Isolation, Chromosomal Mapping, and Expression of the Mouse Tyrosinase Gene

Byoung S. Kwon, Ph.D., Asifa K. Haq, M.D., Mark Wakulchik, M.S., Daniel Kestler, Ph.D., David E. Barton, Ph.D., Uta Francke, M.D., M. Lynn Lamoreux, Ph.D. , j. Barry Whitney JII, Ph.D., and Rutb Halaban, Pb.D. Dcp;l.rtmenr of Microbiology :md Immullology . .and -W.:alrher Oncology Center, Indiana University School of Medicine, Indi.:m .. polis. Indiana. and Molecular Genetics Laboruory. Guthrie Resea.rch lnstitute, Sa)·n~. Pennsylvania (BSK.AKH.MS.DK); Department of Human Generics, Yale University School of Medicine, New Haven, Connecticll( (DEB.UF): Department of Biology, Texas A & M University, College Station. Texas (M LL); Department of Cdl and Molecular Biology. Medic.! College of Georgia. Augusta, Georgia UBW); and Department of Dermatology. Yale University School of Medicine. New H 2ven, Connecticut. U.S.A. (RH)

Using a human tyrosinase eDNA probe. we have isolated mouse tyrosinase genomic clones and used them [0 map the mouse tyrosinase locus and to analyze the promoter sequence of the tyrosinase gene. Southern blot analyses of DNA from somatic cell hybrids, interspecies backcross mice, and albino deletion mice have revealed that the locus for mouse tyrosin­ase resides at or near the albino locus on mouse chromosome

Genetic control of melanin pigmentation has been studied extensive.ly in rotC!: and is reported to involve over 50 gene loci and more than 130 alleles [I}. T y­rosine is thr- initial substrate" for the formation of boch the brownfblack pigments. eumelanin [2], and

the red/ yellow pigments. pheomelanin [3). In the conversion of tyrosine to either type of melanin, the initial step is the enzymatic conversion of tyrosine to dihydroxyphcnylalanine (DOPA) by tyrosinase (E.C. 1.1 4. 18.1 ) [4].

Recent evidence supports early observations (5) that the c-Iocus on mouse chromosome 7 is the sitc for the srructural gene for ryro­sinase. First, we have shown thac the eDNA encoding human ryro­sinase maps at or ncar the c-locus [6J and at human chromosome 11 [71. The laner is known to share linkage-homology with mouse chromosome 7. Second, three independent scientific teams have isolated mouse ryrosinase cDNA having nucleotide sequences highly homologous to human tyrosinase 18.9,10). Oneof the mouse cDNA (10) produced acrive tyrosinase upon transfection into 01 fi­broblast cell line. Human tyrosinase cDNA was reisolated recently from human melanoma cDNA library on the basis of homology with a synthetic oli~onucleotide whose sequence was derived from our published dau 16,11]' The expression of this ty rosinase cDNA in mouse fibroblasts brought about tyrosinase activiry and melanin

M.wuscripr received November )4. 1988: accepted for publication April 25, 1989.

Reprint requests to: Byoung S. K won, ph.D., Indiana University School of Mcdicin(', 635 Barnhill Drive, MS 255, Indi':lnapolis, IN 46202-5120

AbbreviatioDs: DOPA: dihydroxyphenylalanine I.EMX: isoburylmethyl Xanthine MSH: melanocyte stimulating homlone RFLP: restriction fn.g ment length polymorphism

7. There were three TATA-e1ements, bur only one CAT-ele­ment, and the C AT-element appeared to be paired with the third TATA-e1ement, located at the position farthest up­st ream. Mouse ryrosinase mRNA is approximately 2.4 Kb in size. The amount of tyrosinase mRNA reflects the levels of tyrosinase acti vity in nonnal melanocytes and Cloudman 5-91 melanoma cell line.] l"v£51 DermaroI93:589- 594, 1989

synthesis. These data give direct evidence that the protein encoded by the: c-locus cDNA carries the tyrosinase catalytic activity. A murine cDNA clone, originally thought to be a tyrosinase cDNA

fI2], was shown to map to the brown locus on mouse chromosome 4 13 and to code for a protein with no tyrosinase activity [lOJ.

Isolation and analysis of melanocyre-srecific genes are of great importance In underscwding disorders 0 hypopigmentation. The tyrosinase eDNA clone has already been used to identify restriction fragment length polymorphisms (RFLPs) in DNA from patients with tyrosinase-negative albinism [14.15J.

The current report describes the use of the human tyrosinase cDNA as a probe to isolate mouse tyrosinase genomic clones. The murine tyrosinase gene probes have been used to map the murine tyrosinase gene 011 mouse chromosome 7 by methods not utilized before , to analyze the promoter sequence of mouse tyrosinase gene, and to study rhe expression of tyrosinase mRNA in nonna! murine mdanocytes and melanoma cells.

MATERIALS AND METHODS

Cultured Mouse Melanocytes Normal mouse melanocyres wete cultwed ftom skins of newborn mouse-strains C57BL/6J and BIO.BR, as described previously [16J. A melanotic C loudman S-91 murine melanoma cell line was grown as described (17).

