Effect of genic substitution on the incorporation of tyrosine into the melanin of mouse skin

ARCHIVES

OF

BIOCHEMISTRY

ASD

Effect of Genie

BIOPHYSICS

Substitution into

63, 562-568 (1962)

on the Incorporation

the Melanin

of Mouse

of Tyrosine

Skin’

D. I,. COLEMX?I;

Rrceivetl

September

12, 1961

The amounts of tyrosinc-2-C” which were incorporated into Ijigment \wrc stutlicd using skin slices from mice carrying different coat-color grnw. Thcl various :dl~~lic gents at the C-locus (which control whether a mouse will bc full-voloretl, :dbino, or of intermediate color intensity) derrfmml tyrosine incorporntion wcording to the s(‘quence C > c”” > cIL > c’ > c. Using this biochemic:d :wsay. t,ho values obtained in th(i heterozygotes Cc and Cc” wc~rc intprmediatc, betwwn thaw found in PC’ \s. cc 01’ c”c’~ homoxygotes. indicating alwnw of dominwncc at I tlis lrvc~l of grnv c,slli,cwion. Substitution of the brown allele (NJ) for black (HI,) incwasctl tyrosinc> incorlwr:ltion twofold. Thv allele A” at the agouti locus pwvc~ntd the Iltilization of no~,rn:~l :~n~o~mts of tyrosine by these skin slicw. The gem’s fol, m:~ltcw dilution (rid) ant1 lwtl~~n (/,/II, 1, which nffwt thr clumping and not the xmount of pigmenl, hat1 no dfwt on t 110:11110i~nt of lposinr incorporated. 01 h(,r tlilut ing gcnw such as pink-cyc> (p/j) :mtl rlll)y (,‘(((‘1! 1 df~~re;ls(~tl the amount of tyrosine which WM utilizd. Thp signilicxnw of t tlcw rwultr ir discussed with respwt to what, is known nbollt thv form:ltion :tn(l final ;;IIUI~~IIW of pigment granules. ISTRODUCTION

Pigmentation in the mouse is dependent on the action ancl interaction of many genes (1). Most of these genes are known to cont,rol the size, shape, number, and distribution of the pigment granules formed (2, 3). Recent studies using the electron microscope indicate that. observed differences in the fine structure of the melanin granule are also attributable to the action of specific genes (4, 5). Other investigat,ions haw revealed that the activities of certain enzymes involved in the pigmentation process are influenced bv these same genes (6, 7). It would be of “interest to extend the previous studies on the biochemistry of pigmentation to include most. of the diverse genotypes available in the color stocks at ’ Supported in part by a grant from the American Cancer Society 1X-19. The maintennnw anti development of special pigment stocks wrrc supl)ol.trd by grants E-76 and E-162 from the -imcrican Cxnwr Society, and C-1074 from the iX:rtional Instituter of Hcdth.

t’he Roscoe R. ,Jackson Memorial I,aboratory and t’o attempt t’o correlate the known differences in morphology with the enzyme activities obscrvcd in the specific genotypes. This report supports the hypothesis that the various alleles at, the C-locus control tyrosinase activity’ and deals with the effect of alleles at the agouti (A), black (Rj, dilute in), and pink-eye (P) loci on the apparent activity of this cnzyiiic. Hrertling p;liw of mice of the wqllirc~tl gc’notylws were obtainetl wtic,w possible from the IJrotluction tlcpartmrnt of the Roscor 13. Jwkson ’ Tyrosinase adi\-ity wyu~ dctcrmined by a I~IXSurement of the number of tyrosinc molrdes incorporated into melanin, and thus the term “tyroIS usctl in this specific ,sensc. It is sinnsc activity” rrcognized that t lrc, low tyrosinc incorporation obwrvctl in some of the spwific~ gc3notype.q c~~ultl result from incre:lsd utilization of intermdiatw bctwcrn tyrosinc~ and melanin hy sidr wadions as well as by an nc%u;d clecrc~nsc in iyrosinnsc :I+ tiriiy.

