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The structure of Fd fragments of G myeloma proteins
Immunochemtstry
Pergamon Press 1967. Vol. 4, pp. 95-99. Prmted m Great Bntam
THE STRUCTURE OF Fd FRAGMENTS G MYELOMA PROTEINS* B.
FRANorom,t F.
OF
PRELLI and E. C. FRANKLIN:
New York Umverslty School of Me$z;+oymatlc
Diseases Study Group,
(Recezved 2 August 1966) Abstract-The Fd fragments of the heavy chams of normal yG globulin and G myeloma protems contam a number of pephdes common to all of them and several vanable peptides. The precise locahzatlon of these pephdes remams to be determmed by sequence studies. In the light of the results of sequence studies with Bence Jones proteins, It appears most hkely that the heterogeneity of the heavy cham 1s largely luruted to restncted stretches of the Fd fragment.
INTRODUCTION THE Fd fragment of rG globulm 1s the part of the heavy chain whrch is primarily involved in determining antrbody specrficity~l~s) and which carries certain well defined genetic factors m man’s) and rabbits.‘495) Consequently, elucidation of its structure 1s essential to an understanding of the genetrc control and brosynthesis of y-globulms and also the nature of antibody specificity. Smce rsolatlon of this fragment from human yG globulin has been drfficult, analysrs of thus fragment has of necessity been mdnect. Previous studies, comparing the peptide maps of Fc fragments and heavy chains of yG myeloma protems, have shown that the Fd fragments, which constitute the NH2 termmal half of the heavy polypeptrde cham, are subject to varratrons(s~7) while the Fc fragments of proteins belonging to the same subclass are strikingly slmdar. (8) The present study extends these observatrons and demonstrates that the Fd fragments from drfferent myeloma proteins, like the lrght chains,(splO) contam a group of peptides common to most of them, and others which vary.
METHODS Normal G immunoglobulms and srxteen G myeloma protems of the We(~sb)‘rr*rs) type were isolated by starch zone electrophoresis.ua) Papam fragments and polypeptide chains were prepared as described prevrously.(r4) F(ab’)s fragments were prepared using pepsin (3 mg/lOO mg protein) at pH 4 for 16 hr.(le) Followmg reduction and alkylation of Fab or F(ab’)s fragments,“*ls) light chains and Fd fragments were separated on Sephadex G-100 erther in 6 M urea or guanidine(r7) or m 1 N propionic acid (1) In only three of twelve instances were pure Fd fragments obtained. Purrty of all fractrons was checked by immunoelectrophoresrs usmg antisera to K and h Bence Jones proteins, and to Fc, Fab and Fd fragments. The latter was prepared by absorption of an antiserum to normal Fab fragments * Supported by U S Public Health Service Grants No. Am 2594, 1431 and 2489. 7 Fellow, Arthntls Foundation $ Career Sclentlst, New York City Health Research Councd I-274. C
95
96
B FRANGIONE, F. PRELLI and E C FRANKLIN
with a pool of six Bence Jones proteins, t6~ The antlgemc subtypes of the heavy chains were determined with antisera to myeloma proteins belonging to the four ~,-chain subclasses and made specific by absorption with myeloma proteins of the other three types and k and A Bence Jones proteins. Gm factors were determined by H. H. Fudenberg. Fingerprinting and special stains were done as described.U4,18~ RESULTS Since it proved difficult to isolate pure Fd fragments from most of the protems, peptides from this region of the heavy chains were usually identified by comparing peptide maps of heavy chains and Fc fragments. The peptides common to both are derived from the Fc fragment, while those present only in the heavy chain originate in the Fd fragment. Identity or non-~denuty of two pepudes was based not only on their posiuon m the map, but also on the results of specific stains for arginine, hlstidme and tyrosme. The number of peptides thus attributed to the Fd fragment represents a m:mmum number since any pepudes common to the Fc and Fd fragments are automatically excluded. In three instances where it was possible to study the isolated Fd fragments, 6 or 7 addiuonal pepudes were identified. However, here too the