H ybrid Cell Lines The construction and chancterization of the mouse x Chinese· hamster hybrids of series 1, EAS. and EBS have been described [18, 19J. Hybrid lines were subeloned and expanded in culture and chromosomes were analyzed by trypsin-Giemsa banding l18] at the time: of DNA-extraction.

Mice Newborn mice of the genotypes 2H/i'H and t.<"/i'H [20J were: provided by Dr. Salome Gluecksohn· Wae1sch (Albert Ein­stein College of Medicine, Bronx, NY). Newborn mice of the genorypcs c-bjc I40>S. cci' / c6H , and c;6H/CI4CoS were generated through breedjng from three mice of the genotypes c;6H/C14CoS that were given to Dr. Lamoreux by Dr. Gluecksohn-Waelsch. T he original stocks were crossed rwice ontO JU/ CtLm-C/C. then once onto

0022_202X/ 89/S03.50 Copyright © ]989 by The Society for Investigative Derm.atolog)'. Inc.

589

590 KWON ET At

CS7IJL/ 6J-C. then for eight generations onto a CS7BL/6J-r h/rh

stock descended from mice provided by Dr. D. Townsend (Univer­siry of Minnesota, Minneapolis, MN).

CS7BL/6J .spterus-C Hblt mice were maintained by J . B. Whit­ney. This stock of mice was at the seventh generation of backcross­ing of the MilS spre(us wild-type albino-locus allele OntO the inbred strain CS7BL/6J-,hHbbp or C57BL/ 6J-C Hbbd~ . Hb~ allele was originally derived from .Mlls casllHiellS. Baekerosses were set up using males heterozygous for the spretus genotype and a second marker on chromosome 7. either an olbhlo-locus allele or .. hemo­globin beta (Hbb) variant. T he albi" ... locus phenocype/ genotype (e/ch. heterozygous, or chich homozygous h.imalayan) was deter­mined visually. Hbb was typed by the method of Whitney (2IJ.

Isolation of the Mouse Tyrosinase Gene Two mouse genomic libraries. derived from NIH 3T3 cell or BALB/ c embryo DNA. werC," screened with the [llP]-labeled human ryrosinasecDNA probe Pmel 34 (6]' The BALB/c embryo library kindly provided by Dr. Philip Leder of Harvard University. contained a partial Hae III genomic fragment with EcoR 1 linkers in a charon 4A vector. The NIH 3T 3 genomic library (purchased from Stratagene, San Diego. CA). contained EcoR I genomic fragments in a ..llix vector (Srrata­gene).

Genomic DNA Blot Analysis High molecular-weighr DNA from newborn mice, from adult mouse spleens, or from hybrid ceUs were prepared as described previously [22.23]. Restriction-endonu­clease digests of DNA were electrophoresed in 0.8% agatose gel at 4 tIc. The gel was denatured with 0.5 M NaOH plus 1 M NaCl and neutralized with 1 M Tris HCI, pH 8.0. plus 1 M NaCI. The DNA was transferred to Gene Screen Plus (NEN, Boston, MA) as de­scribed by Southern [24]. The blot was hybridized with the ("PJ-Ia­beled eDNA in a solution composed of sixfold concenera ted SSC (0.15 M sodium chloride, 0.015 M sodium ci trate), fivefold con­centrated Denhardt's solution, 0.5% SOS (sodium dodecylsulfate), and 100 ,ug/ mI denatured salmon sperm DNA. The filters were then washed three times at room temperature for 10 min each in twofold concentrated sse plus 0. 1 % 50S and two times at 65 DC for 45 min each in D. l-fold concentrated sse plus 0.1 % SDS. The intensity of the bands in auroradiograms was measured by a densi­tometer at 500 nm (Beckman DU-SB spectrophotometer) .

RNA Blot Analysis Poly(A)+ mRNA from Cloud man 5-91, C57BL/ 6J . and BIO.DR mclanocytes were fractionated on 1.2% formaldehyde denaturing agarose gels, transferred [0 Gene Screen Plus membranes. and hybridized to (l2P]-labeled probes. The filters were then washed three rimes in twofold concentrated sse plus 0.1 % SDS at room temperamre and three times at 42"C in O.l-fold concentrated sse plus 0. 1 % 50S. The filters were autoradio­graphed at -70°C. Pmel 14-2. a eDNA clone that was isolated from a human melanocyte eDNA library [25]' was used as a control probe to show that each lane contained almost the same amount of RNA.

DNA Sequencing DNA restriction fragments. subcloned in M 13 vectors [261, were sequenced by the dideoxy chain termination tech­nique f27]. with modifications made to accommodate 2'-deoxya­denosine S'-(a-["SJthioJ tripho,phate (28J.

Tyrosinase Activity T yrosinase activity was measured in cell exnae[s as described previously P9]. A unit of tyrosinase was de­fined as the activity of enzyme that cau.lyzed the oxidation of 1 pmol of ryrosine in 1 min.