TYROSISASE

ACTIVITY

IS

MOUSE

SKIM

d

5

5G3

Memorial Laboratory. Mice of the other mutant genotypes required for these studies were producc~tl bq a planned breeding program in the wsenrch volony of Drs. E. S. Russell and H. C:. Wolfe and wrro gencroualy supplied to the nuthor. Thcx breeding pairs of mice wcrc c~hecked each day. anti t Irv (late of birth of wch litter wl8 rworclc~cl. METHODS

\ i

3’

4’ AGE

5 IN DAYS

6

7’

1’

cd.1 (nnbb(‘C’) strains of mice. I&ximum activity occurred between 4 and 6 days of age in both black and brown genot’ypcs. Befort this period the amount of tyrosine incorporation increased rapidly, while at lat’er times t’hr rnt’e of incorporation was deprcsscd. Ko detectable incorporation could be demonstrated in adult mice q+ich had fully developed hair. Fitzpatrick and Kukita (7)) studying the changes in t,yrosin:w:e activity during the hair cycle in mice, obtainctl similar results. They found no dctectable tyrosine incorporation unt,il the al)pcarance of pigmented melanocytes in the hair bulb, which occurred 4 days after plucking. The activity increased to :I maximum betwcn 6 and 14 days, and I)y 24 days ttfter plucking no tyrosinase activity was tltrmonstrable. Since the time of assay prol-ecI so critical, all sut)scquent studies were carried out wit,11 mice \vhich wrc 5 t ‘A days of age. The efYect of age or stage of hair growth on tyrosine incorporation ~‘8s deterillined using mice ranging in age from 1 to 9 days. Figure 1 shows the relative rat,es of tyrosinc incorporation into skin slices from thr C57UL/B,J ~rrnRRC”f’) and C57 RR/

Effects

of dllelic Substitrttion nf the C’-Locus

The alleles of t,lic albino ( (‘1 locus limit the amount of pigment in the hair by contjrolling the cnzymc tyrosinasc I 8) The

TABLE

Allele

I

Genotype”

Tyrosine incorporation’ corrnlsinfin.

Wild Wild X :tlbino ChinchilLs Chinchilla Chinchilla X albino Himalayan Himalayan X wild Extreme dilni.ion Slbino Roiled ski11

an C’C’ ml (‘c a(1 ,.rh(.ch

aa “““‘“pp ml Ph”pp au (.hC’l u,ccCr” JLc..Ill (.e(.e aa cc na (‘C’

1200 617 142 350 151 225 676 98 17

zb zk f i f x!z f It +

13 f

36 33 15 5 li 15 4i 11 5 8

(1Only mutations under st.udy and those deviitt ing from wild type are listed. All animals were homozygous bl:tck (1311) and homozygous nc~rldilute (on). h Figures represent countjs/min./mg. of dried protein + standard error of the mean.

int,ensit’y of color of mice with thcsc various alleles ranges from full color in the wild type (CC, to no pigment, in the albino (cc). Table I shows the amount of radioactive tyrosine incorporated int’o the pigmcnt granules of black mice carrying the various alleles of the C-locus. Essentially no tyrosine was incorporated into boiled skins. -4s would be expected, the incorporation of tyrosine into unboiled preparations ranged from almost none in the albinos to a maximum (1200 * 36 counts/mm) in the fully pigmented black mice. Although the genotype Cc is indistinguishable from the normal wild t,ype (CC), the amount of incorporation of tyrosine was only SO% of that, seen in CC. This suggested that at this biochemical level there was no dominance, and further that the threshold incorporation required for full pigment’ation ~vas about 600 counts/min. Mice homozygous for the chinchilla allele (c”~~c”‘~)are distinguishable usually from both CC and Cc, and have an over-all tyrosine incorporation of 442 * I.5 counts/mm The recessive gene for pinkeye (pp) dilutes normal hair pigmentation and greatly reduces the pigment in the eye, Icaring only a faint rim of color in the iris. This gene also reduced the amount of radio-

