isolation and characterization of epidermal dna and rna ... · solution 2 (12). i solation of e pid...
TRANSCRIPT
T HE JounNAt~ OF 1 NVEsTI GA'J'JV0 DEIU.IATOLOOY
Copyrig h t© 1971 by The Willinms & Wil k ins Co . Vo l. 56, No. I
Printed t"n U .S.A.
ISOLATI ON AND CHARACTERIZATION OF EPIDERMAL DNA AND RNA FROM GUINEA PIG SKIN"·
D. M. KRAMER, PH.DJ , M.A. PATHAK, PH.D. AND UTE GUNGERICH. B.S.
ABSTRACT
DNA and RNA were isolated from mammalian epidermis in a relatively small scale procedure. The high purity and native state of the DNA isolated is reflected by its molar absorptivity E (P), its t hermal hyperchromicity and its hyperchromicity upon DN ase t reatment and by its sedimentation profile as well as by its profile in a cesium chloride density gradient. The very low content of protein and RNA, as well as the data of DNA determination, indicate that this method permits the isolation of a highly purified product. This is further substantiated by the determination of UV absorption spectra and by analysis of the base composition .
The mammalian skin DNA showed the following proper ties : Mol. wt . = 3.7 x 10"; melting point = 80.7"C; E(P) = 6150 ; molar N/ P ratio = 3.67; thermal hyperchromicity = 40.9% ; hyperchromicity upon DNase treatment = 38% ; sedimentation coefficient == 16.3; boyant density in CsCl = 1.6969 g/ cm' . The guanine, cytosine, adenine and thymine content was 19.0, 20.5, 29.0 and 28.5 moles% respectively.
Epidermal RNA was isolated and characterized in terms of phosphorus content, RNA content , and hydrolysis by RNase. By our results, a minimum content of 3 mg DNA per 1000 mg epidermis (minus stratum corneum) appears reasonable.
Along with our studies of the molecular mechanisms of cutaneous photosensitization by psoralens (I) and of sunburn reaction caused by 290 to 320 nm radiation, it became necessary to isolate deoxyribonucleic acid (DNA) from mammalian epidermis in a highly pure fo rm. The method of choice was that reported by Kirby and Cook (2), which had to be modified to overcome interferences caused by an abundance of kerat inic proteins and polysaccharides present in the tissue.
D NA from rat skin had been extracted with sodium dodecylsulfate, but no analytical data are given to characteri ze t he DNA (3). Using various methods, DNA and ribosomal ribonucleic acid (r-RNA ) were isolated from mouse skin as well as from dennis, epidermis, and basal
Received March 30. 1970 ; accepted for publication August 18, 1970. P resented at the Thirtyfi rst Annual Meeting of the Society for Investigative Dermatolog,v, Inc., June 20, 1970, Chicago, Illinois.
This work was supported by Grant No . 5R01 CA 05003, National Cancer Institute , DHEvV, and by a grant of "Deutsche Forschungsgemeinschaft".
*From the Photobiology Un it, Depar tment of Dermatology, Harvard .Medi cal School, Massachusetts General Hospital, Boston, l\1assachusetts 02114. (Reprin t requesls to Dr. Pathak.)
t Present address: Physiologisch-Chemisches Institut der Johannes Gutenberg Uni versitiit, Mainz, \Vest Germany.
55
cells by Goshman and Heidelberger, who also established the identity and analytical purity of the nucleic acids isolated; however, only li ttle detail ed information is given concerning the isolation procedures and t he yields ( 4) . For whole rat skin, DNA contents of 64 }1-g (5), 71 }1-g (6), and 84 to 92 }1-g (3) per 100 mg wet skin have been reported. Autoradiographic studies with organ cultures of human skin following the incorporation of thymidine-t ritium into DNA have revealed that DNA is synthesized in t he nuclei of basa l cells as well as in those cells above the basal layer (7). Analogous studies with hairless mice have shown that the synthesis of DNA is highest in the basal cells. Using the fluorimetric method with 3, 5-diaminobenzoic acid, which is one of the most sensitive and specific ways to determine DNA in t issues (9), 82% of the total skin DNA was found to be located in t he epidermis and l So/o in the dermis ; no DNA was found in the st ratum corneum (10) .