RESULTS

Chromosomal Mapping of the Tyrosinase Locus with So­matic Cell Hybrids The human ryrosinaie eDNA Pmd 34 was used to map the chromosoma.l location of the structural gene for tyrosinase in the mouse. DNA &om a panel of 11 mouse x Chinese­hamster somatjc-cell hybrids was analyzed by Southern blot hybrid­ization. A strongly hybridizing 4.7-Kb mouse-specific fragment was detected in &oR I digests of mouse DNA used as a control and

5 .2-

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

::> :r ,. u 2: 2 3 4 5 6 7 8 9 10 11

4 .7- __

.. .... .. -

Figure 1. Southern blot analysis ofEcoR I-digested Chinese hamster (CH) . mouse (MMU). and mouse x Chinese hamster hybrid (ftHlt·s 1- 11) DNA using Pmcl34 as a probe. A major fragment of 4.7 Kb was detected in mouse DNA and in all hybrids retaining mouse chromosome 7 (lam~s 1- 9 a"d 11). Two other large mouse fragments were tOO weak to be scored in the hybrids. Cluncs(" hamster DNA generated a m.ajor fragment of 2.7 Kb and minor fragments of 4.0 Kb, 2.9 Kb. and 2.3 Kb. all of which arc visible in most of [he hybrid lanes. Siu markers of 4.7 and 5.2 Kb are indicated.

in DNA of all hybrids retaining mOllse chromosome 7 (Fig 1). All other mouse chromosomes were ruled our as possible si tes of hybridization with Pmel 34 by at least rwo discordant hybrids (Table I).

Isolation of the Mouse Tyrosinase Gene and Genetic Map­ping Near the C-Locus Two genomic dones. both of which contained the 5' end of the mouse tyrosinase gene. were isolated by screening NIH 3T 3 cell and BALB! c mouse genomic libraries with human tyrosinase eDNA probe Pmel 34: the fixMTY -2 clone from the NIH 3T31ibrary contained a 7.0-Kh insert, and the ChMTY-l clone from tbe BALBlc library contained a 5.2-Kb insert. The relationship between the twO genomic clones, partial restriction map. and {he map position of the 5' coding region of mouse tyrosin­ase gene are shown in Fig 20. The 4.7-Kh EeoR 1 fragment and the conriguous 2.3-Kb EcoR J intron fragment from fixMTY -2 were referred ro as PTY-1 and PTY-2, respectively. The partial restric­tion map agreed with chat of Ruppert et al [30]. Nucleotide se­quence analysis revealed that PTY -1 contai.ned the nrst exon of the mouse tyrosinase gene. We found tha t the 5' end of5.2-Kb BALBle genomic fragment was at the HaelIl site located at nucleotide 54 of the coding region of the mouse tyrosinase gene (Fig 2b). Figure 2b shows the nucleotide sequence of 819 bases of the 5' coding se­quence in the first exon and 300 bases of 5' promoter region of the mouse tyrosi nase gene. which is contained in PTY -1 . The homol­ogy of nudeotides between the human and mouse sequences is approximately 85% in the coding region. This comparison also revealed that the Pmel 34 eDNA probably did not contain [he first nucleotide A of {he first ATG codon. The complete eDNA se­quence of mouse tyrosinase and deduced amino acid comparison bet'A'een human and mouse ryrosinases have been published [91.

Within the 300 bases of the 5' prommer region. three TATA box-related elements were identified at positions - 111 (TATATA). -166 (ATAAAA). and -248 (ATATAT). and one CAT box-related element. at position -281 (CCAAT) (Fig 2b). Recently Ruppert et al [301 identified twO transcription start-sites of mouse tyrosinase gene at positions -81 and -1 36 from the nucleo­tide A of the first ATG codon. The first TATA clemen[ at position -11 1 shown here corresponds to 30 nucleotides upstream of the first transcription stan-site at position - B 1. and the second TAT A element at position -166 corresponds to 29 nucleotides upstream of the second transcription start-site at position -136. The third TAT A box-related element (position - 248) appears at an unusual

VOL. 93, NO. 5 NOVEMBER 1989 MAPPING OF Tt-lE MOUSE TYROSINASE GENE 591

Table I. Correlation o f Sequences D etec ted by Pmel 34 with M ouse C hromoSQme in Mouse X C hinese H amster Somatic C ell H ybrids

Mouse Chromosomes

Hybridiz2tion/ Chromosome 2 3 , S 6 7 B 9 10 II 12 13 14 IS 16 17 I B 19 X

+1+ 5 7 5 3 2 , B S 2 4 0 S 2 , 7 7 6 3 S 6 - I- I 2 I I I 2 2 2 I 2 I 2 2 I I 2 I I I +1- 3 2 , 6 7 , 0 , 6 5 9 , S , 2 3 5 , 2 - (+ 0 0 0 0 0 0 0 I 0 0 I 0 1 1 1

TO!:1/ discord:!.n~ , 3 , 6 8 , 0 , 6 6 9 S S , 3 2 3 6 S 3 Total ",formative 10 II 11 10 11 9 10 9 10 11 11 11 9 10 II 10 II 10 II 10

- The numhl:r$ o(h)'b~l(is [h~[ are concord:!n! (+/+ or / ) and diJcorwm (+/- or - /+) with the pre~nce of the molUC ryrOSlnllS( ~qucnGe are given (or each chtomolOme. H vb~d~ 11\ wllleh a pal'll1:.ut1.T dUCllnOliQfm.· W'U m'lclunll.,. ft :l.rr:mged or W1.li pt~m III fewer Ihan 10% of (("Us wete eJ[c\uded.