active tyrosine which was incorporated into the skin. Values for :inimals of genotypes C”7’cl’np~J and c”“cpp wfrc included in this table to demonstrate thr intermediate valucs obtainctl in the heterozygotc~. In t,his case the two genotypes ( f*““F and cc”!~) are easily distinguished visually. Again, the heterozygote ((‘c”), although indistinguishable in color from the homozygote (CC’ 1, incorporated an amount of r:ulioactirc tyrosinc intermcdiatc between the amounts incorporatcti by the respective hoinozygot,es I CC and c7’C”). These findings suggest tlliit each allele independent~ly of the other cooltributes its own tvpc or amount of tprosinase. Extrcmc dilution (8”~ had a very low act,ivity (98 * 11 countz/min.) and a very small amount of visual pigmentation. The mutant :~ll~le~ d”‘=l”’ irt, tllc agouti locus shoultl not have cffcctcd the incorporation since this gene does not, exhibit its control until about 8 days of agr. The Himalayan mouse ( chcli ) was of spccial intcrcst since the pigment in this ~IIOL~SC tends to be limitctl to t,hc extremities aftci the first hair cycle (9). Prior to t’his t,hc trigmerit is uniformly dist,ributctl throughout t,he entire coat. If Himalayan inice are raised in a colt1 environment the pigmcntat,ion remains significantly tlnrkcr and, conversely, in a warm environment becoiues much lighter. This suggested that in this genotype the enzyme (tyrosinase) responsible for pigmentation was heat labile. To test this hypothesis skin slices were prcpared from both &lay-old normal i C’(‘) and May-old Himalayan (PC”) and hcatetl in a water bath at .55”C. for periods up to 1 hr. before tlic usual inrubat,ion with laI~~letl tyrosinc. After I hr. of heating tlic incorporation of tyrosinc int,o the skin from normal mice (1(1 was decreased by 10% (1080 vs. 1200 counts/niin.) , whereas the amount of incorporation into the skin from the Himalayan mouse decreased about 70% (68 vs. 225 counts/min.). Most of tliis change occurred in the first 20-30 nun. of heating. If the temperature of heating was incrcascd to 68”C., marked losses of a&vi@ were noted in both genot’ypes. This thermolability of the cnzymc produced under t,hc: influence of the Himalayan allele suggests that this allele and thus, by inference) all

alleles at the C’-ZOCUS,control the structure and not the quantity of the enzyme produced. Proof of this will require the isolat.ion of the mutant tyrosinases from the skins of mice carrying the various alleles of the C-locus and a det’ailcd comparison of them by physical-chemical means. Different species of tyrosinasc molecules, one of which was heat labile, have been demonstrated by HoroAz and Fling in ,Veurosporn (10). EWECTS

OF ALLELIC SUBST~T~-TIOS AT THE B-Locrw

The ~-locus in mice controls the color of the pigment produced, causing the animal to be black (RR), cordovan (h’b”), or brown (bb) . This locus influences the shape of the granules. Black mice produce larger granules which were presumed to t:ontain more pigment. Thus the results in Table II, which show more tyrosine-Cl” incorporated into brown animals than into black, are rather surprising (2680 vs. 1200 countr/min. ). Cordovan was intermediate in uptake as well as in pigmentation. The allele blncklight (11” 1, which is dominant to black, was not significantly different from black in the incorporation of tyrosine in thrsc 5day-old animals. The cross bctwccn brov;n and bl:~ck produces black micr wit.11 granules identical in shape to those of black mice. Similarly, the incorl)oration of tyrosine n-as the same as that seen in homozygous black mice (ZjI3 1, and in this case conventional gcnrtic doininancc was observed. When brown chinchilla mice are compared with black chinchilla mice, the incrcnsed incorporation of tyrosinc into the brown genotype (bbj was not evident. Thus the chinchilla allele (,c’“) can also exert a quantitative control over tyrosine uptake. This suggests that, the structural change in the tyrosinase molecule must be sufficient to interfcrc with t’hc production of normal granules. Smaller granules arc observed in hairs and vyrs from chinchilla mice (2, 5j. EFFECT OF THE AGOTTI LOCW TYROSINE: INCORPORATIOS

OS

The series of alleles at the agouti locus arc responsible for the yellow banding seen in hair from wild type (A.4 1 mice. This

EFFECTS

OF

CHILLA

(cc*)

THE

YELLOW

(;ENES

INC~RIWRATI~N .kLLELES

Ox

is THE

‘)

EXHIBITED OF

ASD

?;ORMAL

THE

c:HIS-

TYROSINE

BY THE

]3LA(‘K

Tyrosine incorporationb c,,rrn,s,w1in. Urown Cordov:tn Black Black-light Black X hrox-n Yellow hrowu k7ellom black Brown chinchilla Black chinchilla chinYellotv brow-n chilLi

” All mice were homozygous nonilut,e b Figures represent counts/min./mg. protein + stnnd:trd error of t hc nmtn.

