volume 14 Number 2 1986
Nucleic Acids Research
Nonepidermal members of the keratin multigene family: cDNA sequences and in situ localization
of the mRNAs
Bernhard Knapp, Martin Rentrop, JOrgen Schweizer and Hermelita Winter
Institute of Experimental Pathology, German Cancer Research Center, Im Neuenheimer Feld 280,
6900 Heidelberg, FRG
Received 22 October 1985; Revised and Accepted 13 December 1985
ABSTRACT
A keratin set which consists of a type I 47kd and a type II 57kd protein
occurs as a major constituent of the keratin patterns of various Internal
stratified epitheHa of the mouse. We have Isolated specific cDNA clones
of the two+complementary keratin subunits from a cDNA library constructed
with polyA RNA of mouse tongue epithelium and present the complete nucleot1de and deduced amino a d d sequences of the 57kd protein and about 75% of
the corresponding data of the 47kd protein.
The comparison of the sequence data with those of known epidermal keratin mRNAs coding for the two types of keratin proteins reveals a fundamentally
Identical and type-specific organization of the mRNAs Into both highly conserved and variable domains. In order to avoid cross-reactions wtth other
members of the keratin multigene family, appropriately
taylored
S-labeled
cDNA probes comprising the low and non-homologous 31 coding and noncoding
domains of the mRNAs were used for 1n situ hybridization to tissue sections. The localization and distribution of the corresponding transcripts
Indicates a strongly compartmentalized keratin expression 1n mouse tongue
epithel1um.
INTRODUCTION
Keratins, a multigene family of water-Insoluble proteins are the constituent proteins of Intermediate filaments (IF) present 1n almost all epithelial cells. The complexity of the keratin polypeptide patterns Increases
from simple to stratified non-kerat1n1z1ng tissues and 1s most complex 1n
kerat1n1z1ng terminally differentiating epithelia (1-5). There 1s strong
evidence that at least two complementary, oppositely charged keratin proteins are required for keratin IF assembly 1n vivo (6,7).
Recently, sequence data from both cDNA and genomic clones of epidermal
keratin proteins have extended our knowledge on the structural organization
of this class of IF proteins (8-20). There 1s now general agreement that
both addle type I proteins and basic to neutral type II keratin proteins
possess a common structure 1n which a highly conserved central a-helical
domain 1s flanked by rather variable non o-hel1cal carboxy and amino termi-
© IRL Press Limited, Oxford, England.
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Nucleic Acids Research
nal regions. At the nucleotide level, the mRNAs of both types of keratins
within one species reveal a high degree of divergence within their 3' and
(as far as determined) 5' noncoding regions (12).
These data stem rather exclusively from epidermal keratin subunits of
different species. In the present paper we report on the Isolation and characterization of cDNA clones of nonepidermal members of both keratin subfamilies. The complementary type I 47kd and type II 57kd proteins are coexpressed 1n Internal, stratified mouse epithelia and represent the main
keratin subunits of palate, cheek (21), esophagus, forestomach (22) and
tongue (23). Comparisons with presently known sequence data provided evidence of generally Identical structural properties of keratin proteins
1n epithelia displaying different forms of cell differentiation and enabled
us to prepare highly specific cDNA probes for the study of details of epithelial differentiation by means of 1n situ hybridization.
METHODS
Cloning of cDNAs and screening procedures
PolyA+RNA from heat-separated mouse tongue epithelium was Isolated as
described (24). Double-stranded cDNA > 600 bp was prepared from 10 ug of
polyA+RNA (25), blunt-end Ugated Into pUC8 plasmid which was used to
transform E. coll 0M 214 cells (26). The l i b r a r y was screened for keratin
cDNA clones (27) with 32P-labeled enriched mRNAs (26,28) coding either for
the 57kd or the 47kd keratin protein. Clones strongly hybridizing with one
of the enriched keratin mRNAs were selected and screened by positive hybrid selection (26). The clones pkt57-l and pkt47-l containing the largest
Inserts were selected for further investigation.
