Cloning and Sequence of cDNA for Human Placental Cytokeratin 8. Regulation of the mRNA in Trophoblastic Cells by cAMP
Ritsu Yamamoto, Lee-Chuan Kao, Craig E. McKnight, and Jerome F. Strauss III Departments of Obstetrics and Gynecology and Pathology and Laboratory Medicine University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104
A 1735 bp cDNA for human placental cytokeratin 8 is described which encompasses the entire coding sequence as well as 33 and 250 base pairs of 5'and 3'-untranslated region, respectively. The level of cytokeratin 8 mRNA in various fetal tissues and placentae of different gestational ages was determined as were the effects of 8-bromo-cAMP on cytokeratin 8 mRNA in primary cultures of cytotrophoblasts and JEG-3 choriocarcinoma cells. Cytokeratin 8 mRNA was abundant in fetal small intestine, placenta, pancreas, lung, liver, and kidney. Levels of cytokeratin 8 mRNA in placenta increased slightly during pregnancy. 8-Bromo-cAMP suppressed cytokeratin 8 mRNA in primary cultures of cytotrophoblasts, whereas the cAMP analog increased mRNA levels in JEG-3 cells, revealing differential regulation of this mRNA in normal and transformed trophoblastic cells. (Molecular Endocrinology 4:370-374,1990)
gested a role in differentiation (2). Of particular interest to us is the early appearance of Endo A, also referred to as TROMA-1 antigen, equivalent to human cytokeratin 8, in the mouse 8 cell embryo and the later expression of this gene in the trophectoderm (3, 4). A partial cDNA for human cytokeratin 8 has been described by Leube et al. (5) but little is known about the expression of the human gene in trophoblast. Here we report the molecular cloning and sequence of a full-length cDNA for human placental cytokeratin 8. The divergent regulation of cytokeratin 8 mRNA in normal and transformed trophoblasts by cAMP is also described.
RESULTS AND DISCUSSION Structure of Cytokeratin 8 cDNA Figure 1 presents the nucleotide and deduced amino acid sequences of the cytokeratin 8 cDNA. The clone encodes 1735 nucleotides with an open reading frame starting at position 34 terminating in a stop codon at nucleotide 1485. The coding sequence is for a polypeptide of 52,000 mol wt with an associated 33 nucleotide 5'-untranslated region and 250 nucleotide 3'-untranslated sequence. The amino acid sequence is consistent with the structure of intermediate filament proteins (6). The nucleotide sequence of our placental cytokeratin 8 cDNA is identical to the partial cDNA (1085 nucleotides) reported by Leube et al. (5) derived from the vulvar carcinoma cell line A-431, with the exception of six base pairs in the 3'-untranslated region. Leube et al. (5) identified their partial cDNA by screening at low stringency with a bovine cytokeratin 8 cDNA. Using a hybrid-selection translation assay, they confirmed that the human cDNA encoded sequences of cytokeratin 8. A search of the Genbank and European Molecular Biology Laboratory data bases with the sequence of our clone revealed significant similarity to only the mouse Endo A cDNA (6), which is the equivalent of human cytokeratin 8.
INTRODUCTION
Intermediate filaments (8-10 nm) are prominent components of the cytoskeleton (1). Five major classes of intermediate filaments have been described including cytokeratin, desmin, glial filaments, neurofilaments, and vimentin. Cytokeratins, the largest and most diverse class, are differentially expressed in epithelial cells. On the basis of electrophoretic and immunological properties, peptide mapping, and gene sequences, cytokeratins have been subdivided into two families; type I or acidic and type II or basic. Intermediate filaments are formed from heterodimeric complexes of the two types of cytokeratins. Although the exact function(s) of intermediate filaments are not known, the conserved expression of intermediate filaments during development has sug0888-8809/90/0370-0374$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society
370 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 08:50 For personal use only. No other uses without permission. . All rights reserved.