2680 2290 1200 1170

rt f zt A

51 87 36 60

1060 460 182 14“ 157

f 18 f 11 f 17 3.z 15 f 5

(DD). of dried

control is best’ described as a trigger mechanism which shift,s the nature of the color of pigmentation revcrsibl\- in agouti I A;1 I : within t’lic space of one or tn-0 niedullary cells, from eumelanin (black-brown) to pll:rrorllclaIiin ( yellow,l (2 1 The recessive> condition ( 00 ) allows the hairs prorlucctl to bc ~)igliicntctl throughout wit11 cwiiclanin according to the dictates of the othcl color genes. The most dominant allvlc (&‘,I is lethal when liomozygous, but in combination with any other allele of agouti permits full vixbilit,y but changes the pigment in tlic hair completely to the ycllon- phacomclanin. As seen in Table II, t.his allele cuts don-n the incorporation of tyrosine into pignivnt to approximately one-third that found in non-agouti animals. This reduction in tyrosine incorporation was of the saint ordrr regardlws of the genetic constitution at either the 13-10~~sor the (‘-locus r460/1200 for RBCJC, 1060/2680 for Db(‘(‘ and 1401460 for IZRC’~C”~I. The other alleles of the agouti locus were not amenable to study since they exert, their action at about 8 days after birth which was least tlic t’inie of convenient Assam by this procedure. The pigment ( phneonwlanin 1 isol:lt)rd from yellow granules Iins

566

COLEMAN

been shown to differ in its physical propert,ies from t,hc eumelanin found in normal black or brown pigment granules. These incorporat’ion studies would suggest t,hat the normal scqucncc of events leading to eumelanin formation was interrupted or diverted by the presence of the yellow allele which possibly results in a smaller polymer than that normally formed. EFFECTS OF OTHER COLOR GEI\TE~

The gene ruby (rwu) 1 which causes slightly diluted hair pigmentat’ion and ruby eyes, only slightly diminished t,hc amount of tyronine incorporated into pigment, in zv’tro (1030 I+ 70 vs. 1200 t 36 counts/min.). This allele has been shown to delay the development of mature pigment granulcs in the eye (5) and presumably could have the same action in the skin. The gene for pink-eyed dilution (pp) is similar to ruby but has a much more marked diluting effect on the hair pigmentation and reduces pigmentat,ion in the eye even more extensively. This gene permit,tcd something less than one half the normal amount of tyrosine to be incorporated into hair pigment 1538 t 16 vs. 1200 t 36 counts/min.). Studies using the electron microscope have shown that this gene causes radical changes in the internal structure of the developing pigment’ granule (4j. The t’wo non-allelicdiluting genes, maltrsc dilution (dd) and leatlcn 1Inln) , mimic each other morphologically. They both allow normal amounts of pigment to be formed but cause abnormal clumping of the granules (2, 3, 5’). This clumping is responsible for the apparent decrease in pigmentation as observed visually. Cndcr the conditions of our assay, pigmentation proceeded normally in both these genotypes, 2660 t 60 counts/min. for dilute browns ( tJd bb j and 2840 2 74 countsjmin. in brown leadens (00 Inln) as compared with 2680 * 51 countsmin. for normal brown mice I Ob DTI LnLn). It has been showi that the alleles of maltew tlilmion (tl and d’) were implicated in the normal functioning of the enzyme phenylalanine hydroxylase which may bc related to this abnormal clumping in the dilute animals (11).

The incrcasctl cnzyino activity seen in brown animals when comp:~~l with black animals is in contrast to what one woul~t expect on casual obeerration. This sugg&cd that thcrc may bo a cont,rol ~TL~~izw which limited the amount’ of tyrosinc which could be incorporated int,o pigment. To study this, un-tyrosine (0.5 PC.) was injected into 4-day-old black and brown mice. Aft’er 24 hr. these miw Tvcrc sacrificed and the tyrosinc incorporation determined as dcscribed previously. Since considerable protein synthesis coul~l be expected under these conditions! cont,rol cxperimcnts were run with albino mice of the AKR,‘.J Arain IUU BB w ) which cannot produce any pigment,, After correcting for protein synthesis ( 131 +- 4 counts/min./mg. of tlrietl protein) brown animals were found to incorporatc~ 77 * 7 counts/min. into pigment whereas that incorporated by black mice was only 40 i- 6 counts/min./mg. protein. This iinplies that the II-locus does not accomplish its cont.rol of pigmcnt~ation by limiting substrat,e concentrations in 7li7’0 as previously suggested (6). Again the period bet~wccn 4 ant1 5 days was most active with rcspcct to pigmcnt~ formation. Tryptophan has been auggt&xl as the normal lnwursor of the yellow pigment in inice (7)) but, in similai c~xperimcnt,?;n-hen tryptoplian-<‘lJ (0.5 PC.) was injected into yellow and albino mice the incorporation of radioactivity was no greater in the yellow than that seen in the albino. Tyrosinc uptake by these yello~v animals I AuflBH) was only slightly less than that observetl for normal black miw I 29 vs. 40 count~s,/min.). This, in conjunction with t’hc in 7litm tlat:L, suggests that tyrosine. not trgptophan, is the major prccursor of phacomelanin. INHIBITION