All experiments were carried out according to the guide-lines for recombinant DNA published by the "Bundesm1n1ster fur Forschung und Technologie"
of the Federal Republic of Germany.
RNA blot analysis
Ten ug polyA+RNA from different tissues were electrophoresed 1n 1.4%
agarose gels containing 15 mH methylmercury hydroxide (28), transferred to
nitrocellulose f i l t e r s (29), and hybridized overnight at 42"C (50% formamide) with the
32
P-labeled Inserts (30) of pkt57-l and pkt47-l. Blots were
washed at 68"C 1n 0,lxSSC/0,ll SDS and autoradiographed.
DNA sequence analysis
Restriction site mapping was carried out according to Smith and B1rnst1el
(31).
752
Suitable restriction fragments were subcloned Into the H13 mplO and
Nucleic Acids Research
mpll vectors (32), and sequencing was performed by the dideoxy chain terminator method of Sanger et al. (33). Both strands of the two clones were
completely sequenced.
The translation of the two cDNA clone sequences Into amino a d d sequences,
the nucleotide and protein sequence comparisons, and molecular weight determinations were performed using the programs of Osterburg et al. (34).
For the prediction of the secondary structure of the two keratin proteins
both, the probabilistic methods of Chou and Fasman (35,36) and Gamier et
al. (37) as well as the heptade convention analysis (10,38,39) were used.
In situ hybridization
In situ hybridization to tissue sections was performed according to the
method described by Lawrence and Singer (40) with minor modifications.
Briefly, 5 |jm frozen sections of mouse tongue were hybridized with Slabeled cDNA probes (specific activities 3-4x10 cpm/ug). As a control for
the specificity of hybridization tissue sections were treated with RNAse
(100 ug RNAse A and 100 units RNAse Tl/ml) prior to hybridization. After
hybridization slides were washed and dried by passage through ethanolseries. Subsequently slides were dipped 1n Kodak NTB2 emulsion and exposed
for 4-21 days at 4*C. Slides were developed in Kodak D19 and sections were
stained with hematoxyl1n-eos1n. Details of the 1n situ hybridization technique will be published elsewhere.
RESULTS AND DISCUSSION
Isolation and identification of cDNA clones of the 57kd and 47 kd keratin
proteins
A cDNA library was constructed with po1yA+RNA from mouse tongue epithelium and screened with enriched mRNA probes coding for the 57kd and 47kd
proteins. These probes were obtained by size fractionation of polyA+RNA
from tongue epithelium using methylmercury hydroxide-agarose gels. Strongly
positive recombinants Identified with these mRNA probes by means of colony
hybridization were further screened by positive hybrid selection (results
not shown). These screening procedures yielded the plasmids pkt57-l and
pkt47-l with Inserts of about 2200 bp and 1300 bp, respectively.
Size of mRNAs of the 57kd and 47kd keratin proteins and tissue-specific
expression
The size of the mRNAS coding for the two keratin proteins was determined
by Northern blot analysis. Methylmercury hydroxide-agarose gel fractionated
polyA+RNA from mouse tongue epithelium was hybridized under stringent con-
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Nucleic Acids Research
b
c
d
ttb
—4,8-
X
pkt
57- 1
Pkt
47-1
F1g. 1 . Identification 1n different tissues of mRNAs coding for the 57kd
ana 47kd keratin proteins. Ten |jg of polyA RNA from mouse tongue epithelium
(slots a ) , forestomach epithelium (slots b ) , footpad epidermis (slots c)
and l i v e r (slots d) were separated by gel electrophoresis on 1.4% agarose
gels containing 15 mM methyl mercury hydroxide. The RNA was blotted onto
nitrocellulose paper and the blots were hybridized with T-labeled Insert
DNAs of plasmid pkt57-l and pkt47-l, washed at 68'C with 0,lxSSC/0.1% SDS
and autoradiographed. The 16S/23S and 18S/28S rRNAs (Indicated 1n kilobases)
were run on the same gel as size markers.