posslble presence of small amounts of contaminants made evaluation of some of the faint spots difficult, and consequently precluded a precise esumate of the actual number of pepudes. Figure 1 represents the peptide maps of the heavy chain, Fc fragment and the isolated Fd fragment from one G m ( f + ) protein (Pre) chosen because of the availability of a pure Fd fragment. The pepudes marked by white dots represent the Fd fragment peptides obtained by subtracuon This protein was unusual in lacking a peptide (No. 2 in Fig. 2 and Table 1) common to all the other proteins studied. In addition, there are seven dark pepudes marked by ( × ) in the isolated Fd fragment and the Fc fragment. These appear to have a smular locauon m both preparations and may be identical in composition. Similarly, m each of the three isolated Fd pieces, peptides resembled some of those present m the Fc fragment. In one protein (Sch), five of the darker pepudes m the fingerprints of both the Fab fragment and the heavy chain appeared similar to certain pepudes present m the hght chain. Comparison of the peptide maps of the s~xteen proteins showed six pepudes, marked by the squares m the composite map in Fig. 2, to be common to all Fd fragments from G m ( a + ) molecules. The same pepudes were present in nine of eleven Gin(f+) Fd fragments (Table 1). Two of the G m ( f + ) proteins lacked one and two of the common peptides, respecuvely. A seventh common peptide (No. 1) in Fig. 2 was present in each of the G m ( f + ) Fd fragments. Tlus pepude could not be ldenufied m G m ( a + ) Fd fragments by the indirect techmque since it appears in almost the same posmon as the Gm(a) pepude characteristic of the Fc fragments of G m ( a + ) molecules, u4~ and is, therefore, automaucally excluded. A single punfied Fd fragment isolated from a G m ( a + ) myeloma protein was available for study. It contamed a dark pepude in the region of the 'a pepUde', and when mixed with the Fd fragment from a G m ( f + ) molecule, It failed to gwe rise to a new pepUde. These findings suggest that pepude No. 1 is also common to the G m ( a + )
& %
f
W
(a) FIC 1 Peptlde maps of (a) Heavy chain of Pre, (b) Fc fragment of Pxe, (c) Fd fragment of Pre • Represents Fd piece peptldes obtained by subtraction x Represents addmonal Fd piece peptldes which appear common also to the Fc fragment
(Facing p 96)
t&
FIG
l(b)
~O
0
06
0
,..~
Fie. 1(c)
The Structure of Fd Fragments of G Myeloma Protems TABLE 1 Fd FRAGMENT PEPTIDES OF We(y2b) MYELOMAPROTEINS PEPTIDE N U ~ R
--
I
S
I
4 3 2 I 0 9 8 ? 6 5 4 3 2 ;
:
:
UNIOUE pEPTIDES
1
~-
COMMON
PEPTIDES
I - BUR 2 - FRI :5 - NAG
[]
4 - PRE
~
I
E~
5 - BAC
I /
I
6 - SCHW 7 - FRA 8 - BRE 9 - SCHE I0 - voo
E~
I I - BOR
E~
Om (o +)
12 - DEE 1 3 - CHUR
14 " TRU 15 - BUR
16 " CAB I0 6 9
TOTAL 16
6
5 10 12 5 15 ~6 16 16 15 15 i i
NORMAL
PEPTIDE MAP OF Fd FRAGMmrrs
+ :20 .
.
.
.
.
.
.
.
.
.
.
×----
8
!15
.elI@
too
'~l
3~[ 3
~1~
9®
71~
]4| []
COMMON PEPTIDES
•
ARGININE +
@
HISTIDINE +
~) TYROSINE +
FIG. 2. Schematic diagram of composite peptlde map of Fd fragments showing those peptldes present m more than five of the proteins stuehed.
97
98
B FRANGIONE,F. PRELLIand E. C FRANKLIN
molecules; defimte proof for this, however, will have to await studies of a larger number of purified Fd fragments from Gin(a+) proteins. In ad&tion to this group of common pepudes, a series of peptides characteristic of each Fd fragment could be identified m the pepude maps. Some of these were common to several proteins, while others were seen only occasionally. Fig. 2 hsts only those pepudes present m at least five proteins; others present in only one, two, three or four proteins are not included. The number of variable spots ranged from five to eight. Fd fragments from normal ~,G globuhns had the same seven common peptides charactensuc of G m ( f + ) molecules and some of the variable ones. However, some of the latter were faint and their number was smaller, probably due to the great heterogeneity of this fragment. The appearance of the pepude maps of Fd fragments from G m ( f + ) and ( a + ) molecules was not related to the hght-cham type.