,wnn I 1~ 1

""".

j

1 j t

.. ,. >it ~

m·. m·' , tco"! t )lh4 tll

,. m •• . .. t.oermoo' ~ -al

_lfIl ~ ~ ~ t..;;wr-J'C' m::nrNJT 'lJrC!'T.¢II: ~ -ill

-lHI m:tJCJNC:~~C\'tI:tTIID.~~~ _\Iro\

- 1'£1 M;N:IU11C IJf:r1tKNC ~~ tJDrNNC.ut:n:AIIICf CN;TtrI(J%;' - 11

It_ t)'f'tIIIU- I -lC CD:: CI'C a:r r:n r.t TIC 'Ttl: t:IC at: TCC ICr TIt CI(; Kr. m:: a:r OX OQ' T1t 39 _ ".~. ~ ... . .. ~ _.~ ... _ .... ~~A _ . ~.

t;;;'1j1 ~ a:r~~=~m::~~~aGm~~~'TtI:m~~TCC~ ill •...•.... ... ~ .. . ....... .. ~.~.- .~ - .... .•.... ~ ... ~ -__ a __________________ ~

Ul .. l tICJ .. t .lCt ~ .. t ... .. .............. " ..... t ... C •• '" ...... UIO •• ____ m _a_m_m _________ m

at .... r;; ....... le • • J. ..... r;; •• r;; ...... .A ............. . . T ... . . C... 2IWI ma _____________ m __ m __ m

2011 .. C .. T .. G .................. ... .. . ..... ........ . .. .. . l . ..... . . c :m _____ m_m _________ m ___ _

• .. .. -..... ~ .•.. ~ .... ~.~ ....• .. •... ~ ... -_ ___ m_m _______ mm ____ m ill

• ....... ~ .~._ .... ~u .• .. ~ .~.~ ... ~ ............ _ m ___________________ _ _

ill .... .A . .. .. T ... .. ..... 1 .............. c. .. t ........ . .. . ..... C 1«1

~ -------------------- --... . ~ ..........••... ~.~ ... ~ ..• .... ~ .- ... ... -_ ________ m __________ m _

5011 .. C ......... • C • •• r;; .... .. . ........ C .c ..... .A ..... r;; .. . ... ...... 6ClI _ _____ a __ a ____ mm _____ _

fIIll . ........... ", .0> .............. t ..... l ...... .. I, n . ... ...... 61!0

___________ m _________ _

!'6'1 ..... T.fA c. ... . J. •. T , .r;; ..... G ......... r;; ....... t ..... T ... ....... ••• no _ ___________________ a _

111 ........ t ......... ... ...... ..... . T ••• " .... ct '" .. l fA. ... .. . lIID

m ~mm~~~nmmn~~~m~~~~m~ 0 1111 . ..... ... .. ... . ..... C .... ... .. •• c .... ,. ":,1'1:

Figure 2. a; partial restricrion map of twO tyrosinase genomic dones. The FixMTY -2 done was isolated {l orn :m Ni H 3T3 cell genomic library and tht" chMTY· t clone was isolated from a BALB/ c mOllS(" genomic libnry. T he di2gom:dly srripc=d :!.rea refers rO the first exon of mouse tyrosi n:l..Se gene. h: p2friaillucleoride sequence of mouse genomic clone PTY - t ;l.Ild sequence compl rison berween the mouse PTY -I and hum-all tyrosi nase eDNA done PUlel 34. The two nucl eotide sequences were al igned for maximum simil.;r­iry by introducing g:!.ps as indicaled by hyphem. The dou ed nucleotide resi. dues Are identical berween PTY -1 and Pmel 34. whereas the residues in the mause sequence that 2re different arc shown below the mouse sequence . Th~ Hae III sire at which [he chMTY -1 ,",unt St:l.rts is underlined. Positions of patenti;tl TATA and CCAAT box sequences arc ave-rlined.

,.

distance from both of the twO transcription start-sites. The only CAT box·related clement identified in this region appears at a point 33 nucleotides upstream of the third TATA-reb.ted dement. The signilicanCC" of multiple TATA-e1em enrs in the tyrosinase gene is not yet known.