ditions with 32P-labeled pkt57-l and pkt47-l Inserts (F1g. l a ) . Each probe
hybridized with a single mRNA species containing 2250 ± 100 and 1750 ± 50
bases, respectively. As expected (22), mRNAs corresponding to tongue mRNAs
could be Identified when polyA+RNA from mouse forestomach epithelium was
subjected to blot analysis (Fig* l b ) . In contrast under the highly s t r i n gent hybridization and wash conditions, epidermal and l i v e r mRNAs do not
show cross reaction with pkt57-l and pkt47-l Inserts (F1g. 1 c,d).
cDNA sequences of pkt57-l and pkt47-l
The nucleotide sequence of the pkt57-l Insert, which represents the
f u l l length transcript of the mRNA 1s shown 1n Figure 2. The size of this
single Insert Is 2205 nucleotides. I t Includes the complete 3' end and extends far (117 nucleotides) Into the non-coding 5' end of the mRNA. Thirty
five nucleotides upstream from the ATG i n i t i a t i o n codon appears a putative
ribosome binding consensus sequence CTTCTG which 1s frequently encountered
1n eukaryotic mRNAs (41,42) and has also been described 1n the mRNA coding
for the human 50kd keratin (14). The non-coding 3' end of the sequence comprises 492 nucleotides and contains a polyadenylation signal AATAAA (43)
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Nucleic Acids Research
1 CCT OCC ATA OCC OCC OOC TTO OCA OGA CCC AOC CAC OCT CAT Q M CAT ACT CTT GTO ACC OCC GAO OTT OTA AAQ
rt
ACT TCT QTT CTA AOC fCC I TO CAO CTO CCT CCT CTC TCA OCC ATQ ATC CCC AOA CM] TCA AQT OTC CQC OCA OCC
N
J W TCC ATO AOC CTO OCT COS TCC TOC CAA OOC OOC OOC TAT OOO QOT OCC OCA OOC TTC OOC GTT OOA OOA TAC OCT
tor last tor Va I Ala Ol f toi Cya Ola Qly Oly Cty Tyr Oly Oly Ala Oly Ql r Ptv« Cly V*l Qly Qly Tyf Qly
i r > OCT aoo TTT OUT OCC oar oar r r r oac oar oac m OQA OQC TCC TTC AAC oai OCA OCA oar ccr aac r r c CCA
A l * Qly
4M
arc
Val
pfc* Qly A l a
Oly
Qly
P*«
aiy
dy
TOT CCT OCT OOA oar ATT CA<I QAA arc
Cy« P r * A l a Q l y O l y M a O l a O l * V a l
Qly
Pi»* Qly
Qly
tor
Ptw A«a Qly
M X ATC AAC CAQ AOC CTO era
Thr I I * A m a i a tor L*a L * *
Afg Qiy
aty
Pr» Oly
PW
Pr»
ACA OCT e r r c*o era OAO ATT OAC
Thr P r a L * - * Qlf> V a l O l a I t * A a *
H
lit
IM
•-Hit
•It
CCA QAQ ATC CAO AAA ATC COC ACC OCG CAO COT QAO CAO ATC AAQ ACC CTC AAC AAC AAA TTC OCO TCC TTC ATC
P r a O l * I I * Q l a Lys I I * A r § Thr A l a Q l a A r | Glw Q l " l l a L y * Thr L a * Aaa Aaa Lya P M A l a Ear P * * I I * TCI
AOC AAT OAC AAA OOT COC CTA CAO TCA OAC CTA AAO ATO ATQ CAO OAC AOT CTQ OAO OAC TTC A M I ACC AAO TAT
tor A M Aaai Lya Q l y A*m * • • • O l * Bar Qlw L*a Lya bhit ktol Q l * A*# t * r V a l Q l a A * p P h * L y a Thr Ly» Tyr