371
Human Cytokeratin 8 cDNA and Trophoblast
Distribution of Cytokeratin 8 mRNA in Fetal Tissues Analysis of total RNA prepared from 17- and 21-week fetal tissue revealed cytokeratin 8 mRNA in small intestine, placenta, pancreas, lung, liver, heart, and kidney with little mRNA in adrenal gland and muscle (Fig. 2). This distribution is in keeping with the previously noted expression of cytokeratin 8 in simple epithelia (1-8). Placental Cytokeratin 8 mRNA at Different Stages of Pregnancy Placental cytokeratin 8 mRNA increased slightly between 10 to 37V2 weeks of pregnancy (Fig. 3). In contrast, levels of /3CG (hCG) mRNA declined dramatically, whereas levels of chorionic somatomamotropin (hCS) mRNA increased. Regulation of Cytokeratin 8 mRNA in Trophoblastic Cells by cAMP With time in culture, the amount of cytokeratin 8 mRNA increased in isolated cytotrophoblast cells (Fig. 4). Treatment of these primary cultures with 8-bromocAMP (1.5 ITIM) for 24 h promotes expression of genes encoding hCG and increases levels of mRNA for the cholesterol side-chain cleavage system (9). However, during the 24 h of treatment with the cAMP analog the rise in cytokeratin 8 mRNA was prevented so that levels were 10% of those in control cells cultured for 24 h (Fig. 5). 8-Bromo-cAMP also resulted in a 60% reduction in actin mRNA levels, as we have found in previous studies (9). In contrast, JEG-3 choriocarcinoma cells responded to 8-bromo-cAMP with a 3-fold increase in cytokeratin 8 mRNA (3.1 ± 0.5-fold, mean ± SE, n = three separate experiments) and levels of actin mRNA were not altered. As previously reported, a and /3hCG subunit mRNAs increased in JEG-3 cells 10- and 18fold, respectively (9). Cytokeratin expression accompanies retinoic acid and (Bu)2cAMP-induced differentiation of F9 embryonal carcinoma cells (10). In this system, cytokeratin filaments recognized by TROMA 1 appear in association with a reduction in the rate of cell proliferation. The JEG-3 cells are similar to F9 embryonal carcinoma cells in that increased cytokeratin 8 mRNA levels accompany enhancement of differentiated functions {i.e. increased hCG secretion) promoted by 8-bromo-cAMP. Cytokeratin 8 mRNA levels in isolated cytotrophoblasts increase during the initial hours of culture as the spherical cells attach to the substrate and flatten out. Although the primary cultures of cytotrophoblasts from term placentae also respond to 8-bromo-cAMP with increased hCG secretion like JEG-3 cells, cytokeratin 8 mRNA levels are suppressed and actin mRNA levels also are lowered. This is reminiscent of the response of rat granulosa cells to gonadotropins and their cAMP second messenger (11). When stimulated, these cells round up and there is an associated change in the organization of the cytoskeleton which includes dimin-
ished synthesis of vinculin, a-actinin and actin. The reduction in actin synthesis is due at least in part to a decline in actin mRNA. Like rat granulosa cells, the cytotrophoblastic cells from term placentae round up in response to 8-bromo-cAMP and undergo cytoplasmic differentiation (12, 13). The divergent response of cytokeratin 8 mRNA in JEG-3 cells and primary cultures of cytotrophoblastic cells to 8-bromo-cAMP may reflect cell differences related to the normal and transformed phenotypes. JEG3 replicate whereas the term cytotrophoblastic cells do not. Another difference is that JEG-3 cells do not merge to form syncytia whereas cytotrophoblastic cells undergo a process of morphological differentiation in which they aggregate and subsequently fuse to form large multinucleated cells (13-15). It is of interest that the suppression of cytokeratin 8 mRNA levels by 8bromo-cAMP does not apparently impede cellular aggregation and fusion as these processes occur in the presence of the cAMP analog (14). The cytokeratin 8 cDNA clone described in this report was isolated during screening of a human placental expression library with labeled oligonucleotides to detect clones encoding DNA binding proteins. Does binding of DNA to cytokeratin 8 have physiological significance? Traub (16) has reviewed the literature describing nucleic acid binding to intermediate filament proteins. Some of these proteins have a high affinity for nucleic acids including rRNA, single-stranded DNA, and supercoiled DNA. This affinity, which appears to be driven by electrostatic as well as other forces, may account for our detection of the cytokeratin 8 clone in screening for DNA binding proteins. Traub (16) has questioned whether the interaction between intermediate filaments and nucleic acids might have importance in the control of DNA replication and gene transcription. This is an intriguing notion which deserves further attention.