A decrease in DNA content of mouse epidermal cells undergoi11g keratinization has been observed (11). As far as tlw RNA content of skin is concerned, values repor ted in the li terature are about twice as high for RNA as t hey are fo r DNA (6).
In our present study \re describe t he isolation
56 T HE J OURNAL OF I N VESTIGATIVE DERMATOLOGY
of DNA and of RNA from epidermis of "albino" gu inea pigs. Little or no t issue of the dermis was involved in our procedure.
MA'l'E IUALS AND ME'l'HODS
R eagents . Kirby solution 1: A mixture consisting ol' 1 g sodium t ri-isopropylnaphthalcnesulJonate (Eastman-Kodak) and 6 ml butanol-2-ol was prepared to wh ich were ::tdded 100 ml water, 6 g soclium-4 am1nosali cyl::rte, and 1 g NaCl (2). Kirby solution 2 : phenolcresol mixture wa. made by adding 55 ml water, 70 ml m-cresol, and 0.5 g 8-hydroxyquinoline to 500 g of phenol. Phenol, as well as cresol, has to be eli t ill ed before making K irby solution 2 (12).
I solation of epidennal DNA and R NA (1). The adult "albino" guinea pigs (700-900 g) were immobili zed on a wooden board. The ent ire back of each animal was epilated (13). After 48 hours the animals were sacrifi ced under et.her anest he:ia. kin of t he epilatcd dorsum measuring 7.5 X 17 .5 em (135 em") was removed and immediately dipped i11to 25 ml of co ld K irby solution 1 to inhibit the action of skin deoxyribonuclease (DNase) . The epidermis, including the horny layer, was then separated from t be underlying t issue by t he stretch and scrape method (14); during thi scraping operation, 2 ml of K irby , solu lion J was applied to the surface of the skin. The epidermal t issue was transferred inlo t he 25 ml of cold Kirby solut ion 1 mentioned above. About 0.9 to 1.3 g of epidermal t issue (wet weigh t) wa. obtained from a 135 em" skin area. The t i sue was then homogenized , using 7 to 9 strokes, in a glass homogen izer of the PotLer type. Small amounts of Kirby solut ion 1 "·ere added for rinsing purpose: dming this operation ; the fi nal volume of homogenate per animal epidermal t issue was 30 ml. This volume is rather critical, since smaller vo lumes (e.g., 20 ml) were found to interfere wit h t he pbenul-cr ol step (no top pha:e could be obtained), and larger volumes will interfere with the first precip itation . tep (no DNA will be precipi tated).
The purification process was carri ed out according to Kirby and Cook (2) with the following modifications : (a) Much longer cent ri fugat ion t imes (60 minutes), higher g va lues (20,000 g), a nd repeated centrifugation · were required to obta in a clc:n top phase from the phenol-ere-
sol tep. The first phenol-cresol step produced 16 to 18 ml of top pha e, and after the second phenol-ere. ol step 11 to 13 ml of clear top pha e was obtained . (b) For the first p recipitation step of DNA and RNA, 0.8 to 1.2 volumes of butoxyeth::mol were needed, nncl the mixture had to stand overnight at 5°C.