DISCUSSION
The results of this study indicate that, of the twelve to fifteen peptides detected m the Fd fragments of the We(y2b) type myeloma proteins, at least SLx are shared by all but a few of the proteins, and a seventh common peptlde was always ldenufied m the common region when the molecule was Gin(f + a - - ) . Since this peptlde appeared m almost the same posmon as the Gm(a) peptide m the Fc fragments of G m ( a + ) molecules/s~ and since several others m the peptide maps of pure Fd fragments corresponded to pepudes found in the Fc fragment, the posslblhty that there are addmonal common pepudes in the Fd fragments cannot be excluded and wdl have to await studies with addmonal pure Fd fragments from proteins dlffenng m Gm type. The remaining pepudes charactensuc of the Fd fragment differed for each myeloma protein. However, at least one-third of these pepudes were common to more than one-half of the proteins. It appears that the d~fferences m the primary structure of heavy chains of different myeloma proteins, and presumably anubodles, may reside m a relauvely small poruon of this chain. The precise locahzauon of the common and variable regmns on the Fd fragments is unknown On the barns of studies with Bence Jones proteins, t9,1°~ it xs reasonable to think that the common pepudes are clustered in one region, possibly the C-terminal end of the Fd fragment, wlule the variable ones may occur in the NH2-terminal end. Such a region of relatively constant composition ~s consistent w~th the existence of certain genetic factors, such as Gm(f) or Gm(z), ttg~ and antigenic determinants common to wrtually all Fd fragments from We proteins A region of varmbdlty, probably near the antlgen binding site, is suggested by the affinity labehng experiments of Doohttle and Smger,(20~ showing the label to be associated with a heterogeneous group of large pepudes in both chains The locahzauon of several Fd peptldes, including the extra pepude found m G m ( f + ) molecules, m the same posmon as some Fc pepudes rinses the posmbfllty that the structure of part of the Fd fragment may resemble a stretch on the Fc fragment. Slmdarmes were also noted m some instances m several Fd fragment pepudes and spots in the homologous lmmunolog~cally pure hght-cham maps Further work will be reqmred, however, to prove their identtty.
The Structure of Fd Fragments of G Myeloma Protems
99
REFERENCES t FLEISCHMANJ B , PORTER R R and PRESS E. M , Bzochem. jY 88, 220 (1963) 2 FRANEK F. and NEZLIN R. S , Foha mwrobtol. Praha 8, 128 (1963). a KRONVALL G , Vox Sang 10, 303 (1965). 4 TODD C. W , Btochem. b~ophys Res Commun. 11, 170 (1963) FEn,~STEn~I A., GELL P G. H and KELUS A , Nature, Lond 200, 653 (1963) 6 FRANGtONXB and FRANKLIN E C , y exp Med 122, 1 (1965) 7 FRANGtONE B , PRELLI F and FRANKLIN E C , Proc Fedn A m Socs exp. Bzol. 2.5, 374 (1966) s FRANGION~B , FRANKLIN E C , FUDENBERGH H. and KOSHLAND, M E , J exp. IVied. 124, 715 (1966) 9 HILSCHMANN N and CRAIG L C , Proc natn Acad Sez, U S A 53, 1403 (1965) t0 TITANI K , WmTLEY E , JR, AVOZERDO L and PUTNAM F W , Science 149, 1090 (1965) tl TERRY W D and FAI-IEYJ L , Sczence 146, 400 (1964) 12 GREY H. M and KUNKEL H G , J exp Med. 120, 253 (1964) la KUNKEL H G , Zone electrophores*s m methods of bzochemwal analyses (Edtted by GLICK D ) Vol 1, p 41 Intersctence (1954) 14 MELTZER M , FRANKLIN E C , FUDENBmtG H H and FP.ANCIONXB., Proc natn Acad Sc, U.S A S1, 1007 (1964) t5 NlSONOn~ A , WlSSLER F C and LIPMAN L N , Sczenee 132, 1770 (1960). 16 HEIMER R , Immunochemzstry 3, 81 (1966) t7 SMALL P A and LAMM M E , Bzochem~stry S, 259 (1966) is EASLEY C W , Bzochtm bzophys Acta 107, 386 (1965) 10 LtTWIN S D and KUNKEL H G , Nature, Lond 210, 866 (1966) 20 DOOLtTTLE R F and SINGER S J , Proc natn Acad Scz, U S A $4, 1773 (1965)
Pergamon Press 1967. Vol. 4, pp. 95-99. Prmted m Great Bntam
THE STRUCTURE OF Fd FRAGMENTS G MYELOMA PROTEINS* B.
