. Biochem. Genet. 12, 467-473. SARICH V. M. (1972) Generation time and albumin evolution. Biochem. Genet. 7, 205-212. SIMPSON G. G. (1945) The principles of classification and a classification of mammals. Bull. Am. Mus. Nat. Hist. 85, 1-350. STRATTON L. P., DUFFY L. K. & PORCHET A. (1977) Com-

parative solubilities and electrophoresis of microtine hemoglobins. Comp. Biochem. Physiol. 56B, 321-327. UEDA S. & SCHNEIDER R. (1969) Rapid differentiation ot polypeptide chains of hemoglobin by cellulose acetate electrophoresis of hemolysates. Blood 34, 230-235. VA~ VALEr~L. (1974) Molecular evolution as predicted by natural selection. J. molec. Evol. 3, 89-101. WEISER E., YEH C. & MAZUR A. (1976) Nonuniform biosynthesis of multiple hemoglobins in the adult rat and guinea pig. J. biol. Chem. 251, 5703-5710. ZUCKERKANOL E. (1976a) Evolutionary processes and evolutionary noise at the molecular level: I. Functional density in proteins. J. molec. Evol. 7, 167-183. ZUCKERKANDL E. (1976b) Evolutionary processes and evo. lutionary noise at the molecular level: II. A selectionist model for random fixations in proteins, d. molec. Et;ol. 7, 269-311.