For t he second and t hird precipitation steps, t he gel was dissolved in 3.5 ml of 0.1 M sod ium acetate buH'er, pH 6, con taining sodium chloride (3 % W/ V) ::tnd .·odium benzoate (20% \T)TjV), and t he DNA was precip itated with 0.2 volume of butoxyethanol. For the :eparation step of DNA and RNA wilh 3 M NaCI at oac, a volume of 2.5 ml of NaCl solut ion was taken. The RNA was sedimen ted b.r centrifugat ion at 10,000 g for 20 minute.· at oac, and the supernatant that contained the DNA was removed. The RNA was washed twice with 1.5 ml of ice-cold buffer solu tion, pH G.O, containing 3 M NaCl and sodium acetate and ,,·as dissolved in 0.1 lVI ·odium acetate butTer, pH 6. This RNA solution was also dialyzed against t he same buffer .
The supernatant from the 3 M NaCl step was processed for DNA, u ·ing t he procedure described by Kirby and Cook (2). This involved centrifugation over 6 M N aBr (2 ml), precipi t::tt ion with ethoxyetbano l, dissolut ion in sodium acetate buffer, and dialysis.
l<'or spectrophotometric studies the solu tion of DNA in 0.1 M sodium acetate buffer, pH 6 wa used. For analytic::tl determinations involving weighing operations, t he DNA again was precipitated with ethoxyethanol after its sod ium acetate buffer solution had been made 3 M with respect to NaCL This DNA was wa ·heel twice with cthanol-wnter-:oclium acetate (75: 25 :2, vj v / w) solution and then with a mixture of i o- , propanol-water (first Oo/o , then 96% isopropanol) . This was followed by three washings in ether and drying under reduced pressu1·e at room temperature (15). This procedure gave us a form of DNA (sodium salt) with a water content of 8 to 10%.
M ethods of characteTization of DNA. Ultraviolet (uv) spectra were recorded in a PerkinElmer 350 spectrophotometer. Phosphorus determination was carri ed out using t he method of Weil-Malberbe (16), according to perchloric acid treatment of Allen (17). N itrogen was determined with a modified Kjeldabl procedure (1 8). For protci11 determination according to
I SOLATION OF EPIDERMAL DNA AND RNA 57
Lowry's m ethod (19), 1200 fJ.g of D NA were taken . The R TA content was determined by W ebb's method (20) , and t he DNA content accordin .,. to Kissa ne's fluorim etric m ethod (9) ; herrin~ sp erm D NA (sodium salt, from Fa. 1viack, Ill ertissen, 1;v est Germany) w:ls taken as a r eference standard. This st::~ nd ard DNA prepared by Z::~hn 's heat dete rgen t m ethod (15) contained 1% protein , 0.7 7< RNA a nd 8% H"O ; molar ab orptivity (E [P] ) at 260 nm fo r nucleic acid based on one gr::~m atom of phosphorus per liter ' in standard sa line ci trate, (0 .15 M NaCI, O.OlS M t ri-sod ium. citrate) pH 7, was 6150 (±100) and molecular weight e tablished by lirrht -scatteri ng was 1. X 10' (21).
"'Melting point and t hermal hyperchromicity of the epiderma l DNA were measured in Dr. P. M. Doty's laboratory at Harvard Un iversity, using a Beckman DU spect rophotometer equipped with a utomatic program med acce sories for temp erature elevation and recording (22). The sedim entation coefficien t was determin ed in nn analytical ult racen trifuge (Spinco l\Iodel E, Beckman) at 260 nm . Centrifugat ions " ·e re ca rri ed out at 18000 r.p.m. These measu remen ts \I"CI"C
done in Dr. U. H agen's laboratory at t he In "Lit ut f i.ir Strah lenbiologie, K ern f orschu ngszent rum K arls ruhe, \~T . Germany. The .·cdimentation coefficients obta incd at d iffercnt conccn trations were extrapo la Led to ze ro concen1 r~11 ion. whi ch results i11 8~0 . From t he rela t ion bet\\"crn S~0 a nd int rinsic viscosity, established by E igner and Doty (23) and by Weinert and Hagen (2-1), i.he avera,.e molecula r weight 11·as ca lcula ted acco rd-"' . ing to t he equ ation given b~, Mandelkern, I<ng-baum, Scheraga and Flory (25).