FRANorom,t F.
OF
PRELLI and E. C. FRANKLIN:
New York Umverslty School of Me$z;+oymatlc
Diseases Study Group,
(Recezved 2 August 1966) Abstract-The Fd fragments of the heavy chams of normal yG globulin and G myeloma protems contam a number of pephdes common to all of them and several vanable peptides. The precise locahzatlon of these pephdes remams to be determmed by sequence studies. In the light of the results of sequence studies with Bence Jones proteins, It appears most hkely that the heterogeneity of the heavy cham 1s largely luruted to restncted stretches of the Fd fragment.
INTRODUCTION THE Fd fragment of rG globulm 1s the part of the heavy chain whrch is primarily involved in determining antrbody specrficity~l~s) and which carries certain well defined genetic factors m man’s) and rabbits.‘495) Consequently, elucidation of its structure 1s essential to an understanding of the genetrc control and brosynthesis of y-globulms and also the nature of antibody specificity. Smce rsolatlon of this fragment from human yG globulin has been drfficult, analysrs of thus fragment has of necessity been mdnect. Previous studies, comparing the peptide maps of Fc fragments and heavy chains of yG myeloma protems, have shown that the Fd fragments, which constitute the NH2 termmal half of the heavy polypeptrde cham, are subject to varratrons(s~7) while the Fc fragments of proteins belonging to the same subclass are strikingly slmdar. (8) The present study extends these observatrons and demonstrates that the Fd fragments from drfferent myeloma proteins, like the lrght chains,(splO) contam a group of peptides common to most of them, and others which vary.
METHODS Normal G immunoglobulms and srxteen G myeloma protems of the We(~sb)‘rr*rs) type were isolated by starch zone electrophoresis.ua) Papam fragments and polypeptide chains were prepared as described prevrously.(r4) F(ab’)s fragments were prepared using pepsin (3 mg/lOO mg protein) at pH 4 for 16 hr.(le) Followmg reduction and alkylation of Fab or F(ab’)s fragments,“*ls) light chains and Fd fragments were separated on Sephadex G-100 erther in 6 M urea or guanidine(r7) or m 1 N propionic acid (1) In only three of twelve instances were pure Fd fragments obtained. Purrty of all fractrons was checked by immunoelectrophoresrs usmg antisera to K and h Bence Jones proteins, and to Fc, Fab and Fd fragments. The latter was prepared by absorption of an antiserum to normal Fab fragments * Supported by U S Public Health Service Grants No. Am 2594, 1431 and 2489. 7 Fellow, Arthntls Foundation $ Career Sclentlst, New York City Health Research Councd I-274. C
95
96
B FRANGIONE, F. PRELLI and E C FRANKLIN
with a pool of six Bence Jones proteins, t6~ The antlgemc subtypes of the heavy chains were determined with antisera to myeloma proteins belonging to the four ~,-chain subclasses and made specific by absorption with myeloma proteins of the other three types and k and A Bence Jones proteins. Gm factors were determined by H. H. Fudenberg. Fingerprinting and special stains were done as described.U4,18~ RESULTS Since it proved difficult to isolate pure Fd fragments from most of the protems, peptides from this region of the heavy chains were usually identified by comparing peptide maps of heavy chains and Fc fragments. The peptides common to both are derived from the Fc fragment, while those present only in the heavy chain originate in the Fd fragment. Identity or non-~denuty of two pepudes was based not only on their posiuon m the map, but also on the results of specific stains for arginine, hlstidme and tyrosme. The number of peptides thus attributed to the Fd fragment represents a m:mmum number since any pepudes common to the Fc and Fd fragments are automatically excluded. In three instances where it was possible to study the isolated Fd fragments, 6 or 7 addiuonal pepudes were identified. However, here too the posslble presence of small amounts of contaminants made evaluation of some of the faint spots difficult, and consequently