The tv.·o mouse genomic DNA probes. PTY -1 and PTY -2. were used to idL'ntify restriction fragment length polymorphism (RFLP) in DNA isola ted from partially congenic mice that differed from each orher in the region of the :tlbino locus. £CoR I-digested DNA (rom mice o( the C57BL/ 6J,spretus- C Hblt stock was compared with EcoR I.digested DNA from the parent C57BL/ 6J.rb HbbP st rain . W bile PTY-l detected a single hybridizing band o f 4.7 Kb from an the mouse DNA employed (data nOt shown) , 2.n RFLP was dC'tr'cced when the pa rrially congenic C57BL/ 6).spretus-C Hbbo. C57BL/ 6J.ch HbbP. and C57 BL/ 6J·C·Hbb were compared using probe: PTY·2 (Fig 3). Each o f the tested offspring that received the MilS sprrtliS-derived C -Iocus marker on ch ro mosome 7 had the 3.7 Kb spmus form of tbe polymorphic tyrosinase EcoR I fragment

2 3 45 6 7 89 'D1l 121314 1516T7 Mark!!,

21

5.2

1 .•

Figure 3. Southern biOI aI12lysis of £CaR I-digested mouse genomic DNA hybridlud with PTY-2. DNA w:u isol2ted from mice carrying different alleles at (he C-Iocus 25 indicated. umes 1-14 and 16- 17 cont;linet:i DNA from mice pmenn.;i1y recombinanl between the RFLP muker :lind markers known to residt" in chromosome 7. SP: presence of the non-;tlbioo wild-rYf)C C 21 1e1e and the Hblt allele from Mus sprrtu.s; H: presence of the himalayan mutllH '" 21 1ele; cas: presence of [ht" non-:!.Ibina wild-IYpe Callele fromMUJ C(JStDIlt'US. LJllt' 1: SP / H; L:HI~ 2; H/ H: Lon("): H/ SP; LaPl( 4: H/ H; 1..0/1(' 5: cas/ H: La'It' 6: C2S/ SP; l..A,u 7: cas/ H; Lint 8: cas/ SP; LAnt 9: cas/ SP; LAnt 10: cv;.lcas~ umr 11 : H/H~ LallI.' 12: H/ H: unt 13: H/ SP; Lant 14: H/ H; Lan~ 15: C57BL/ 6; Llu(" 16: H/ SP; LAtif 17: H/ H. Siztmarkm are Hitld IJi·EcoR I fragments of A pllllge DNA. Minlu signs indic:!.f,e 3.7 and 2.3 Kb bands.

592 KWON ET AL

(lanes 1,3,6,8,9, 13,and 16) . None of the 11 possible recombinants between the albino locus and the spretus RFLP was observed.

If the chromosomal locus homologous co the PTY-2 probe had not been located on chromosome 7. then half recombinants and half non~recombinants would have been obtained in the rwo test­crosses. The binomial probability of obtaining an 11 : 0 ratio under these conditions is less than 0.00 I and that of obtaining as : 0 ratio is under 0.05. We therefore conclude that the RFLP detected by PTY -2 is on chromosome 7 close to Hbb and C. Thus, it is probable that the RFLP identified by PTY -2 is located in the region of the chromosome 7 that has been backcrossed for seven generations from MIH sprellls onto the inbred C57BL/6] strain of Mus musculus. That region of chromosome 7 contains the albino and the Hbb locus.

Southern Blot Analysis of Albino Deletion Mutants Cl 4CoS

and c6H represent large deletions. extending in opposite directions from and incl ud ing the albino locus on chromosome 7 [20]. Mice homozygous for either of these deletions die before or shordy after

21.7 -

5.2 -

1.6

Figure 4. Southern blot analysis of genomic DNA of albino deletion mu· tams. Twenty microgr:nllS of each mouse DNA of various genorypes was digested with EcoR I. electrophoresed on 0.8% agarose gel. transferred to GcneScreen Plus membrane. ~nd hybridized to PTY -2. Lone 1: rd'/cd'; umt 2: r"/(I~: umd: , 14C05 /cI'H; Loue4: cd'/~H; LAnd: c'H/c'H; LAnr 6: r"/~. A faint band (-4.7 Kb), seen in fallts 1, 2, 4, alld 6, is due to hybridization with PTY -1 because PTY·2 was slightly contaminated with vrY·l during elution of the fragmellts from the gel. Rehybridization of the same blot with a melanocyte.specific probe Pmd 17~1 [251 confirmed that aU lanes con· tained a similar amount of DNA (data not shown). The intensity of e.ach band in the autoradiogram was measured by a densitometer at 500 nm. MiliUS sign indicates 2.3 Kb b.and.

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

birth. Mice that are heterozygous for the overlapping cl.cos and c'H alleles, however, survive. The portion of chromosome 7 that is missing from these heterozygous mice is relatively small and in­cludes the albino locus [20]. As shown in Fig 4. ct 4CoS/c6H DNA (la ne J) contains no sequences that hybridize to PTY -2. However, DNA isolated from mice that were heterozygous for one of the deletioru did hybridize with the probes. As expected. the intensity of the 2.3-Kb bands from the heterozygous mice (lane 2) was approxi­mately half of tha t observed in lane 1, which contained DNA from homozygous chinchilla mice that carried rwo C-Iocus alleles. Simi­larly. DNA from mice homozygous for another C-Iocus deletion (c'") did nor hybridize with rhe probe (/.,,, 5), while the DNA from ~H/r-h mice (lane 4) produced bands that were labeled at half the intensity observed in the control (la'if 6) . The same results were obtained with the PTY -1 probe. The data again indicate that PTY·1 and PTY -2 map near or at the albino locus, :md thus the albino locus on mOuse chromosome 7 cOlltain the structural gene for tyrosinase.

