FAX: (312) 996-1414. men [16, 29]. Recently, a partial cDNA for the glycoprotein in the baboon was cloned and sequenced [30]. In the pres- ent investigation, we ...
BIOLOGY OF REPRODUCTION 51, 685-694 (1994)
Complementary Deoxyribonucleic Acid Cloning and Molecular Characterization of an Estrogen-Dependent Human Oviductal Glycoprotein' EDWARD B. ARIAS,3 HAROLD G. VERHAGE, 4 and RANDAL C. JAFFE 2'3'5
Departments of Physiology and Biophysics,3 Obstetrics and Gynecology,4 and Genetics University of Illinois at Chicago, Chicago, Illinois 60612 ABSTRACT A 120-kDa oviduct-specific glycoprotein is synthesized and secreted into the oviductal lumen during estrogen dominance in the human. The objective of this investigation was to clone, sequence, and characterize the cDNA to this core protein. Rapid amplification of cDNA ends was used to clone a contiguous 3' cDNA end and 5' cDNA end. The total length of the cDNA was determined to be 2.2 kb by sequence analysis and exhibited a 92% sequence identity with the comparable overlapping baboon cDNA (1.2 kb). A high degree of homology was found to the N-terminal sequence of hamster oviductin and the partial sequence of a homologous baboon and bovine oviduct glycoprotein. Northern blots revealed a single mRNA species of 2.4 kb. Using RNA from various tissues of an estrogen-treated baboon, we found that the mRNA for the oviductal glycoprotein was present only in the oviduct. Hybridization was detected to an mRNA of similar size from oviductal tissue of the baboon, hamster, and mouse and to an mRNA of slightly smaller size in the rabbit, cow, and cat but not to any mRNA species from rat oviductal RNA. Slotblot analysis showed that the message was present in significantly greater (p < 0.05) concentrations in RNA from oviductal tissue from the late follicular stage than from the early follicular, early or late luteal, or postpartum stages. In conclusion, we have isolated the complete cDNA for a human oviductal glycoprotein. The presence of significantly greater amounts of the mRNA during the late follicular phase of the menstrual cycle is consistent with the proposed estrogen control. The mRNA for the oviductal glycoprotein is present only in the oviduct of an estrogen-treated baboon, and a cross-hybridizing RNA is found in oviductal RNA from various mammals.
INTRODUCTION
men [16, 29]. Recently, a partial cDNA for the glycoprotein in the baboon was cloned and sequenced [30]. In the present investigation, we took advantage of the high nucleotide sequence homology among primates to clone. and sequence the full-length cDNA for this protein synthesized by the human oviduct. cDNA segments of the protein were also used to demonstrate tissue specificity, species homology, and menstrual cycle variations of its message.
The mammalian oviduct must provide the appropriate conditions for the reproductive processes of gamete transport, fertilization, and early embryonic development. Under the control of the ovarian steroids, the oviduct undergoes morphological changes in ciliation and cell height and functional changes in secretory activity [1]. Early investigations in the mouse [2,3], hamster [4], rabbit [5,6], sheep [7-9], pig [10], cow [11, 12], baboon [13-15], and human [16] detected and characterized tissue-specific glycoproteins in the oviductal lumen. Subsequent studies confirmed the specific association of some of these secretory glycoproteins with the zona pellucida of ovulated ova or embryos [17-24] as well as a correlation between secretion of these oviductal glycoproteins and the estrogen-dominated stage of estrous [25, 26] and menstrual [15, 16] cycles. In the human, the oviductal epithelium synthesizes and secretes a rich glycocalyx composed of a variety of glycoproteins [27, 28]. During the estrogen dominance of the mid- to late follicular and periovulatory phases of the menstrual cycle, a high-molecular-weight (120 000-130 000) glycoprotein is synthesized by the secretory cells of the baboon [15] and human [29] oviductal epithelium and secreted into the lu-
MATERIALS AND METHODS Materials Kits for 5' and 3' rapid amplification of cDNA ends (RACE) were purchased from Gibco BRL (Grand Island, NY); pBluescript II SK(-), and TA Cloning kits were purchased from Stratagene Cloning Systems (La Jolla, CA) and InVitrogen, Inc., (San Diego, CA), respectively. Oligonucleotide primers were obtained from National Biological Systems (Plymouth, MN). Kodak XAR-5 film was a product of Eastman Kodak (Rochester, NY). Magic Plasmid Purification, Magic PCR Purification, and fmol PCR Sequencing kits were purchased from Promega (Madison, WI). The random primer labeling kit and molecular weight standards were purchased from Boehringer-Mannheim (Indianapolis, IN). Electrophoresis supplies, Prep-a-Gene kits, Zeta-Probe blotting membranes, bis-acrylamide, N;N,N',N'-tetramethylethyleneamine, and acrylamide were purchased from Bio-Rad Labs. (Richmond, VA) and Fisher Scientific (Itasca, IL). Amersham Corp. (Arlington Heights, IL) was the supplier of [32P]dCTP (3000 Ci/mmol) and [y32P]ATP (3000 Ci/
Accepted May 24, 1994. Received March 9, 1994. 'Supported by NIH grants HD20571 and HL07692. The nucleotide sequence reported in this paper will appear in GenBank with the accession number U09550. 'Correspondence: Randal C. Jaffe, Ph.D., Department of Physiology and Biophysics (M/C 901), University of Illinois at Chicago, 901 South Wolcott Avenue, Chicago, IL 60612-7342. FAX: (312) 996-1414.