OAA QAO OAO ATC AAC AAO COT ACA OCT OCA OAO AAT OAC TTC QTO OTC CTC AAO AAA OAT QTO OAT OCT OCC TAC
• r « oco QAQ era ace
CAQ ATO CAO ACA CAT arc TCA OAC ACA TCT OTA QTO era TCC ATO OAC AAC AAC COO AAC era
A l a Q l * La« A l a O l a tm\ O I B Thr H t a V a l Sar A«# Thr t a r V a l V « l L*a %•! hail Aap A « J I A « M AI% A M L * «
H i t
TCC TOO TAC C M ATC AAQ OTC CAO CAO CTC CAO ATO TCA OCT GAC CAA CAT OQA OAT AOC CT« AAO ACC ACC AAO
II**
AAT QAO ATC TCA OAA CTC AAC AOO ATO ATC CAO AOQ CTQ COO OCA OAfl ATT OAfl AAC ATC AM) AAO CAO AOC CAQ
1*M
ACT CCO C M OCA TCT OTO OCT OAT OCA OAO C M COT OQA O M CTC OCC CTC AAA OAT OCC TAC AOC AAA OOC OCA
D M
OAA CTQ OAC ACT OCA CTO CAO AAQ OOC AAO OAO OAC CTO OCC COO CTO CTC AflQ OAC TAC C M OCT CTC ATO AAC
« . . L . * O l - T»r A l a L a - Q l - L y . A l a L y . O l * A « L a , A l a A T , L « L - A,,
A * . Ty. O l . A l . L . u ^ t o ^ A «
Iltt
QTC AAO CTQ OCC CTO OAT OTO OAO ATT «0C ACC TAC AQO AAO CTQ CTO OAO OOC OAO QAQ TOC AOO ATO TCT OQA
V a l L y * L*a A l a L*a A * » V a l O l a I I * A l a Thr Tyr A j f Lya L * « L*a Q l a O l y O l * O l * Cya Ax» a t * l t « r O l y
\$—
OAA TOC AAO AST OCT OTO AOC ATC TOO OTA OTT OOC OOC AOC CAO CAT TOO OQC ACT OOC CTT COC CTO OQT AOC
t\\
1 t T » OOT. TTT TOC TCC OQT TCT OQT TCT QOA AOT OOC TTT QQA TTT QOT QOT OOC ATT TAT OOC OQT TCT OQT TCC AMI
1 T 2 i AOQ QTC AOC OQT TCC TOA TAT CTC CTQ TOC OAT CTT CAC TTC ACC TCC ACT CTT CCT OOT OCT CAT CTT QTT ACT
1|M
CTT CCC CCT CTQ CCT TOO TCC CTC AOT TCC AOQ AOQ ATC TTC TTO QCC ATO OTO TOA CCT CTO TTC TAT TOO AAC
liri
CTQ OTA OCT TCT TCT CAA CTT OQO TCA ACT OOC CCC CCA CCC CCT CCA TOC GAO OOO TOT CTO ATT TCA CCC CAA
1IU
TOO ACA QAO OOO ATC AAO AOC AOA AAC TCC TTC TOC AOA TOT ACA CTO TCA CTC TCA OCT CCT CTC OTC ACA COO
Itlt
OCT TCC ACC TCA TCC TOO AOA TCT ATO TTC TAC AAT CAT OCT CAT TCT CCA TCA CCT OOC AOC AAC OOQ CTC TOC
1 1 M CAQ OTC AAC AAQ OAC TOC TTO OTQ TCC COT OCA ACC CTO CTC* CTC TCT CTA CTC ATC CCC AAT AAA ATO CTT TTO
JIT*
TCA TTC AAA AAA AAA AAA AAA AAA AAA AAA
F1g. 2. Complete nucleotide sequence of the pkt57-l Insert and deduced
amino a d d sequence of the 57kd keratin protein
of mouse tongue epithelium.
The nucieotide sequence 1s shown 1n the 51 to 3' direction of the mRNA
strand. Nucleotides are numbered from the first base of the cDNA Insert at
the left side and amino a d d s are numbered at the right side. The stop
codon Is marked by three asterisks. Arrows denote the deduced demarcation
of the central o-hel1cal domain. HI, VI, N and H2, V2, C Indicate the proposed threefold subdivision of the non-a-hel1cal end domains (19). Both the
putative polyadenylation signal and the putative ribosome binding consensus
sequence are underlined.