D etermiJl <ttion of buoyant dens ity in ces ium chloride was carri ed out according 1o Yinograd et al. (26) in a Beckman-Spinco . 1Iodel L-50 centrifuge, using a type 40 rotor at 32,500 r.p .m. Average dens ity of t he cesium chl oride solution, conta ining 150 p.g DNA/ ml , 1\"::t .· 1.7005 p;j cm' . Centrifugation t ime was 140 hom;:; . The dcnsit..v gradi ent was fractionated using a tube p iercer device obtained from Measuring and , cicntific Equipment Limited, London, England. Optical density of the efflu ent at 260 nm " ·as reco rd ed with a U vi cord II (LKB Instrum ·n1·s, Inc.,
tockbolm, Sweden). In addit ion , t he optical density of each fraction was measured in a Ze iss PM QII spectrophotometer. The refra ctive index was measured in a Zeiss-Abbe rcfr:t ctome-
ter. From t hese data, the density of t he DNA "·as ca lculated from t he 50% concentration poin t of t he DNA profile, after its in te,. ration.
Analy is of ba e composit ion by uv spectrophotometric determination after formic acid hydrolysis, pnper chromatography and elution with 0.1 N H CI, \WlS ca rri ed out acco rding to Wyatt (27).
RE SU LT .
900 (±200) f.tg epidermal DNA and 1600 (±350) ~,g RNA were isolnted f rom a 135 em" dor al aren of one guin cn pig. From 20 a nimals a totnl yield of 1 mg DNA was obtained. Remova l of the horny layer by ten sequential app li cation of an ad hesive tape in two living anima ls prior to sac rifice r esulted in a t wofold increase of DNA yield and n sharp decrense of RNA yield ( lawn Lo 400 tJ.g).
Ana1yt.ica1 and p hys ic;l l lata of t he epidermal DNA are summarized in t he Table.
These cbta indicate a high degree of chemical purit.y and naLive state of t he DNA isolated. Its rather . ymm etricn1 profil e in t he cesium chloride den.·ity grad ien t inclicntes an ab ence of major a moun ts of ..:atelli te DNA or of clennturatcd D~A (Fip;ure 1).
The DNA isolated wa ea.- ily hydroly ~ed by D Nase·>:·. The nbsorpti on pea k at 260 nm in 1he ces ium chloride density gradi en t disappea red upon DNase 1rc;~tment·x·. The DNA wa table to RNnse t reatment-\". These fi nclin o-s add .1dcl it iona1 suppor t for the iden t it~' a nd puriLy of t he isola ted DNA. From the buoyant dell ity in ce·ium chloride (1.6969 g/ cm' ) a o·uanine-cyto ·inc content of 37.7 mole % ca n be estimated. This vn lue satisfacLorily agrees with a guanine-cyto-in e content of 3 .6 m ole percent ca lculated
from t he melt in o- point, acco rding to Lhe relation given by Scllilclkraut :1 ncl Lifson (28) . The melting p rofi le of the DNA is given in F igure 2. By dete rmination of the base composit ion, u ing Wyatt's m ethod (27), we fo und 19 mole % guanine, 20.5 mole 'Yo cytosine, 29 mole % adenine and 2 .5 mole 7o t hymine; t he recovery of moles of bnses per mole of phosphn te present in the hydrolysate being 0.97. No nracil was detected on the paper chromatorrrnms.
* dcoxv ribonucl cnsc-A from beef pfln ercas, Sigma. DN-EP, 2000 K uni lz uni ls/ mg. . ,
j· ribonu clease-A from beef pancrefl s S1gma XII A. 100 Kunilz unit s/ mg.