685
686
ARIAS ET AL.
mmol). SDS, urea, dithiothreitol, 13-mercaptoethanol, agarose, and all other inorganic chemicals of molecular biology grade were products of Sigma Chemical Co. (St. Louis, MO). Collection of Tissues Human. Human oviducts were obtained from normal cycling patients undergoing hysterectomy or elective tubal ligation. Informed consent was obtained from all patients in compliance with the Institutional Review Board at the University of Illinois at Chicago. Upon removal, the oviducts were immediately transferred to sterile culture medium. A small piece of oviduct was fixed and examined histologically [1] to confirm the menstrual stage as determined from patient history. Tissues were weighed, frozen in liquid nitrogen, and stored at -70°C until further processed. Baboon. Baboon (Papio anubis) oviducts were obtained from intact, ovariectomized animals or from estradiol-treated ovariectomized animals as previously described [30]. A single ovariectomized baboon was treated for 14 days with estradiol. The animal was then administered a lethal level of anesthesia, and tissues were harvested from many major organs. All tissues were portioned into 1-2-g sections, frozen in liquid nitrogen, and stored at -70 0C. Other. Bovine oviductal tissues used for the present study were the same as previously described [11]. All other mammalian oviductal tissue RNAs were the same as previously described [30]. Northern and Slot Blots Total RNA was isolated from frozen tissues by the guanidinium isothiocyanate method of Chirgwin et al. [31]. For Northern blots, 10 jig of total RNA was electrophoretically separated on a 1.2% formaldehyde-agarose gel and subsequently transferred by capillary blotting onto Zeta-Probe blotting membranes [32]. Slot blots were prepared on a Minifold II Slot-Blotter (Schleicher and Schuell Co., Keene, NH) according to the manufacturer's protocol. Ten micrograms of total RNA was loaded into each well. All membranes were baked for 2 h at 800C in a vacuum oven and prehybridized at 65 0C (high stringency) or 50°C (low stringency) for 16-20 h in prehybridization solution; this consisted of 6-strength standard sodium citrate (SSC), 5-strength Denhardt's solution, 0.5% SDS, and 200 g/ml denatured salmon sperm DNA [33]. Hybridizations were carried out in the same manner with the addition of 1 x 106 cpm/ml [ot32P]dCTP-labeled human or baboon cDNA restriction fragments. Human and baboon oviductal glycoprotein cDNA fragments were purified from a 1.5% agarose gel with a Prep-a-Gene kit and labeled to a specific activity of 1 x 109 cpm/xg with a random primer labeling kit. The membranes were washed sequentially in double-strength SSC/ 0.5% SDS for 5 min and double-strength SSC/0.1% SDS for
15 min, both at room temperature, and 0.1-strength SSC/ 0.5% SDS for 1 h at the same temperature used for hybridization (65°C or 50°C); they were then air-dried and exposed to Kodak X-Omat film with a du Pont Cronex intensifying screen (E. I. du Pont, Wilmington, DE) at -80 0C. Sequential hybridization of the blots was performed after the membranes were boiled for 15 min in 0.25% SSC and 0.1% SDS to remove bound cDNA probe, the efficiency of which was checked by autoradiography. The human P-actin was obtained from the ATCC (#65281; Rockville, MD). 