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Nucleic Acids Research
I GIG A M I ATT CCT G A C TGG C A T C T C AAA CAO AGC CCA OCT ACT C C A O A O COG G A C TAC ACT OCT TAT TAT A A G ACC
t% ATC G A C G A C C T C C O G A T C AAG A T T C T C QAA G C C ACC A C O GAC AAC AAC C O O ATC A T C C T O G A G ATT GAC AAT O C C
II* Gto Qlu Lau Araj lla Lyi II* L«u Gl w Ala Thr Thr Asp Asn Asn Arg II* Its Lau Olw lla Asp A a n Ala
SO
IbO AGG CTG OCT C C A O A T G A C TTC A C C CTC AAG TAC GAA AAT GAG C T Q A C C TTO COC C A O A O C Q T G G A Q G C C O A C ATC
Af s L s u AI a AI a A s p f^ 9P P^i t Ar s Lau Lys Tvf QI u A sftGI u L su Thr Lf u Af s1 OIft^s f Va I GI u AI a A s p lla
22fc <VSC OOC CTG COC AOfi G T G C T G G A T OAG CTO ACT CTG OCT WC1 ACT O A C C T O GAA A T C f*ftQ ATT O A C AQC C T G AAT
A«n Gly Lau Art Af s Val Lay A s p Glu Ltu Thr Ltu Ala Lya Thf Asp Law Qlu las I G i n lla O l u •*• Lau A»n
i GQC C A O GTC
Gly G i n Val
37* AAT G T G GAG ATG O A C O C C ACC CCT OOC ATT OAT CTG ACC CGT G T G C T G GCT GAG ATG AGG G A G C A O TAT GAA OCC
Aan Val Glu ••••• t A s p A l a Thf Pro Glv M a Asp Lau Thr Af s Val Lau Ala Glu y § t Ar sj O l u G i n Tyr G l u Ala
460 C T Q GCA GAO AAC AAT C O G A G G G A T GCT GAG GAA TGG TTC C A O A C C A A C AGT O C A C A O C T C AAC A A O GAA O T A TCC
Lau Ala Glu Lya Asn Af aj Af % A s p Ala Glu Glu Tfp Pha Gin Thr Lys tar Ala G l u Lau Asn L y s Glu Val fin
ft?& TCC AAC GCT GAA A T G A T C C A G A C C AOC AAG ACA GAO ATC ACA GAA C T C A G O C G C ACA C T C C A O O G C C T O G A O ATT
S a r A s n A l a G l u M s I l l a G i n T h f b a r L y s T h f G l u l l a T h f G l u L a u A f sj A i l T h f L a u O l n G l ^ L a u G l u l l a
; AAA GCC OGC CTQ GAA ACT ACQ TTG OCA GAO ACA GAG TGC COC TAC GCC CTG
Lya Ala Gly Lau Glu Gar Thr Lau Ala Glu Tbr Qlu Cya Arf Tyi Ala Lau
*S
too
125
130
175
2OO
223
TbO CAO AAC CAG GAA TAC AAO ATG CTO TTG G*C ATC AAA ACA AGO CTG QAA CAO GAC ATC QCC ACC TAC COC ACC CTG
Qln Aan Gin Glu Tyi Lya Mat Lau Lau Asp lla Lya Thr Arg Lau Glu Qln Glu lla Ala Thr Tyr Arf Bar Lau
275
• 2S CTA GAO GGC CAO CAT OCT AAC ATG ACC GCC TTC AAC TCC OOA GCA AAT AAC ACT ACT ACC TCC AAC OCC TCC CCC
Lau Glu Qly Gin Aap Ala Lya Mai Thr Qly Ptta Aan Car Gly Qly Asn Asn Thr Thr Thr Bar Aart Gly Gar Pro
300
• 00 TCC TCC AAT TCC OGT COC CCA OAT TTC CCA AAO TAT TAA CTC ACC CTO GCO TCC CTT CCC TGG CCT TCA GCA GCC
• 7B ACA GOA GQA GAG CTO ATG TCO CCT QTA OQA TGA GAG OAC TOC ACC ATO TOA TCC CCA GTC CCA TTO TCC CCC CAG
lOaO GAC CCA TGG CTC TGA CAA CCC CTG AO0 TGG OCC CTT CCC CAT CTC TTC TGA TTG GAA TCT TTC TTC TCC CTA CCC
I12§ CAT TCT CTC TAT TCT TTO GCT TCT CAG TGT CTC TAC AAC AAA GAA JM3 ATO OTT TCA ATA AAC TTC TCT OAT TTC
1200 CTO GAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA
I2T& AAA AAA AAA AAA AA
Fig. 3. Nucleotide sequence of the pkt47-l Insert and deduced amino a d d
sequence of the 47kd keratin protein of mouse tongue epithelium. The stop
codon, the end of the o-heiical domain and the putative polyadenylation
signal are Indicated as described 1n legend to Figure 4.