precluded a precise esumate of the actual number of pepudes. Figure 1 represents the peptide maps of the heavy chain, Fc fragment and the isolated Fd fragment from one G m ( f + ) protein (Pre) chosen because of the availability of a pure Fd fragment. The pepudes marked by white dots represent the Fd fragment peptides obtained by subtracuon This protein was unusual in lacking a peptide (No. 2 in Fig. 2 and Table 1) common to all the other proteins studied. In addition, there are seven dark pepudes marked by ( × ) in the isolated Fd fragment and the Fc fragment. These appear to have a smular locauon m both preparations and may be identical in composition. Similarly, m each of the three isolated Fd pieces, peptides resembled some of those present m the Fc fragment. In one protein (Sch), five of the darker pepudes m the fingerprints of both the Fab fragment and the heavy chain appeared similar to certain pepudes present m the hght chain. Comparison of the peptide maps of the s~xteen proteins showed six pepudes, marked by the squares m the composite map in Fig. 2, to be common to all Fd fragments from G m ( a + ) molecules. The same pepudes were present in nine of eleven Gin(f+) Fd fragments (Table 1). Two of the G m ( f + ) proteins lacked one and two of the common peptides, respecuvely. A seventh common peptide (No. 1) in Fig. 2 was present in each of the G m ( f + ) Fd fragments. Tlus pepude could not be ldenufied m G m ( a + ) Fd fragments by the indirect techmque since it appears in almost the same posmon as the Gm(a) pepude characteristic of the Fc fragments of G m ( a + ) molecules, u4~ and is, therefore, automaucally excluded. A single punfied Fd fragment isolated from a G m ( a + ) myeloma protein was available for study. It contamed a dark pepude in the region of the 'a pepUde', and when mixed with the Fd fragment from a G m ( f + ) molecule, It failed to gwe rise to a new pepUde. These findings suggest that pepude No. 1 is also common to the G m ( a + )
& %
f
W
(a) FIC 1 Peptlde maps of (a) Heavy chain of Pre, (b) Fc fragment of Pxe, (c) Fd fragment of Pre • Represents Fd piece peptldes obtained by subtraction x Represents addmonal Fd piece peptldes which appear common also to the Fc fragment
(Facing p 96)
t&
FIG
l(b)
~O
0
06
0
,..~
Fie. 1(c)
The Structure of Fd Fragments of G Myeloma Protems TABLE 1 Fd FRAGMENT PEPTIDES OF We(y2b) MYELOMAPROTEINS PEPTIDE N U ~ R
--
I
S
I
4 3 2 I 0 9 8 ? 6 5 4 3 2 ;
:
:
UNIOUE pEPTIDES
1
~-
COMMON
PEPTIDES
I - BUR 2 - FRI :5 - NAG
[]
4 - PRE
~
I
E~
5 - BAC
I /
I
6 - SCHW 7 - FRA 8 - BRE 9 - SCHE I0 - voo
E~
I I - BOR
E~
Om (o +)
12 - DEE 1 3 - CHUR
14 " TRU 15 - BUR
16 " CAB I0 6 9
TOTAL 16
6
5 10 12 5 15 ~6 16 16 15 15 i i
NORMAL
PEPTIDE MAP OF Fd FRAGMmrrs
+ :20 .
.
.
.
.
.
.
.
.
.
.
×----
8
!15
.elI@
too
'~l
3~[ 3
~1~
9®
71~
]4| []
COMMON PEPTIDES
•
ARGININE +
@
HISTIDINE +
~) TYROSINE +
FIG. 2. Schematic diagram of composite peptlde map of Fd fragments showing those peptldes present m more than five of the proteins stuehed.
97
98
B FRANGIONE,F. PRELLIand E. C FRANKLIN
molecules; defimte proof for this, however, will have to await studies of a larger number of purified Fd fragments from Gin(a+) proteins. In ad&tion to this group of common pepudes, a series of peptides characteristic of each Fd fragment could be identified m the pepude maps. Some of these were common to several proteins, while others were seen only occasionally. Fig. 2 hsts only those pepudes present m at least five proteins; others present in only one, two, three or four proteins are not included. The number of variable spots ranged from five to eight. Fd fragments from normal ~,G globuhns had the same seven common peptides charactensuc of G m ( f + ) molecules and some of the variable ones. However, some of the latter were faint and their number was smaller, probably due to the great heterogeneity of this fragment. The appearance of the pepude maps of Fd fragments from G m ( f + ) and ( a + ) molecules was not related to the hght-cham type.