MATERIALS AND METHODS Cloning of the Cytokeratin 8 cDNA The cytokeratin 8 clone was isolated from a human placenta Xgt11 expression library prepared from poly(A)+ RNA isolated from a placenta of 34 weeks gestational age (Clontech, Palo Alto, CA) in the process of screening for DNA binding proteins using 32P-labeled oligonucleotide probes. The oligonucleotide used was a synthetic 43 base pair sequence containing the cAMP response element of the 5'-flank of the ahCG gene. Protein replica filters were prepared and screened according to the methods of Vinson et al. (17). Filters were first submerged in binding buffer [25 mM NaCI, 4 ITIM MgCI2, 0.5 ITIM dithiothreitol, and 25 mM HEPES (pH 7.9)] supplemented with 6 M guanidine hydrochloride and further washed in sequential dilutions of the binding buffer. The filters were then incubated in blocking buffer consisting of binding buffer with 5% Carnation nonfat dry milk and later replaced with binding buffer supplemented with 0.25% dry milk. For screening, the filters were incubated in binding buffer with 0.25% nonfat dry milk containing nick-translated concatemerized 32P-labeled 43-mer oligonucleotide probe (1 x 106 cpm/ml). After 2 h of hybridization at 4 C with gentle agitation, the filters were washed
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 08:50 For personal use only. No other uses without permission. . All rights reserved.
MOL ENDO-1990 372
Vol 4 No. 3
ACC rCTACCTCCACTCCTGCCTCCACC
ATC
46
CTG
ACC
CAO
AAG
TCC
TAC
AAG
Lyi
Sci
Tyi
Lys
83
CCC
COO
OCC
TTC
AGC
AOC
CGC
Pro
TCC
AGG
Sei
Ati
GTO
TCC
ACC
TCC
TAC
ACG
TCT
OOC
Sci
Gly
AGT
GGG
Ait
Ala
nc
Sci
Sci
An
Set
Tyi
Tin
Sei
Gly
OGT
TCC
COC
ATC
AOC
TCC
TCG
AGC
TTC
TCC
CGA
Gly
Set
AIJ
It
Sci
Sci
Sci
Sci
Phc
Set
Alt
OCC
AOC
ACC
AAC
TTT
COC
OGT
GCC
CTC
GCC
GGC
01,
SIT
Sci
Am
Phc
A18
Gly
Gly
Leu
Gly
Gly
CGG
Clu
Uu
7«S
GTC
CTG
TCC
Val
Uu
Set
CAC
ACC
ATC
Set
lie
ATT
GCC
Atj
80' 118
CCC
134
OTG
Ho
V«l
IW
COC
TAT
GOT
COO
AOC
OCC
ATG
CCA
GCC
ATC
ACC
Gly
T)i
Gly
Gl)
Ab
Sci
Gly
Me.
Gly
Gly
lie
Tht
GTT
ACG
CTC
AAC
CAG
AGC
CTG
CTG
AOC
CCC
TTC
ATC
CAG
GCA
226
OCC
Sci
Sci
Pio
CCC
CTG
CGC
Asp
838
GAT
Uc
Ab
874
ATC
TAC
CAG
910
GCT
COO
AAG
Gly
Asp
ICC
CTO
GAG
Set
Leu
Olu
Val
298
ACC
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OAO
AAO
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CAC.
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lie
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CAC
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ACG
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1539
AGC
AAG
TAT
GAG
CAO
CTC
CAG
GTC
TTC
AAG
ACC
CGC
ACC
AGC
AAO
TCT Val
ACACCTGCCGCAGCCCCTCCCACCCTACCCCTCCTGCGCTGCCCCA(]AGCCTG
GGAAGOAOOCCOCTATGCAGGOTAOCACTGGCAACAGGAGACCCACCTGACG CTCAGCCCTAOCCCTCAGCCCACCTOOGOAGTTTACTACCTCGGGAICCri'CT
AGC
16W
Lys
Asp
Asp
Leu
ATC
ATC
AAC
COG
lie
Uu
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AJa
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1054
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Ab
AIJ
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AAO
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Uu
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OAO
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Glu
OAO
ATT
OAC
CCC
GCC
CGG
CAO
CTO
CGT
GTC
AAG
CTG
GCC
CTC
AAG
CTO
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TAAAACCTCACCTAGCIdCCCAAAAAAAAAAAAAAA
Atg
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ATG
Gin
Asp
Mel
CTG
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AAC
GCC
Ab
"» 2?