5' and 3' RACE RACE [34-37] was performed according to the manufacturer's protocol. In brief, for 3' RACE, an adapter primer was used to synthesize first strand cDNA from late follicular phase human oviductal total RNA. After RNase H treatment, the sequence of interest was amplified using a sense 20mer oligonucleotide primer (5'-TCTACGTAATGAATGATATC-3') derived from the baboon sequence [30] and a universal amplification primer. For 5' RACE, an antisense 20mer oligonucleotide (5'-ACAGGGGTCACAGATGGATG-3') was synthesized that corresponded to the baboon oviductal glycoprotein sequence 423-442 bp 3' of the internal EcoRI site; this was used to generate the first strand cDNA from the total RNA. After incubation with RNase H, the amplified products were purified on a GlassMax (Gibco BRL, Grand Island, NY) spin cartridge and dC-tailed with terminal deoxynucleotide transferase. Polymerase chain reaction (PCR) was then performed using a second antisense oligonucleotide (5'-GGGTCAGGGCCTTCTCTCCA-3') to the baboon oviduct glycoprotein sequence and an oligo dG anchor primer. Cloning and Sequencing of 5' and 3' cDNA Ends Both RACE products were cut with EcoRI (internal site) and Sal I (anchor primer site) and ligated with T4 DNA ligase into a similarly cut pBluescript II SK(-) plasmid vector. Three independent clones were selected that contained the EcoRI-truncated 3' cDNA end (1.0 kb), and five clones were selected that contained the 5' cDNA end (1.2 kb). Complementary DNA inserts were sequenced by the Sanger dideoxy method [38] as adapted for PCR using Taq polymerase (Promega). The full-length sequence was defined through use of at least three independent clones in both directions. Serially designed oligonucleotide primers (National Biological Systems) of 20 bases were used to complete the sequence. PrimerExtension Primer extension studies employed an antisense 20-mer oligonucleotide (5'-GCCCAGTrGGTGAAATAACA-3') specific to a region of the 5' RACE fragment 88-107 nucleotides 3' of the first nucleotide. The oligonucleotide primer was end-labeled with [y-32P]dATP using T4 polynucleotide
ESTROGEN-DEPENDENT HUMAN OVIDUCTAL GLYCOPROTEIN cDNA
687
FIG. 1. Northern blot analysis of total RNA isolated from human and baboon oviducts. A Northern blot of total human RNA (10 pg) prepared from ectopic pregnancy or late follicular stage (Day 13) oviducts was probed with an [a-P32]dCTP-labeled 5' fragment of the human oviductal glycoprotein cDNA insert under stringent conditions (65°C). The blot was then stripped of label and reprobed with an [U-P 3 21dCTP-labeled 230-bp fragment of the baboon oviductal glycoprotein cDNA. A single mRNA species (2.4 kb) is recognized by both cDNA probes only in the oviduct RNA from the late follicular phase of the menstrual cycle.
kinase. The 5' end-labeled primer was then incubated with 10 Kzg of the total human oviductal RNA and extended with reverse transcriptase (Superscript RT, BRL) as described by Ausubel et al. [39]. The product was electrophoresed on an 8% sequencing gel along with a sequencing reaction that served as a size marker and then autoradiographed. StatisticalAnalysis Differences among groups were determined by ANOVA after arcsine transformation of the ratio of oviductal glycoprotein mRNA to 0-actin mRNA, and differences between groups were determined by the Student-Newman-Keuls test using the program SigmaStat Oandel Scientific, San Rafael, CA).
RESULTS Molecular Mass of the Human Oviductal Glycoprotein mRNA The 1.2-kb 5' cDNA end of the human oviductal glycoprotein and a 230-bp piece from the baboon oviductal glycoprotein cDNA [30] were used to sequentially probe a Northern blot containing human total RNA from Day 13 (follicular stage) oviductal tissue and from oviductal tissue contralateral to an ectopic oviductal pregnancy (6 wk after the last menstrual period). The 1.2-kb human cDNA probe hybridized strongly to a 2.4-kb message in the lane containing Day 13 oviductal total RNA (Fig. 1). No hybridization was detected with the total oviductal RNA of the ectopic pregnancy tissue sample. The blot was then stripped and
688
ARIAS ET AL.