Northern Blot Analysis To investigate the size and abundance of tyrosinase transcripr, poly(A)+ RNA from primary cul tures of mela­notic C57BL/ 6] and BlO.BR melanocytes and mela.notic Cloud­man 5-91 mouse melanoma cells were fractionated on denaturing agarose gels and h}'bridized to rhe mouse gene probe PTY-1. As shown in Fig 5. mouse tyrosinase mRNA was approximately 2.4 Kb in size and was highly abundam in C57BL/6] and BI0.DR melano­cytes (lanfS 2 and 3). The melanotic S-91 Cloudman melanoma cells contained less tyrosinase mRNA than BIO.BR or Cs7BL/ 6] mela­nocytes (la ue 1). The tyrosinase activities ofB 1 O.BR and C57BL/ 6] melanocytes were 400 ,uU/mg protein. while that of Cloudman S-91 cells was 5.2 ,lIU/ mg protein.

DISCUSSION

W e describe here the isolation and chromosomal mapping of the mouse tyrosinase gene. obtained by screening mouse genomic li­braries with POle! 34 human cDNA sequence [6]. The nucleotide sequence at the 5' coding region ohhe tyrosinase gene was highly homologous between mouse and man. and the mRNA detected by the murine gene probe was similar in size to human tyrosinase mRNA. The levels of tyrosinase gene tr:mscripts reflected the levels of tyrosinase activity in normal melanocytes and Cloudman 5-91 melanoma cell lines. T he levels of ryrosinase mRNA were signifi-

1 2 3

28-

Figure S. Expression of tyrosinase mRNA in normal murine melanocytes and melanoma cells. Five micrograms of poly{A)+ RNA from Cloudman S-91 (UJllt I) , C57BL/6J (Iaru 2), and BIO. DR (Iant. J) melanocytes were e1ccrrophoresed 011 a 1.2% fonnaldehyde denaturing agarose gel , blotted, and hybridized to the PTV -1 probe.

VOL. 93, NO. 5 NOVEMBER 1989

candy reduced in the mdanoma cdls by .:I: factor of 10 or more, suggesting that the down-regulation of tyrosinase activity in some melanoma ceUs is controlled, in part, at the levd of transcriprion.

Recently , several reports have described rhe isolation of mOuse tyrosinase cDNA [8-1 O, 12J. Because thecDNA sequenceofShiba­hara's done {12] was different from the others. there was ambiguity as to which clone encoded tyrosinase, and whether or nor there was mort than one tytosinase gene. The problem of determining which of the dones coded for a protein with tyrosinase activity was further aggravated by the fact that all the clones were melanocyte-specific. encoding highly abundant mRNAs, and the deduced amino acid sequences revealed that an were gtycoproteins with similar molecu­lar sizes and with consen ted positions of histidine and cysteine resi­dues [6]. Some of th.e proteins cross-reacted with antibodies raised to others. For example, anti-tyrosinase antibodies detected not only cON A clones ptoducing tyrosinase, but also another set of unrelated dones. Pmel17-1 [251. as well as a clone encoding anas-yetunjden­tificd protein (13]. One monoclonal antibody raised against gp7S, known as 2G 1 O. also reacted with ryrosinase. immunoprecipitatin.g 40% of tyrosinase activity from cell extracts and a monoclonal anti­body against the protein encoded by the b locus immunoprecipitated tyrosinase and Pme117-1 protein (Balaban et al. unpublished obse[~ varion).

Studies by Muller et al r10l. in functional analysis of tbe eDNA after transfection of plasmid carrying these clones into tyrosinase­nega tive cells, demonstrated that the protein encoded by the c, and nor by the b locus. directed the synthesis of a protein wi th ryrosinase activity. Muller's data, those described in this paper. and those re­vealing structural alterations in ryrosin2se proteins in vanoLlS ,-locus mut2nts [31] demonstrate that the ( locus encodes the structUral gene for tyrosinase and that the ryrosinase gene is present as a single copy.

The enzymatic activity of the b-Iocus proteins was recendy shown to be a catalase, and immunologic studies have demonstrated that gp7S appeared to he identical with the b~locus protein (Hala­ban, Moellman. and Straight, unpublished).

Tyrosinase is a key enzyme in mdanin synthesis. and levels of tyrosinase activity in human meianocyres and in some melanoma cells are regulated by tbe levels of tyrosinase nlRNA. as described here. However, other gene producrs are undoubtedly critical for melanin synthesis, such as those involved in the formation of pre­mdanosomes and melanosornes and in the transport of tyrosinase ro these subcellular organel les. For example, mutation at the pink~ eyed dilution in mice drastically reduces the pigmentation of coat­color {1]. Some of these proteins that affect pigmentarion might be Pme! 17-1 protein [25] or the protein encoded by the brown locus. Mclanotropin (MSH). known for its action on increasing tyrosinase activity and melanization in mouse melanocytes. also increased the abundance of tyrosinase protein 1321 and mRNA 19]. and the mRN A levels of Pmd 17-1 protein f25J . Therefore. the aV:lilability of probes specific to the gene products involved in pigmentation is importa.nt for studies of the albino phenotype in cases where che defect is not in tyrosinase.