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GAC
CAG
Leu
AAG
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Leu
1198
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Glu
GCT
II6-1
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CAG
1450 CTG
TCC
GTC
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GAG
1018
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314
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GTT
Set
OAC
478
CTC
GTC
I486
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1126 442
TCC
CTG
Gin'
Ab
1090 TTG 41*
CGC
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946 262
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730
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AAG
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Asp
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TCT
CAT
OAA
OCA
TAC
ATG
AAC
CTC
AAC
AAG
ACC
Lys
TlT
1270
ACC
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Ab
Tyi
1342
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ACC
CTG
Sci
Uu
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OTA
CTC
CCC
ACA
GA
Ritsu Yamamoto, Lee-Chuan Kao, Craig E. McKnight, and Jerome F. Strauss III Departments of Obstetrics and Gynecology and Pathology and Laboratory Medicine University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104
A 1735 bp cDNA for human placental cytokeratin 8 is described which encompasses the entire coding sequence as well as 33 and 250 base pairs of 5'and 3'-untranslated region, respectively. The level of cytokeratin 8 mRNA in various fetal tissues and placentae of different gestational ages was determined as were the effects of 8-bromo-cAMP on cytokeratin 8 mRNA in primary cultures of cytotrophoblasts and JEG-3 choriocarcinoma cells. Cytokeratin 8 mRNA was abundant in fetal small intestine, placenta, pancreas, lung, liver, and kidney. Levels of cytokeratin 8 mRNA in placenta increased slightly during pregnancy. 8-Bromo-cAMP suppressed cytokeratin 8 mRNA in primary cultures of cytotrophoblasts, whereas the cAMP analog increased mRNA levels in JEG-3 cells, revealing differential regulation of this mRNA in normal and transformed trophoblastic cells. (Molecular Endocrinology 4:370-374,1990)
gested a role in differentiation (2). Of particular interest to us is the early appearance of Endo A, also referred to as TROMA-1 antigen, equivalent to human cytokeratin 8, in the mouse 8 cell embryo and the later expression of this gene in the trophectoderm (3, 4). A partial cDNA for human cytokeratin 8 has been described by Leube et al. (5) but little is known about the expression of the human gene in trophoblast. Here we report the molecular cloning and sequence of a full-length cDNA for human placental cytokeratin 8. The divergent regulation of cytokeratin 8 mRNA in normal and transformed trophoblasts by cAMP is also described.
RESULTS AND DISCUSSION Structure of Cytokeratin 8 cDNA Figure 1 presents the nucleotide and deduced amino acid sequences of the cytokeratin 8 cDNA. The clone encodes 1735 nucleotides with an open reading frame starting at position 34 terminating in a stop codon at nucleotide 1485. The coding sequence is for a polypeptide of 52,000 mol wt with an associated 33 nucleotide 5'-untranslated region and 250 nucleotide 3'-untranslated sequence. The amino acid sequence is consistent with the structure of intermediate filament proteins (6). The nucleotide sequence of our placental cytokeratin 8 cDNA is identical to the partial cDNA (1085 nucleotides) reported by Leube et al. (5) derived from the vulvar carcinoma cell line A-431, with the exception of six base pairs in the 3'-untranslated region. Leube et al. (5) identified their partial cDNA by screening at low stringency with a bovine cytokeratin 8 cDNA. Using a hybrid-selection translation assay, they confirmed that the human cDNA encoded sequences of cytokeratin 8. A search of the Genbank and European Molecular Biology Laboratory data bases with the sequence of our clone revealed significant similarity to only the mouse Endo A cDNA (6), which is the equivalent of human cytokeratin 8.