DISCUSSION
The results of this study indicate that, of the twelve to fifteen peptides detected m the Fd fragments of the We(y2b) type myeloma proteins, at least SLx are shared by all but a few of the proteins, and a seventh common peptlde was always ldenufied m the common region when the molecule was Gin(f + a - - ) . Since this peptlde appeared m almost the same posmon as the Gm(a) peptide m the Fc fragments of G m ( a + ) molecules/s~ and since several others m the peptide maps of pure Fd fragments corresponded to pepudes found in the Fc fragment, the posslblhty that there are addmonal common pepudes in the Fd fragments cannot be excluded and wdl have to await studies with addmonal pure Fd fragments from proteins dlffenng m Gm type. The remaining pepudes charactensuc of the Fd fragment differed for each myeloma protein. However, at least one-third of these pepudes were common to more than one-half of the proteins. It appears that the d~fferences m the primary structure of heavy chains of different myeloma proteins, and presumably anubodles, may reside m a relauvely small poruon of this chain. The precise locahzauon of the common and variable regmns on the Fd fragments is unknown On the barns of studies with Bence Jones proteins, t9,1°~ it xs reasonable to think that the common pepudes are clustered in one region, possibly the C-terminal end of the Fd fragment, wlule the variable ones may occur in the NH2-terminal end. Such a region of relatively constant composition ~s consistent w~th the existence of certain genetic factors, such as Gm(f) or Gm(z), ttg~ and antigenic determinants common to wrtually all Fd fragments from We proteins A region of varmbdlty, probably near the antlgen binding site, is suggested by the affinity labehng experiments of Doohttle and Smger,(20~ showing the label to be associated with a heterogeneous group of large pepudes in both chains The locahzauon of several Fd peptldes, including the extra pepude found m G m ( f + ) molecules, m the same posmon as some Fc pepudes rinses the posmbfllty that the structure of part of the Fd fragment may resemble a stretch on the Fc fragment. Slmdarmes were also noted m some instances m several Fd fragment pepudes and spots in the homologous lmmunolog~cally pure hght-cham maps Further work will be reqmred, however, to prove their identtty.
The Structure of Fd Fragments of G Myeloma Protems
99
REFERENCES t FLEISCHMANJ B , PORTER R R and PRESS E. M , Bzochem. jY 88, 220 (1963) 2 FRANEK F. and NEZLIN R. S , Foha mwrobtol. Praha 8, 128 (1963). a KRONVALL G , Vox Sang 10, 303 (1965). 4 TODD C. W , Btochem. b~ophys Res Commun. 11, 170 (1963) FEn,~STEn~I A., GELL P G. H and KELUS A , Nature, Lond 200, 653 (1963) 6 FRANGtONXB and FRANKLIN E C , y exp Med 122, 1 (1965) 7 FRANGtONE B , PRELLI F and FRANKLIN E C , Proc Fedn A m Socs exp. Bzol. 2.5, 374 (1966) s FRANGION~B , FRANKLIN E C , FUDENBERGH H. and KOSHLAND, M E , J exp. IVied. 124, 715 (1966) 9 HILSCHMANN N and CRAIG L C , Proc natn Acad Sez, U S A 53, 1403 (1965) t0 TITANI K , WmTLEY E , JR, AVOZERDO L and PUTNAM F W , Science 149, 1090 (1965) tl TERRY W D and FAI-IEYJ L , Sczence 146, 400 (1964) 12 GREY H. M and KUNKEL H G , J exp Med. 120, 253 (1964) la KUNKEL H G , Zone electrophores*s m methods of bzochemwal analyses (Edtted by GLICK D ) Vol 1, p 41 Intersctence (1954) 14 MELTZER M , FRANKLIN E C , FUDENBmtG H H and FP.ANCIONXB., Proc natn Acad Sc, U.S A S1, 1007 (1964) t5 NlSONOn~ A , WlSSLER F C and LIPMAN L N , Sczenee 132, 1770 (1960). 16 HEIMER R , Immunochemzstry 3, 81 (1966) t7 SMALL P A and LAMM M E , Bzochem~stry S, 259 (1966) is EASLEY C W , Bzochtm bzophys Acta 107, 386 (1965) 10 LtTWIN S D and KUNKEL H G , Nature, Lond 210, 866 (1966) 20 DOOLtTTLE R F and SINGER S J , Proc natn Acad Scz, U S A $4, 1773 (1965)