YTUDIE~

A1 slight dccrcasc in the incorporat,ion of labeled tyrosine into pigment was noted when a large amount of unlabeled L-tryptophan was added to the reaction mixt,ure. Since studice showed that this rnaterial was not acting as a prccursor to pigment, it must be functioning as an inhibitor to normal utilization of tyrosinc. Plienylalanine

was another related compound which had been shown to be a competit,ive inhibitor of tyrosinase from tumors (12). To test these compounds in this assay system, separate experiments were set up using normal black skin (n&B ) in which tryptophan and phcnylalaninc were added in amounts up to 100 tinics the molar concentration of L-tyrosinc. Fifty per cent inhibition n-as not rcachcd in this system unt,il the molar conccntration of tryptophan to tyrosine rcacliecl 100: 1. Phenylalaninc was found to bc a somewhat bcttcr inhibitor, in that a molar concentration of only 70: 1 was sufficient to proclucc a iiO”;- inhibition. The amount of inhibition obtained by phcnylalaninc, how(‘~(‘1’. was considerably less than that, r(‘portcld pr(xviously using isolated tyrosinase froiii t8unior:: (12‘1. In our studic~s n-ith a similar mammalian tyrosinase preparat’ion, thca relative inhibitory action of phcnylalanine n-as found to increase in proportion to t11c amount of dihydroxyplicnyl:danine ( DOPA) in the rcact,ion mixture. No increase in incorporation of tyrosine into skin slices was obtained when the animals were irradiated with ultraviolet light prior to assay nor did the addition of various levels of cupric ions to the reaction medium have any significant stimiilating action on the rate of incorporation. DTSCTWSTOK

The importance of using tyrosine as the subst,ratc for tlic enzyme tyrosinase rather than the more active dihydroxyphenylalanine (DOPA) has been adequately pointed out (7,1. The assay used in this study is similar to the radioautographic tyrosinase mothoc1 used previously with mouse hair bulbs, malignant melanoma, and embryonic retinal pigment epithelium (7) and, in general. these data agree w-cl1 with those published using t,liiy radioautograpl~ic techniquc. 130th mclanic (black-brown’) and phaeomclanic (yellow) hair bulbs oxidized tyrosinc. Han-ever, the present, data suggest that tyrosinc is an actual Ijigmcnt prc’cursor to both pigments, whereas Fitzpatrick and Kukita suggcst#cd that t,yrosine only arted :1s s l)recursor in zGtro and artcd in 74lw in an clcctron transport sys-

tem \yhich was responsible for the oxidation of tryptophan, the actual pigment precursor. These authors, in other respects, obt,ained results similar to those presented here. They fount1 the greatest tyrosinase activity in brown mice (bb) or brown mice with maltest dilution (tld,l I~ss incorporation was seen in black (RB 1 and yellow I ;l!‘nHB) liiicc. Pink-cycd dilution (l),io1 significantly dccrcased the amount of incorporation in black cl{Nj mire, whereas albinos hat1 no dct~c~ctableactivity. In ot)lirr st,utlies using the rate of oxygen uptake as a incasure of tyrosinnse activity, ratSlier than some measurement of actual pigment formed, mice l~on~ozygous for pinkeyed dilution (pp) wcrc found to have highc)r activity than the non-pink-eyed (~1)) aniiiinls in t)otli the I)OPA 0xitl:istl test using DOPA as substrate and the tyrosinasc test using t’yrosincb as substrate (6). It must be emphasized 11~~ that most of the oxygen is consumed before any actual pigment is produced, and this incrcascd oxygen uptake could rcprcscnt nonspecific “tyrosine or DOPA oxidases” which (10 not. normally lead to pigment. Xaturally occurring melanin is a polymer of indolc-5,6-qiiinonc which col)olymerizcs with protein to forni :L mcltinin granule. This indole-. ,&quinonc arises from the oxidation of tyrosinc by the enzyme tyrosinase (13). The protein matrix of the pigment granule is felt to contain the tyrosinase act,irity (,7) and melanin is consitlere~l to polymerize directly on to it, eventually blocking the active enzyme sites so that ;t mature granule no longer has tyrosinase activity. Moyer, in his stuclics of granule fine structure in the eye, has pictured granule foriiiat,ion a$ having four distinct stages (3). The fibers whicll first. appear in stage 2 granules are considered to have tyrosinase actiI.ity since melanin is tlsposited on these to form st’age 3 fiber:: which are further niclanized to form stagcl 4 granul~~s. After this no further mclanization occurs, and the underlying protein m:itrix is obscured. I-sing this as a model of n-hat’ iy occurring in the skin, it is suggested that, the skin of the :i-(lay-old mouse contains most of tlic grnnulrs at stage 2, which are readily