INTRODUCTION
Intermediate filaments (8-10 nm) are prominent components of the cytoskeleton (1). Five major classes of intermediate filaments have been described including cytokeratin, desmin, glial filaments, neurofilaments, and vimentin. Cytokeratins, the largest and most diverse class, are differentially expressed in epithelial cells. On the basis of electrophoretic and immunological properties, peptide mapping, and gene sequences, cytokeratins have been subdivided into two families; type I or acidic and type II or basic. Intermediate filaments are formed from heterodimeric complexes of the two types of cytokeratins. Although the exact function(s) of intermediate filaments are not known, the conserved expression of intermediate filaments during development has sug0888-8809/90/0370-0374$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society
370 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 08:50 For personal use only. No other uses without permission. . All rights reserved.
371
Human Cytokeratin 8 cDNA and Trophoblast
Distribution of Cytokeratin 8 mRNA in Fetal Tissues Analysis of total RNA prepared from 17- and 21-week fetal tissue revealed cytokeratin 8 mRNA in small intestine, placenta, pancreas, lung, liver, heart, and kidney with little mRNA in adrenal gland and muscle (Fig. 2). This distribution is in keeping with the previously noted expression of cytokeratin 8 in simple epithelia (1-8). Placental Cytokeratin 8 mRNA at Different Stages of Pregnancy Placental cytokeratin 8 mRNA increased slightly between 10 to 37V2 weeks of pregnancy (Fig. 3). In contrast, levels of /3CG (hCG) mRNA declined dramatically, whereas levels of chorionic somatomamotropin (hCS) mRNA increased. Regulation of Cytokeratin 8 mRNA in Trophoblastic Cells by cAMP With time in culture, the amount of cytokeratin 8 mRNA increased in isolated cytotrophoblast cells (Fig. 4). Treatment of these primary cultures with 8-bromocAMP (1.5 ITIM) for 24 h promotes expression of genes encoding hCG and increases levels of mRNA for the cholesterol side-chain cleavage system (9). However, during the 24 h of treatment with the cAMP analog the rise in cytokeratin 8 mRNA was prevented so that levels were 10% of those in control cells cultured for 24 h (Fig. 5). 8-Bromo-cAMP also resulted in a 60% reduction in actin mRNA levels, as we have found in previous studies (9). In contrast, JEG-3 choriocarcinoma cells responded to 8-bromo-cAMP with a 3-fold increase in cytokeratin 8 mRNA (3.1 ± 0.5-fold, mean ± SE, n = three separate experiments) and levels of actin mRNA were not altered. As previously reported, a and /3hCG subunit mRNAs increased in JEG-3 cells 10- and 18fold, respectively (9). Cytokeratin expression accompanies retinoic acid and (Bu)2cAMP-induced differentiation of F9 embryonal carcinoma cells (10). In this system, cytokeratin filaments recognized by TROMA 1 appear in association with a reduction in the rate of cell proliferation. The JEG-3 cells are similar to F9 embryonal carcinoma cells in that increased cytokeratin 8 mRNA levels accompany enhancement of differentiated functions {i.e. increased hCG secretion) promoted by 8-bromo-cAMP. Cytokeratin 8 mRNA levels in isolated cytotrophoblasts increase during the initial hours of culture as the spherical cells attach to the substrate and flatten out. Although the primary cultures of cytotrophoblasts from term placentae also respond to 8-bromo-cAMP with increased hCG secretion like JEG-3 cells, cytokeratin 8 mRNA levels are suppressed and actin mRNA levels also are lowered. This is reminiscent of the response of rat granulosa cells to gonadotropins and their cAMP second messenger (11). When stimulated, these cells round up and there is an associated change in the organization of the cytoskeleton which includes dimin-
ished synthesis of vinculin, a-actinin and actin. The reduction in actin synthesis is due at least in part to a decline in actin mRNA. Like rat granulosa cells, the cytotrophoblastic cells from term placentae round up in response to 8-bromo-cAMP and undergo cytoplasmic differentiation (12, 13). The divergent response of cytokeratin 8 mRNA in JEG-3 cells and primary cultures of cytotrophoblastic cells to 8-bromo-cAMP may reflect cell differences related to the normal and transformed phenotypes. JEG3 replicate whereas the term cytotrophoblastic cells do not. Another difference is that JEG-3 cells do not merge to form syncytia whereas cytotrophoblastic cells undergo a process of morphological differentiation in which they aggregate and subsequently fuse to form large multinucleated cells (13-15). It is of interest that the suppression of cytokeratin 8 mRNA levels by 8bromo-cAMP does not apparently impede cellular aggregation and fusion as these processes occur in the presence of the cAMP analog (14). The cytokeratin 8 cDNA clone described in this report was isolated during screening of a human placental expression library with labeled oligonucleotides to detect clones encoding DNA binding proteins. Does binding of DNA to cytokeratin 8 have physiological significance? Traub (16) has reviewed the literature describing nucleic acid binding to intermediate filament proteins. Some of these proteins have a high affinity for nucleic acids including rRNA, single-stranded DNA, and supercoiled DNA. This affinity, which appears to be driven by electrostatic as well as other forces, may account for our detection of the cytokeratin 8 clone in screening for DNA binding proteins. Traub (16) has questioned whether the interaction between intermediate filaments and nucleic acids might have importance in the control of DNA replication and gene transcription. This is an intriguing notion which deserves further attention.