58 THE JOURNAL OF I NVESTIGATIVE DERMATOLOGY
TABLE
Analylical and phy.s·ica l data of epidermal DNA from alb·ino g'ttinea pig
Data
DNA co nte nt (sodium sal t) RNA eo nte nt Protein co ntent H,O co nte nt
P hosphorus con Lent Nitr ogen
!)1%
0 .2% 0.2% !J.O%
8 .5% 14. 1% l. GG 3.67
Remarks
Weigh t loss upon drying in vacuo at (i0°e .
Weigh t rat io N/P Molar rat io N /P E(P) at 260 11m Abso rbance rat ios at:
6150 ± 100 In standard sali ne-cit rate b uffer, pH 7.0 In stand ard saline cit rate buffer, pH 7.0
esse buffer) 280 nm/260 nm 260 nm/230 nm
0 .516 2.40
Buoya nt dens ity in esel Sedimentation coefficient Average molec ul ar weigh t Melt ing poin t
l. G969 g/cm3 16.3 At 20oe, in SSe buffer
From S 020 3 .7 X 10°
80.7 oe In sod ium acetate buffer , 0.1lVI , pH 6.0 In sod ium acetl\le buffer , 0.1 M, pH G.O In 0.1 Msodium phosphate buffer, pH G.O
Thermal hyperchromi ciLy at 260 nm Hyperchromicity upon DNase-hydrolys is
40 .9% 38. 0%
1 .00--------------- 1.4 3
0 .75
0 .50
0 .4 5
" 1.42 ~ h.
"" "' ..,
1.41 ~ ~ I::
" ~ 1 .40 .... ~
0.40 '----- 1-'-0 _____ 2_,_0 _ _ _ _ _} 1.39
FRACTION NUMBER
FIG. 1. Equilib riu m sedimentation of epidermal DNA in a cesium ch loride density gradient. Soli d li ne: optical density at 260 nm; dotted line : refractive index at 20°C. For deta il s sec methods and material.
The concentration pro.fil c of the DNA upon sedimentation in an analytical ult racentrifuge is shown in Figure 3 and gives further evidence that our preparation consists mainly of DNA in the native state.
0.63 ,/
0 .61
!: 0 .59 I <: I
~ I
0.57 I
"' I h.
"" 0 .55 ;...--rm
"- I h. 0 .53
~ i ~ 0 .51 i ..., / ~ i:::
0.49 I ~
0.47 I 0.45 ...... , .·~- ·- ······ · ···
/
50 60 70 80 90
TEMPERATURE ( •c)
Fw. 2. M el ting profi le of epiderma l DNA (21 !Lg/ ml) in 0.1 M sodium acetate buffer , pH 6. Au ton:iatically controll ed rate of temperature eleva lion was 1 oc per 15 minutes.
The epidermal RNA showed a phosphoru content of 8.5%. It was stab le to DNase treatment and was easily digested by ribonuclease-A, when assayed in Kalnitzky's test system (29). Using Webb's method for RNA determination
I SOLATION OF EPIDERMAL DNA AND RNA 59
(20), and a standard RNAt for reference, t he epidermal RNA content was fou nd to be 106% of the total RNA isolated.
DISCUSSION
Our results shew that with a few modifications of Kirby and Cook's method a well characterized DNA in the native state can be isolated from guinea pig epidermis at a relatively small scale procedure. The average molecular weight of our preparations (3.7 X 10") is lower than molecular weights given by Kirby for preparations of DNA from rat liver or rat spleen (6 X 10"). As Kirby pointed out (2), a decrease in molecular weight may be caused by prolonged times of shaking which we had to do with our modified procedure (e.g. 40-60 minutes shaking of the phenol-detergent system instead of 20 minutes). There is, however, li ttle or no doubt that our preparation contains DNA mainly from the cells of the viable epidermal layer which follows from our results of in vivo radioactive labelling of the DNA. After topical application (30) as well as intraperitoneal (31) injection of radioactive thymidine in vivo, the DNA showed radioactivity at a high incorporation level, even when we sacrificed the animals two hours after injection of thymidine-tritium. After hydrolysis and chromatography, radioactivity was found only in the thymine spot .