CAGACCATTGAG ATO TOG AAS CT TTO CTG TGG OTT 00G CTO OTT CTT GTG CTO AAA CAC CAC GAT GT OCT CC CAT AAA CTC OTO Het Trp Lys Leu Leu Leu Trp Val Oly Leu Val Leu Val Leu Lys His Hi Asp Oly Ala Ala HiS Lys Leu Val
87 25
TGT TAT TTC ACC AAC TGO OCA CAC AOT COG CCA OOC CCT OCC TC ATC TTG CCC CAT SAC CTO SAC CCC TTT CTC TOC ACC CAC Cys Tyr Phe Thr Asn Trp Ala His Ser Arg Pro Oly Pro Ala Ser Ile Leu Pro His Asp Leu Asp Pro Phe Leu Cys Thr His
171 53
CTG ATA TTT OCC TTT OCC TCA ATO AAC AAC AAT CAD ATT OT OCT AA SAT CTC CA GSATGA AAA ATT CTC TAC CCA GAG TTC Leu Ile Phe Ala Phe Ala Ser Met An Asn ASn Oln Ile Val Ala Lys Asp Lu lGin Asp Glu Lys Ile Leu Tyr Pro Olu Phe
255
AAC AAA CTA AAG GAG ASO AAC ADA GAG CTO AAA ACA CTA CTO TCC ATC GOC 000 TOO AAC TTT GOC ACC TCA AGA TTC ACC ACT Asn Lys Leu Lys Glu Arq Asn Arq Glu Leu Lys Thr Leu Leu Ser Ile Gly Gly Trp Asn Phe Gly Thr Ser Arg Phe Thr Thr
339 109
ATO TTG TCC ACA TTT OCC AAC COT GAA AAG TT ATT OCT TCA OTT ATA TCC CrT CTO AGO ACA CAT GAC TTT GAT GOT CTT GAC Met Leu Ser Thr Phe Ala Asn Arg Olu Lys Phe Ile Ala Ser Val Ile ler Leu Leu Arg Thr His Asp Phe Asp Oly Leu Asp
423
CTT TTC TTC TTA TAT CCT OOA CTA AGA GOC AOC CCC ATO CAT SAC COO TOO ACT TTT CC TTC TTA ATT OAA GAO CTC CTO TTT Leu Phe Phe Leu Tyr Pro oly Lou Arq Oly Ser Pro Met His Asp Arg Trp Thr Phe Lu Phe Leu Ile Glu Glu Leu Lou Phe
507
GCC TTC CGOGAA GAG OCA CTO CTC ACC ATO COC CCG AO Ala Phe Arg Lys Glu Ala Leu Leu Thr Met Arg Pro Arg
CTO CTO TCT OCT OCT OTT Leu Lou Ber Ala Ala Val
OSO GTC CCA CAC ATC OTC CA Oly Val Pro His Ile Val Gln
591
ACA TCC TAT SAT GTG COC TTT CTA OGA AA CTC CT GAT Thr Ser Tyr Asp Val Arg Phe Leu oly Arg Leu Lou Asp
ATC AAT OTC TTO TCT TAT Ile Ann Val Leu aerTyr
TTA CAT GOA ADT TOO GOA AO Leu His Oly Ser Trp Olu Arg
675 221
TTC ACA GOA CAT AAT AOC CCC CTC TTC TCT CTO CCT GAA Phe Thr Gly His ASn Ser Pro Leu Phe Ser Leu Pro Glu
CCC AAA TCT TCO OCA TAT Pro Lys Ser er Ala Tyr
ATO AT TAT TOG ADA AMA CTT Met Asn Tyr Trp Arg Lys Leu
759 249
OGG OCA CCC TCA GAG AO CTC ATC ATO G ATC CCC ACC TAT GOACOT ACC TTT COC CTC CTC AAA OCC TCT AAG AAT GOG TT Gly Ala Pro Ser Glu Lys Leu Ile Met Oly Ile Pro Thr Tyr Gly Arg Thr Phe Arg Leu Leu Lys Ala Ser Lys ASn Oly Leu
843
CAGO CC AGA OCG ATC OOA CCA OCA TCT CCA OGG AAG TAC ACC AAO CAAOAA 0OC TTC TTG OCT TAT TTT GAG ATT TGT TCC TTT Gln Ala Arg Ala Ile O1y Pro Ala Ser Pro Sly Lys Tyr Thr Lys OlGin Olu Oly' Phe Lou Ala Tyr Phe Glu lie Cys Ser Phe
927 305
CGAO AA A CAC TOG ATT GAT TAC CAG TAT GOC C GTC TOG OGA C GAO OO AA GAO TG MT OOC TAT OAC AAT TAT OCC AAC Val Trp Gly Ala Lys Lys Him Trp Ile Asp Tyr Oln Tyr Val Pro Tyr Ala Asn Lys Gly Lys Olu Trp Val Oly Tyr Asp ASn
1011 333
OCC ATC AOC TTC AOT TAC AAD OCA TOG TTT ATA AO CGA OAG CAT TT 000 o0o CC ATG GTO TOG ACA TTGO AC ATG GAT GAC Ala Il Setr Phe Ser Tyr Lys Ala Trp Phe Ile Arg Arg Olu H1 Phe Oly Gly Ala Met Val Trp Thr Lu Asp Met Asp Asp
1095 361
GTC AG GOC ACG TTC TOT 00C ACT OOC CCT TTC CCC CT OTC TAC OTA TTG AAT GAT ATC CTG GTO COG OCT AO TTC AGT TCA Val Arg Oly Thr Phe Cys Oly Thr Gly Pro Phe Pro Leu Val Tyr Val Leu ASn Asp Ie Lou Val Arg Ala Glu Phe Ser Scr
1179 389