MATERIALS AND METHODS Cloning of the Cytokeratin 8 cDNA The cytokeratin 8 clone was isolated from a human placenta Xgt11 expression library prepared from poly(A)+ RNA isolated from a placenta of 34 weeks gestational age (Clontech, Palo Alto, CA) in the process of screening for DNA binding proteins using 32P-labeled oligonucleotide probes. The oligonucleotide used was a synthetic 43 base pair sequence containing the cAMP response element of the 5'-flank of the ahCG gene. Protein replica filters were prepared and screened according to the methods of Vinson et al. (17). Filters were first submerged in binding buffer [25 mM NaCI, 4 ITIM MgCI2, 0.5 ITIM dithiothreitol, and 25 mM HEPES (pH 7.9)] supplemented with 6 M guanidine hydrochloride and further washed in sequential dilutions of the binding buffer. The filters were then incubated in blocking buffer consisting of binding buffer with 5% Carnation nonfat dry milk and later replaced with binding buffer supplemented with 0.25% dry milk. For screening, the filters were incubated in binding buffer with 0.25% nonfat dry milk containing nick-translated concatemerized 32P-labeled 43-mer oligonucleotide probe (1 x 106 cpm/ml). After 2 h of hybridization at 4 C with gentle agitation, the filters were washed
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 08:50 For personal use only. No other uses without permission. . All rights reserved.
MOL ENDO-1990 372
Vol 4 No. 3
ACC rCTACCTCCACTCCTGCCTCCACC
ATC
46
CTG
ACC
CAO
AAG
TCC
TAC
AAG
Lyi
Sci
Tyi
Lys
83
CCC
COO
OCC
TTC
AGC
AOC
CGC
Pro
TCC
AGG
Sei
Ati
GTO
TCC
ACC
TCC
TAC
ACG
TCT
OOC
Sci
Gly
AGT
GGG
Ait
Ala
nc
Sci
Sci
An
Set
Tyi
Tin
Sei
Gly
OGT
TCC
COC
ATC
AOC
TCC
TCG
AGC
TTC
TCC
CGA
Gly
Set
AIJ
It
Sci
Sci
Sci
Sci
Phc
Set
Alt
OCC
AOC
ACC
AAC
TTT
COC
OGT
GCC
CTC
GCC
GGC
01,
SIT
Sci
Am
Phc
A18
Gly
Gly
Leu
Gly
Gly
CGG
Clu
Uu
7«S
GTC
CTG
TCC
Val
Uu
Set
CAC
ACC
ATC
Set
lie
ATT
GCC
Atj
80' 118
CCC
134
OTG
Ho
V«l
IW
COC
TAT
GOT
COO
AOC
OCC
ATG
CCA
GCC
ATC
ACC
Gly
T)i
Gly
Gl)
Ab
Sci
Gly
Me.
Gly
Gly
lie
Tht
GTT
ACG
CTC
AAC
CAG
AGC
CTG
CTG
AOC
CCC
TTC
ATC
CAG
GCA
226
OCC
Sci
Sci
Pio
CCC
CTG
CGC
Asp
838
GAT
Uc
Ab
874
ATC
TAC
CAG
910
GCT
COO
AAG
Gly
Asp
ICC
CTO
GAG
Set
Leu
Olu
Val
298
ACC
CAO
OAO
AAO
Tht
Gin
Glu
\U
TTT
CCC
AAC
Asp
Pra
Am
CAO
CAC.