fifteen nucleotMes upstream from the polyA tall (A24K The polyadenylation
signal 1s followed by a ATGC-sequence occurring at the same position 1n the
mRNAs of the type II mouse 60kd (11, see also F1g.5) and bovine 69kd keratin proteins (13). The central 1572 nucleotides represent the coding region
for the entire length of the 57kd keratin protein.
The nucleotide sequence of the pkt47-l Insert 1s shown In Figure 3. The
size of this Insert 1s 1289 nucleotides, thus comprising approximately 75X
of the calculated length of the mRNA. The reading frame reveals a stop codon TAA which divides the sequence Into a coding region (936 nucleotides)
and a 3' non-coding region (268 nucleotides) containing a putative polyadenylation signal AATAAA followed 17 nucleotides downstream by a 85 nucleotide long polyA tract.
These sequence Informations supplement the 11st of currently available
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Nucleic Acids Research
57kD
1A
47KD >,
MO
jjo
2A
V
c
2S
L2
«
4 0
«4
w
F1g. 4. Structural organization of the 57kd (upper part) and the 47kd (lower
part) proteins. N and C Indicate the non-o-hel 1cal atnino and carboxy terminal regions. 1A, IB, 2A and 2B represent the colled coll regions and L I ,
L12 and L2 the non-co1led coll linker regions of the a-hel1cal domains.
[Adoption of the nomenclature used by Steinert et a l . (11)]. The numbers
denote (from l e f t to right) the entering positions of the Individual structural segments. NO = not determined.
sequence data of epidermal keratins by one representative nonepidermal member of the type I and type I I keratin multigene family.
Structural organization of the 57kd and 47kd keratin proteins
The 57kd protein comprises 523 amino adds 1f the I n i t i a l meth1on1ne residue 1s excluded (Fig. 2). The calculated molecular size of 56,429 Da
(without the unknown phosphate content) 1s 1n good agreement with the size
estimated by gel electrophoresis (21). The amino add sequence of the 47kd
protein (Fig. 3) could be deduced over a length of 312 amino adds and comprises the carboxy terminal part and almost the complete a-hel1cal region.
Secondary structure analysis (10,35-39) of both nonepidermal proteins revealed a high conformity with the structural organization elucidated so far
for epidermal keratin proteins. This 1s especially relevant for the typical
subregions of the central a-hel1cal domain Into four colled coll regions
and three short linker regions which cannot adopt colled colls (l9;F1g.4).
In addition both the non a-hel1cal N and C termini of the type I I 57kd protein can be divided Into three distinct subdomains according to the natures
of their amino add sequences (19). These comprise highly conserved H subdomains on either side of the rod domain, glydne and serine rich V subdomains of varible size and sequence and highly variable, usually basic N and
C termini (F1g.2). In contrast these structural particularities cannot be
detected 1n the C terminus of the type I 47kd protein (F1g.3). The entire
region encompasses only 29 amino add residues. I t 1s therefore the shortest type I protein carboxy terminal part determined so far and confirms
the prediction (19) that especially small type I keratin proteins may lack
the substructuration of their non a-hel1cal end domains.