A CO OTO ACC ACO OCA TO ACT TCT TTA CCA CAL TTT TOO CTO TCA TCT OCT GOT AAT TCT CA AOC ACT GAC CCT G Thr Ser Lou Pro Gin Phe Trp Lou Ser Ser Ala Val Asn Ser Ser Scr Thr Asp Pro Olu Arg Lou Ala Val Thr Thr Ala Trp
1263 417
ACC ACT GAT AOT AAG ATT TTG CCC CCA OGA OGA GAG OCT O00 OTC ACT GAG ATC CAC OG AAO TOT GAA AAT ATO ACT ATA ACC Thr Thr Asp Ser Lys Ile Leu Pro Pro Gly Oly Olu Ala Oly Val Thr Olu lie His Oly Lys Cys Olu An NMt Thr l1e Thr
1347 445
CCT AGA GOT ACA ACT GIO ACC CCT ACA AAGO GA ACT OTA TCC CTT OGA AAO CAC ACT OTA OCT CTA OGA GA AAO ACT GAG ATC Pro Arq Oly Thr Thr Val Thr Pro Thr Lys Olu Thr Val Ser Lou Gly Lys His Thr Val Ala Lou Oly Olu Lye Thr Olu Ie
1431 473
G GT CAT CAA TCT ACT 000 OCA ATO ACC ATO ACT TCT GITO GO CAT CAO TCC ATO ACC CCT 0OA GAO AAO OCC CTO ACC CC OTO Thr Oly Ala Met Thr Met Thr Ser Val Oly His Oln Ser Met Thr Pro Oly Glu Lys Ala Lou Thr Pro Val Oly His Gin Ser
1515
OTO ACC ACT OA CAO A ACC CTG ACC TCT GTO GOT TAT C TCT O ACC CCT OG000AA AA ACC CTO ACC CCT OTO OOT CAT Val Thr Thr Oly Oln Lys Thr Lou Thr Shr Va1 Oly Tyr Oln Ser Val Thr Pro Oly olu Lys Thr Lou Thr Pro Val Oly 13
1599 529
CAO TCT GTO ACC CCT GO AOT CAT CA TCT GTG ASC CCT GOA GA ACO ACT ATO ACC CCT OTC CAT TTT CAO ACT GAG ACC CTT Gin Ser Val Thr Pro Val Scr 1is Gln Br Val Ser Pro Gly Gly Thr Thr Mt Thr Pro Val His Phe Gin Thr Olu Thr Leu
1683 557
AOL CAD AATT A AA ASO AAG OCT SGO OCC COT GAA AO GGO ACT GC CCC TCC AGA AAC ATA TCA OTC ACC CCT Ser Val Thr Pro Arg Oln Ann Thr Val Ala Pro Arg Arg Lys Ala Val Ala Arg Olu Lys Val Thr Val Pro Ser Arg ASn Ii
1767 585
GAA OO CAO AC? ATG CCTTTA AGA Go GAO AAT TTG ACT TCT GOAGOO GC ACT CAC CCC AGO ATO OT AAC TTO GOT CTT CAO Olu Oly Oln Thr M.t Pro Lou Arg Oly Olu Ann Lou Thr Ser Olu Val Oly Thr His Pro Arg Met Oly An LOu Gly Lou Oln
1851 613
ATG OAA OCT GAA AAC AOO ATO ATO CTOG CC TCC AOC CCC GTC ATC CA CTC CCO GA CAA ACT CCT CTA OCS TTT GC AC COT Met LOU Sr r Setr Pro Val 11s Oln Lou Pro Olu Oln Thr Pro Lou Ala Phe Amp Asn Arg Ht Olu Ala Glu Amn Arg Ht
1935 641
TTG TTC CCA TCT ATO GAA ACC ATT CCT CTO TCA ACT CAO TAA CCC Lou Phe Pro Sar Met Olu Thr lie Pro Lou Ser Thr Oln Knd
2032 654
ars
&A
._ fuu'Tt-
A0ChMATAO0qCCULTCTCTTTTCCATA
A
TOTCTTTTCTTCTAAOTGCATOTTOGAAG :m ,TT~r···~·
TCCITTCACTAAAAAACCCAOAAAATCTOCTGT CCTTCTAT
LL'·~
81
137
165
193
277
501
2143 2216
ESTROGEN-DEPENDENT HUMAN OVIDUCTAL GLYCOPROTEIN cDNA
689
reprobed with the 230-bp baboon oviductal glycoprotein cDNA probe. The baboon cDNA probe recognized a message of the same size (2.4 kb) only in the lane containing follicular phase RNA. Full-Length cDNA Sequence and Translated Protein Product
Sequence analysis showed the full cDNA to be 2216 bp in length (Fig. 2). The complete sequence has an openreading frame of 1962 bp (13 to 1975) that codes for a 654 amino acid protein with a predicted molecular mass of 72.9 kDa and a pI of 6.2 as indicated by the program Microgenie (Beckman Instr., Palo Alto, CA). The human oviductal glycoprotein mRNA has a short 5' (12 nucleotides) untranslated region and a 240-nucleotide 3' untranslated region with the consensus AATAAA polyadenylation signal at 