ATC
OAC
AAG
Gin
ALj
Val
AlB
ACC
CTG
AAC
AAC
GCC
TCC
TTC
ATA
AAG
GTA
CCC
TTC
CTG
Phc
Ab
Sci
Pbc
lie
Asp
Lyi
Val
Am
Phc
Uu
GAG
CAC
CAG
AAC
AAO
ATC
CTG
GAG
ACC
AAG
TGG
AOC
Glu
TV
CAG
CAC
AAG
ACG
GCT
CGA
ACC
AAC
Ais
Sci
GAC
Gin CTC
CTC
Lm
CAG Gin
AAC
ATO
TTC
GAG
ACC
TAC
ATC
AAC
AAC
CTT
AGG
Am
Mci
Phc
Glu
Sci
TIT
lie
Am
Am
Leu
Ait
CCG
CTfl
Atg
ere
CAG
ACT
CTC
GGC
CAC
CAO
Leu
Glu
Th
Uu
Gly
Gin
Clu
Lys
Uu
Lyi
CAO
CCC
CAO
CTT
GCC
AAC
ATC
CAC
COG
CTG
CTC
Glu
Uu
Gin
Gly
Uu
Glu GAG
TIC
1539
AGC
AAG
TAT
GAG
CAO
CTC
CAG
GTC
TTC
AAG
ACC
CGC
ACC
AGC
AAO
TCT Val
ACACCTGCCGCAGCCCCTCCCACCCTACCCCTCCTGCGCTGCCCCA(]AGCCTG
GGAAGOAOOCCOCTATGCAGGOTAOCACTGGCAACAGGAGACCCACCTGACG CTCAGCCCTAOCCCTCAGCCCACCTOOGOAGTTTACTACCTCGGGAICCri'CT
AGC
16W
Lys
Asp
Asp
Leu
ATC
ATC
AAC
COG
lie
Uu
Gin
AJa
TCC
CTO
1054
COT
Ab
AIJ
AMI AAO
AAO
Set
Uu
OCA
OAO
Gly
Glu
OAO
ATT
OAC
CCC
GCC
CGG
CAO
CTO
CGT
GTC
AAG
CTG
GCC
CTC
AAG
CTO
AIJ
TAAAACCTCACCTAGCIdCCCAAAAAAAAAAAAAAA
Atg
AAC
ATC
CTC
ATG
Gin
Asp
Mel
CTG
ATG
AAC
GCC
Ab
"» 2?
OAT.
GCC Glu
GAC
CAG
Leu
AAG
TCC Set
Leu
1198
>»
CGC
AC
Glu
GCT
II6-1
Gin
CAG
1450 CTG
TCC
GTC
Sei
GAG
1018
ATG
Asp
314
CAC
GTT
Set
OAC
478
CTC
GTC
I486
Th
ACT
1126 442
TCC
CTG
Gin'
Ab
1090 TTG 41*
CGC
TCT
L>s
Ly,
AAG
.17(1
CTG
AGC
AC
ACA
1591
946 262
iAC
GAG
730
Mci'
Am
ACC
TAC
AAG
Tin
TyT
Lys
CTG
Uu
CTC
Uu
CTO
CAG
CGG
CCC
AC
CAC
GGC Glu
GAG Gly
OAO Glu
AAC
CAG
CAG
AGC Glu
Set
s.° a?
Ml
5X6
AAG L).
f>22
658
AAG
GAG
CGT
ACA
GAO
ATG
CAO
AAC
OAA
TTT
GTC
A>»
Tt»
Glu
Mci
Glu
Am
Glu
Phe
Val
AAO
CTC
GAT
CTC
Asp
Val
GAO
TCT
CAT
OAA
OCA
TAC
ATG
AAC
CTC
AAC
AAG
ACC
Lys
TlT
1270
ACC
DC*
TCG Set
Ab
Tyi
1342
TAC
ACC
CTG
Sci
Uu
Tt»
ATC
OTA
CTC
CCC
ACA
GA