Protein and RNA content of our preparation were found to be lower than 0.2%. We were not able to estimate the polysaccharide content of the DNA but based on Kirby and Cook's observations (2) concerning the purity of t he D JA isolated by their procedure, our preparation appears to be free from polysaccharides. Indirectly, we may point out that our preparation of DNA contains a rather very low content of polysaccharides, since otherwise t he percentages in nitrogen and phosphorus would not match so nicely with a theoretica lly pure DNA.
Kirby's y ields are about 40% of total DNA for rat liver and 65% for rat spleen (2, 32). Estimating the DNA content of gu inea pig epidermis with Kissane's method (9), we found about 1% DNA (wet weight). This means that our yields are about 10% of total DNA (for whole epidermis) and 20% (for epidermi · minus horny layer). This significant drop in yield may be discussed as follows. In our sma ll scale mu lt istep procedure that uses approximately 0.9 to 1.3 g of wet epidermal tissue, t here is loss m
t ribonucleic acid , Sigma, type XI, purified.
FIG. 3. Sedimentation of epidermal DNA. Concen traLion-sedimen La Lion profiles are shown for: (1) at sta rt. (2 ) after 32 minu tes and (3) after 72 minutes of centrifugation. For detai ls see methods and material s.
vo lume of about 40-50 percent . Moreover, our tissue contains DNA in various forms, namely native DNA in t he viable epidermal cells and denaturated or degraded DNA in those cells the nuclei of which are going to be digested and eliminated in the process of the formation of the stratum corneum. Both types of DNA would be determined by Kissane's method, whereas only the native and high molecular weight form of DNA would be isolated with Kirby's procedure. Furthermore, although Kissane's method for estimating the DNA has been shown to be a reliable one for a number of tissues from various organisms, it may not be so specifi c for epidermis t ha t contains an appreciable amount of proteins and polysaccharides and t herefore it is necessa ry that this method be checked by some independent method which is not interfered by polysaccharides. Finally in our procedure t here may be an appreciable loss of DNA along with t he protein removal step. Recently it has been shown by Skinner and Triplet (33) that deproteiniza tion wi th phenol ca n result in the selective loss of DNA satellites. Inspite of all these problems indicated above our yields clearly allow us to suggest t h::tt t he guinea pig epidermis contains a minimum of 3 mg DNA per 1000 mg wet epidermis.
The RNA iso la.ted shou ld be of ribosomal origin, in analogy to the findings of Kirby (2) . The significant decrease in RNA yield upon Scotch taping (removal of horny la)rer) may be due to t he release of some active nuclease, however, this needs further investigations.
The isoh tion procedure of epidermal D 1 A and RNA presented here may become useful in determin incr (a) t he early effects of ultraviolet light on DNA synthesis in mammalian skin ; (b) 1he nature of uv-inclu ced damage in li ving skin
GO THE J OURNAL OF I NVE ST IGATIVE DE RMATOL OGY
and (c) t he nature of repa ir process in 1JY-c1Hmaged skin .
W e a rc ind eb ted lo Dr . 0. Uh lcnbeck of Dr. P . M. DaLy's labomlory , H arvard U ni1·crsi ty, fo r his help. :md Hssistance in recording a n I in terpre ting mel lin g; curves, and lo Dr. U. _H agen , Kcrnfo r~chung~zcn trum K a rlsruhe for sednn cn la l.ion cocftl eicnL meu~uremenls, as well as lo Mr. B eyer and lo Dr~ . M . Geisert a nd B . H cikc o [ Dr . R. K. Za hn 's laborato ry, U ni versiLat M a inz, fo r p ro vi d ing us n·ith Lhe a naly ti ca l d ata con ceming s tand ard D N A and for ce ium chl oride densily gmdi cnL mr :tsurc men l. .
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