melanizcd on exposure to tyrosine in ?~itro. After this stage of development more of the granules are in the later stages and consequently the amount of t;vrosine incorporated IS less. The observation that’ the amount of tyrosinase activity was directly related to the alleles at the C-locus is in agreement with what has been observed visually (,a), and is in keeping with t,he concept that the Clocus controls the production of the enzyme tyrosinasc (8). The heat lability of the enzyme produced in the Himalayan mutant (c”c”) suggests that this gene acts by controlling t’he protein structure of this enzyme ratSlier than by altering its quantity. If this is the case, it is not apparent how a structural change in the protein could influence the size and number of the pigment granules. Possibly t’he abnormal protein cannot be produced at the normal rate, or, more likely, it is unable to conjugate properly with the other proteins dest,ined to form the melanin matrix. Another apparent anomaly is the higher activity found in brown skins both in viva and in vitro, although black appears to contain more pigment on both visual and fine struct,ure examinat,ion. It would seem t,hat there must bc more active sites of melanin synthesis in t,he brown granules which lcad to a greater amount of polymerized tyrosine (melanin) in the granule. Certainly the size, shape, and molecular orientation of both t,he melanin molecule ant1 the granule itself are factors which must be considered also in this regard. The alleles at the agouti (A) locus appear to be controlling an inhibitor of normal melanization. In yellow mice the action of this inhibitor is always present, and it prevents the normal level of tyrosine incorporation into melanin. Histological studies (2) indicate that the granules are smaller and fewer in yellow mice. Possibly,

studies on the fine structure of the developing granules in yellow hair bulbs will reveal whether the protein mat.rix is altcrcd first, leading to abnormal pigment format,ion! or whether ubnormnl pigment formation is the factor which influenws t,he size and shape of t,he granule.

The author wishw to express his npprcciation to Dr. E. S. R~~sscll for her helpful atlvicr and criticism, to Dr. H. G. Wolfe who tlevclopcd and prorided mnny of the mutant stocks used in this study, and lo MISS Merrill Rwr who undertook a RII~VC~ of tyrosinnsc nctiyily in various preliminary stmins of micac (luring her tenure xx :1 Fummcr et,udent at this laboratory. The technical as.sist:rncc of Mr. I~iclwd Col~ll is grntcfull>. acdx~owldged. H EFERENCIC? (ienetiw of the 1. GH~~ZTEBERG, H., ill “The Mouse,” 211tl ed., Chaps. IV and V. Martinus Sijhoff, The Hague, 1952. 34, 146 (194Y). 2. RUSSELL, E. S., Gvr!etics C. I,.. ASI) I~ILVERS, IX-. K. irl “Pig3. MARIXRT, ment, Cell l~iology” (M. Gordon, ed.), 1~. 241. Acdvmi(. I’WSH, zj~~w York. 1959. 4. MOYER, F., Anat. Record 138, 372 (1960). 5. MOWER, F., in “The Structure of the Eye,” p. 469. dcadcmic~ Press, New York, 1961. 011 Biology” CM. 6. FosTeR, M .) it1 “Pigment (;ordon, ed.), 11. 301. Academic Press, Sew Tork, 1959. T. B., AKD Iiryiwr.4, .I.. ill “l’ig7. FITZP.~TRICK, inent Cell Biology” (M. Gordon, cd.), 1). 489. Acadcrniv Press, New York, 1959. 8. 1