It is interesting that the tyrosinase gene comains three TA TA·el~ ements and only onC C AT-element. and that the CAT -element appears to be paired with the TATA-e1ement located farthest IIp­stream. Whether such multiple promoter-eleme,nts playa role in the developmental or cell-specific regulation of tyrosinase gene ex­pression remains to be determined.

Note Added in Proof After the manwcript was typeset, we deter­mined rile further upstream nucleotide sequence of the 5' flanking region of the mouse tyrosinase gene. There are tWO copies of TAT AA elements at the positions - 405 and - 455, which are found in the promoter region of hsp 70 {33). In addition. a sequence with high homology to the consensus elements of the nuclear re­ceptors TRE and RRE (thyroid- and retinoic acid-responsive ele­ments. GGTCATGACC, see Ref 34 for review) is also prescnt at position - 340. TTTCA TGACC, in wh,ich 8 of 10 residucs arc invariants. Yamamoto et al [35J recendy published the 5' flanking

MAPPlNG OF THE MOUSE TYROSINASE GENE 593

sequence of [he mouse tyrosinase gene and his sequence is identical to ours.

When the first exon sequence of rhe Balb/ c tyrosinase gene was compared wirh thar of C57DL/ 6 110], we found chat there was a single G to C nucleotide change at 30B. altering the cysteine in C57BL/ 6 tyrosinase to serine residue at amino acid 103 in Balb/ c. Considering that this cysteine is one of the conserved amino acids between b-protein and tyrosinase 16). this muution might be a cause of the albinism sec::n in Balb/c mice.

Tlte a«fllOn Intlnk Dr . S. CluedtsoJin~Wat:lsch JOT providing tllbino ddelion mitt, Norry C. DatlisJor consultation on s(tltistical're4.tntfflt olthe Jato, and Helttl Kelley, Elaine Wall, and Mary Ki9frfor rypillg thr monl4snipt. ThU work WQj5upportetl in port by grallu frOnt S(heri,lg Corporation and Jrom NTH 1 R2J A12J058-02 (B.S .K.) and 5 R.Ot CA04679 (R.H ., A ,B.L.) . A.K.H. WIl5 supported in paT! by Q dOtlolilJtl jrom LB.M. (OUltgo. NY) .

REFERENCES

I. Silvers WK: The Coar Colors of Mice: A Model for Mammalian Gem~ Action and Interaction. Springer Verlag. New York . 1979

2. Fitzratrick TH, Miy:unoto M . Ishikawa K: The evolution of concepts o mebnin biology. In: Montagna W. Hu F, (eds.). Advances in Biology of Skin. vol. 8. Pergammon Press. New York, 1966. pp 1-30

3. Prot::l G; Recent advanc('s in tbe chemistry of melanogenesis in mam­mals. J In veST DerrnatoI15:122-127, 1980

4 . Pomerant2 SH: T yrosine hydroxylarion catalyzed by mamnuJian ryrosin:llie: an improved method of assay. Biochem Biophys Res Comm 16:188- 194 , 1964

5. eokman DL: Effect of genic substitution on {h(' incorporation of ryrosine into ti1(, melanin of mouse skin. Arch Biochem Biophys 96,562- 568. 1962

6. K won BS. Haq AK. Pomerantz. SH. Habban R: Isolarion and sequence of a cDNA clone for human tyrosinase that maps at the mouse c~albino locU5. Proc Natl Acad Sci USA 84:7473 -7477. 1987

7. Barton DE. Kwon BS. Francke U: Human tyrosmase gene. mapped to chromosome 11 (q I4--q21). defines second region of homology with mouse chromosome 7. Genomjc5 3: 17 - 24, 1988

S. 'Y amamoto H.. Takeuchi S, Kudo T , Makino K. Nakoata A. Shinoda T. Takeuchi T: Cloning and sequencing of mouse ryrosiruse cDNA. Japn J Generics 62:27 1 - 274. \987

9. Kwon BS. W2kulchik M, Haq AK., Halab:m R. Kestler D: Sequence analysis of mouse tyrosinase. eDNA md the effecl of rnelanotropin on its gent' expression. Biochem Biophys Res Comm 153: 1301 -1309.1988

10. Muller G, Ruppert S. Schmid E. Schurz. G: Functional :malysis of alternatively spliced ryrosinase gene transcripts. £MBa 7:2723-2730. 1988

II . Bouchard B. Fuller Bll. Vijayasaradhi S. HoughtOn AN: Induction of pigmentation in mowe fibroblasts by cxpres~ion of hum~ tyrosin~ as< eDNA. J Exp Med 169,2029- 2042. 1989

12. Shibahara S. Tomita Y. SabkuT3 T. N2ger C, Chaudhuri B. Muller R: Cloning 2nd expression of eDNA encoding mouse ryrosimse. Nu~ deic Acids Re.Io 14:241) -2427. 1986