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Nucleic Acids Research
H i T^TCOCCAOk—CA—OTCA*OTOTCCQCOtiMKCTCCCCOOOCTTI*Ca>CTClCKTC*arr*TIOCTOQTOCICtCA*3CG«Ia>XTTT*OCT^^
Q--«ar--<xKM*o«TTCQocoTTa(MoaAT«3MTacToaaTTTOOToccaa^
-CTOOCOO
JIT
TATOO
!••
C TQOCT
r*Toocj^Kca*TTCocwoo**orrrci»c*TTOCToa-T--oa»ociOQ—TAOTaacTTTaocTtcoaTGGtoa—AOC
T QOC TTTTOOTOQTOOCTATOQOQCWOCTOOCT
TOOTOQTOOCT ATOOOOTWOC TOOC TTCCCO
;TTCCCA
TtMATCCT T TCTT TQ*QACT TACATCAACOCC T TOMOAAQMCC ro6*OM: T
rrCAOATOCAAACTCACATCTC*a^AC»TCTOTOOTCCTCTCCATOOTCA*CAACCaTAOCCTC
r
^
TTOgc-aMTGOCCTTQOCCtQd»TAOC(KlriII<^IO-rtnTICICQT1CTO(y^OT(^
f
*
CACCTCCAO--ICTTCCTOQt-aCTCA--rcrTO--TT«rCTTCCCCCTCtaCCTTGG-TCCCICA—OIICC--*CGACOA--ICt-'CrTCCCCATQa-tCTQACCT-<:TaiTCTAT
CACCAAaA*KtTQTCTCTOCTCCCAO*TQTCAT<>^T(kUCCTCAACCA<>TCCTn
—TCCTTCT
TCTQACTTCClCCAQMnCIClA^TJULtfclQCCCCCACAACAAAC
•
OTCAArAAOOACTQeTTCCrQTCCCOTOCAACCCTQCICCICTCTCTACTCATCCC^T11^-•
Fig. 5> Comparison of the nucieotide sequences of the mRNAs coding for the
nonepidermai 57kd keratin protein and the mouse epidermal 60kd keratin protein (11). Large arrows Indicate the positions of the region coding for the
o^heHcal domains, small arrows denote the ends of conserved regions coding
Tor the non a-hel1cal H subdomains (compare F1g. 2 ) . I n i t i a t i o n and stop
codons and the putative polyadenylation signals are In boxes. Asterisks Indicate conserved nucleotides.
Sequence comparisons of nonepidermai and epidermal keratin mRNAs
mRNAs which code for epidermal type I and type I I keratin proteins of
mouse and man (8-11, 14-19) were compared with the mRNAs of the nonepider758
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mal 47kd and the 57kd keratin proteins. In each case, nucleotide sequences
with the highest degree of sequence Identity correspond to those coding for
the <*-hel1ca1 domains of the proteins. The calculated values are 1n the order of magnitude of 70-80% and therefore ressemble those determined for
epidermal keratin mRNAs coding for the same type of proteins (12). Figure 5
which shows the alignment of the 57kd protein mRNA and the mRNA of the mouse
epidermal type II 60kd protein (11) 1s representative for these multiple
comparisons. In this case the sequence Identity of the a-hel 1x-cod1ng region 1s 75%. In addition the alignment reveals highly homologous regions of
constant lengths Immediately upstream (108 nucleotides; sequence Identity
70%) and downstream (39 nucleotides; sequence Identity 53%) of the a-hel1xcoding region. These sequences are common to all known epidermal type II
keratin mRNAs (11,16,19) and code for the non a-hel1cal H subdomains of
this keratin protein subfamily (F1g. 2 ) . They are absent from both type I
epidermal (10,17-19) and the type I nonepidermal keratin mRNA (F1g. 3 ) .
In contrast both the lengths and the sequences of the remaining 5' and 31
regions, encompassing coding as well as noncoding sequences of the mRNAs
are highly variable, unless functionally related epidermal keratin mRNAs of
different species are compared (12). However, Figure 5 shows that the 5' end
regions of type II keratin mRNAs are slightly more conserved than the 3'
end regions. Occasionally they contain rather extended homologous nucleotide
sequences I.e. positions 254-278 of the 57kd protein mRNA which code for a
characteristic type II protein octade (11, 16, 19). As a rule, this 1s not
the case for the 3'end regions which at most exhibit rare stretches of 4-6
conserved nucleotides and therefore seem to be most specific for any given
keratin mRNA.