2173. The deduced amino acid sequence contains four potential N-glycosylation sites (Asn-X-Ser/Thr), two potential N-myristoylation sites, and a variety of potential phosphorylation sites as indicated by a PROSITE [40] search. The absence of a consensus sequence for O-linked glycosylation precludes the determination of a realistic value for the number of serine and threonine residues that are potentially glycosylated. A GenBank database search using BLASTN and BLASTP and direct comparisons using LFASTA revealed that the human oviductal glycoprotein cDNA had a 92% identity and the deduced amino acid sequence had a 91% identity to the 1232-nucleotide and 179 amino acid sequence of the estradiol-stimulated baboon oviduct-specific glycoprotein (GenBank Accession no. M59903). Subsequent to the submission of the present manuscript, Sendai et al. [41] reported the cloning of a bovine oviduct-specific glycoprotein that has an 83% identity of the 5' 1496 nucleotides and a 72.5% identity in a 534 amino acid overlap. Identities on both the nucleotide and amino acid levels of approximately 55% and 46%, respectively, were also found to a variety of Family 18 glycosyl hydrolases [42] and certain mammalian proteins of unknown function that are structurally related to the Family 18 glycosyl hydrolases [43, 44]. Primer extension studies resulted in a single major band 108 bases in length (Fig. 3). This was the precise distance the 5' end of the primer was from the 5' end of the sequence, demonstrating that the sequence included the transcription initiation site. The sequence of this region does not conform to the proposed consensus transcription start site of Corden et al. [45]. To confirm that none of the sequence had been lost during the cloning procedure, the 3' RACE first strand cDNA product was employed as a template to am-
FIG. 2. Full-length sequence of the estrogen-dependent human oviduct-specific glycoprotein cDNA. The full-length cDNA was 2216 nucleotides in length. The cDNA sequence contained an open-reading frame of 1962 nucleotides coding for a 654 amino acid protein with a predicted molecular weight of 72 908 and p 6.21. Potential N-glycosylation sites are underlined and a polyadenylation signal is double underlined.
FIG. 3. Primer extension studies. A single prominent band was observed with a length of 108 nucleotides, confirming the 5' end of the mRNA to be 108 nucleotides 5' of a human gene-specific antisense 20-mer primer (nucleotides 88-107.
plify a region spanning the internal EcoRI site by means of oligonucleotide primers designed from the deduced sequence of the 5' cDNA end and 3' cDNA end. Sequencing of this region confirmed the presence of a single EcoRI site and no further sequence. Tissue Specificity of the Estrogen-DependentOviductal Glycoprotein mRNA
The 1.2-kb 5' human oviductal glycoprotein cDNA insert was used to probe a Northern blot containing total RNA from various tissues of an adult female baboon treated with estradiol for 14 days (Fig. 4). The 1.2-kb cDNA probe de-
690
ARIAS ET AL. _
5 -
--
*
4 .r IC .G 0
22 -
.
1
(4)
(3)
II
V
0
_
I |
(4) I
(6)
I
I
Q) 1