13. Jackson IJ: A eDNA encoding ryrosinast'~rebtcd protein nu.ps to the brown locu~ in mouse. Proc Nat! Aead Sci USA 85:4392-4396. 1988

14. Spriu R. Strunk K. Oening W. King R; RFLP for Taql at the human t)'rosjn.a.~e locus. Nucleic Acids Res t6(20}:9890. 1988

15. K won BS. Haq AK, Wakukhik M, H .llaban R. Witkop C. Liddell A., Kestler D: Molecular genetic ana.iysis of human and mouse aJbinism (abn). J Invesr DermatoI90(4):578. 1988

16. T2mura A. Habban R. Moellmann G. CowanJM. Lerner MR. Lerner AB: Nonnal murine melanocytes in cuJture. In Vitro Cell Develop 8;0123,5 19-522.1987

17. Hahban R. Nordlund J. Francke U, MoeUnunn G. Eisenstadt JM: Supermela.notic hybrids derived from mouse rneianom.:lS and normal mOwe cells. Somatic Cell Genetics 6:29 - 44, 1980

18. Francke U . Lalley PA, Moss W , Ivy F, MinnaJD: Gene m.apping in

594 KWON IT AL

Mus musc-wius by intt=rspecific cell hybridization: assignment of the genes for tripcprida.sc:-l to chromosome 10. dipc:pricbsr-2 to du'O­mosome 18. acid phospha~- l [ 0 chromosome 12. and adenyl­;litc kinase-l EO chromosome 2. Cytogenet Cdl Genet 19:57-84, 1977

t 9. Fr.mcke U. Taggart RT: Assignment of the gene for cytoplasmic su­peroxide dismuwe (Sod-I) to a region of chromosome 16 :md of Hprt to a region of the X chromosomt in the:: mouse. Proc Nad Acad Sci USA 76,5230-5233,1979

20. Gluecksohn-Wadsch $: Genetic control of morphogenetic and bio­chemical di£fC=['f'nciarion: lethal aJbino dderiolU in the mouse. Cell 16:225-237. 1979

21 . Whitney JB III: Simplified typing of mouse hemoglobin (Hbb) pheno­types using cystamine. Biochem Generics 16:667-672, 1978

22. de Martinville B, W yman AR. White R, Francke U: Assignment of fi rST nndam remicrion fT2gmenr length polymorphism (RFLP) locus (DI4S1) to a regio n ofhum:m chromosome 14. Am] Hum Genet 34:216 - 226. 1982

23. G ross-Bellard M. Oudet P. Chambon P: lsol:nion of high-molecular­weight-DNA from mamnuli..an cells. Eur) Biochem 36:32-38. 1973

24 . Southern E: Gel electrophoresis of restriction fragments . Methods En­zymoI68:152-176.1979

25. Kwon BS, Halaban R. Kim GS, Usack L, Pomerantz S. Haq AK: A melanocyte-specific complementary DNA clone whose expression is inducible by melanotropin and isobutyl methyl xanthine. Mol Bioi Med 4,339-356,1987

26. Messing ) . Crea R, Seeburg PH: A system for shotgun DNA seq uenc­ing. Nucleic Acids Res 9:309-322.1981

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

27. Sanger F, Nicklen S. Coulson AR: DNA sequencing with chain-termi­nating inhibitors. Pmc Nat! Acad Sci USA 74:5463-5467.1977

28. Biggin M. Gibson T . Hung G: Buffer gradient gels and 'SS label as an acid to rapid DNA sequence determination. Proc Nul Acad Sci USA 80,3963 - 396S, 1983

29. Halaban R. Pomera.nn S. Marshall S. Lambert OT, Lerner AB: Regu­lacion of tyrosinase in human mdanocytes grown in culture. ] Cell Bioi 97:480 - 488. 1983

30. Ruppert S. Muller G. K won BS. Schun G: Multiple transcripts of the moust tyrosinasc: gcne are generated by alternative splicing. EMBO J 7m IS-2722, 1988

3 1. Halaban R. Moellmann G, Tamur.a A. Kwon BS. KuklinskaE. Pomer­ann SH. Lerner AB: T yrosin.tses of murine mel.:tnocytes with muta· tions ar the albino locus. Proc Nat! Acad Sci USA 85:7241-7245. 1988

32. HaJaban R. Po merantz SH, Marshall S, urner AD: T yrosinase activity and abundance in Cloudman melanoma cells. Arch Biochem Biophys 230,383-387,1984

33. Simon MC. Fisch TM. Benecke B) , Nevins R] , Heintz N : Definition of multiple. funcrionaJly distinc[ T ATA dements. one of which is a targe[ in the hsp70 promoter for EtA regularion. Cc:1I52:723- 729. 1989

34. Beato M : Gene regulation by neroid hormones: Review. Cell 56:335-344, 1989

35. Yamamoto H. T akeuchi S. Kudo T . SatO C. Takeuchi T : Melanin production in cultured albino melanocytes mnsfected with mouse tyrosin25e eDNA.)pn] Genet 64:121-135.1989