Besides the demonstration of fundamentally Identically organized keratin mRNAs 1n epithelia showing different forms of cell differentiation,
these comparisons are of paramount Importance 1n view of the generation of
specific cDNA probes for 1n situ hybridization experiments. cDNA probes
containig sequences complementary to the 3' non-homologous end regions of a
keratin mRNA should exclude the danger of crossreactivity and therefore be
Ideally suited to Investigate the specific cellular location of the corresponding mRNA.
In situ hybridization
Figure 6 Illustrates the results of the 1n situ hybridization to sections of adult mouse tongue using a 35S-labeled 553bp HhaI-H1ndII fragment
derived from the 3' region of the pkt57-l Insert (nucleotides 1553-2106,
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Fig. 6. In situ hybridization of the 553 bp 35S-labe1ed Hha I - H1nd II
fragment of the pkt57-l Insert. The probe was hybridized to a sagittal section of the ventral side (a) and to a sagittal (b), frontal (c) and tangential (d) section of the dorsal side of adult mouse tongue. Similar results
were obtained with a specific probe of the 47kd Insert. Control sections
treated with RNAse prior to hybridization showed only background noise.
Competition experiments with Increasing amounts of unlabelled specific cDNA
fragments abolished the label, whereas unrelated DNA was Ineffective.
BC, basal cells; SC, stratum corneum; CT, connective tissue; IP, Interpapillary region; FP, filiform papillae; DP, dermal papillae (H&E; x200).
I.e. containing parts of the low homologous terminal coding region and the
noncoding region without the polyA tract; F1g. 2 ) . Figure 6a shows a sagittal section of the ventral side of the tongue which 1s covered by a conti-
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nuous, multilayered epithelium (44). It can easily be seen that the label
1s restricted to the epithelial cells of this tissue with negligible background density of silver grains over non-ep1thel1al cells. It should be emphasized that treatment of the sections with RNAse prior to hybridization
did not lead to any detectable label 1n the tissue. Whereas most basal
cells show a level of hybridization that 1s not significantly above background noise, the entire suprabasal compartment of living cells 1s heavily
labeled. The highest density of silver grains 1s found over the spinous
cell layers. The label gradually decreases 1n the granular cell layers and
1s absent from the dead stratum corneum. Unlike the ventral epithelium, the
dorsal epithelium of the tongue shows a rather complex morphology 1n that
numerous papillae - mainly of the filiform type - are distributed 1n parallel rows within an 1nterpap1llary epithelium (44,45). In situ hybridization
with the same specific probe reveals that the corresponding mRNA 1s abundantly present 1n the suprabasal Interpapillary epithelium, however, again
absent from Its basal cell layer as well as from the entire filiform papillae (F1g. 6b-d). As observed for the morphologically related ventral epithelium, there 1s a clear cut decrease of the suprabasal 1nterpap1llary label from the spinous to the granular cell layers (best visible 1n F1g. 6d).
Whether the progressive death of cells at the transition from the granular
to the cornified layer correlates with a simultaneous decrease of keratin
gene expression or 1f there exists a temporal difference between cell death
and cessation of keratin expression remains to be Investigated. Modulations
of epithelial keratin mRNA expression by certain drugs (46,47) and the localization of these alterations at the cellular level by 1n situ hybridization with specific cDNA pobes may be a promising approach to study elements
which control the differential expression of this multigene family.
ACKNOWLEDGMENTS
We thank H.P. Schmitt and A. Alonso for helpful advice and discussion
and C. Frees for skillful technical assistance. We are Indebted to E.
Aratmann for facilities with methylmercury hydroxide-agarose gel electrophoresis. This work was supported by the Deutsche Forschungsgemeinschaft
(W1 489/2-1).
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