Differential Expression of Distinct mRNAs for Ovine Trophoblast ...

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KENNETH P. NEPHEW,3 AMY E. WHALEY,3 RONALD K. CHRISTENSON,4 and KAZUHIKO IMAKAWA2 '3. The Women's Research Institute,3 Department of ...
BIOLOGY OF REPRODUCTION 48, 768-778 (1993)

Differential Expression of Distinct mRNAs for Ovine Trophoblast Protein-1 and Related Sheep Type I Interferons' KENNETH P. NEPHEW,3 AMY E. WHALEY, 3 RONALD K. CHRISTENSON, 4 and KAZUHIKO IMAKAWA 23' The Women's Research Institute,3 Department of Obstetrics and Gynecology University of Kansas School of Medicine, Wichita, Kansas 67214-4716 U.S. Department of Agriculture,4 Agricultural Research Service Roman L. Hruska US. Meat Animal Research Center Clay Center, Nebraska 68933-0166. ABSTRACT An antiluteolytic substance secreted by the ovine conceptus and primarily responsible for maternal recognition of pregnancy is ovine trophoblast protein-1 (oTP-1), a new type I interferon (IFN). The objectives of this research wefe 1) to investigate whether multiple, distinct genes encode oTP-I and other type I IFNs in the ovine genome and 2) to examine expression of oTP1 and other IFN mRNAs during conceptus development. Genes for type I IFNs were isolated from a subgenomic library constructed from Day 25 (Day 0 = estrus) ovine conceptus high-molecular-weight DNA. Six clones were isolated and nucleotidesequenced from -1000 to +900 (bases relative to cap site). Comparisons of inferred amino acid sequences demonstrated that four clones were distinct oTP-1 genes and that two clones, defined as 09 and o012, were related type I IFNs (deduced aa homology of o9 and o12 to oTP-I was 71% and 54%, respectively). The presence of mRNAs encoded by oTP-I and type I IFN genes was examined quantitatively via reverse transcription-polymerase chain reaction (RT-PCR) analysis of total cellular RNA (tcRNA) extracted from Day 13-45 concepti. Total cellular RNA obtained from Day 75 placenta and adult lymphocytes was also analyzed by RT-PCR, coupled with Southern blot hybridization of the PCR reaction products with specific DNA probes. PCR products were sequenced in order to confirm primer specificity, and mRNAs corresponding to two of the four oTP-1 genes and to both related IFN clones (09 and o012) were identified. Furthermore, quantitation of the PCR products revealed that of the two oTP1 genes examined, one was highly expressed on Days 13-20 and transcripts were weakly detectable on Days 30 and 45. In contrast, the other oTP-1 gene examined was weakly expressed on Days 13-20 only. Densitometric analysis of hybridization signals revealed that IFN 09 mRNA was detected in Day 75 placenta but only weakly detected in conceptus (Days 13-45) and adult lymphocytes. IFN o 012 mRNA was abundant in lymphocytes relative to the other tissues examined. Collectively, these results demonstrate the existence of distinct oTP-1 and related type I IFN genes. The data suggest that these genes display differential, tissue-specific expression and developmental regulation during pregnancy.

INTRODUCTION

anisms by which oTP-1 exerts its antiluteolytic effect(s) have recently been reviewed by Bazer et al. [5]. On the basis of similarities in the N-terminal amino acid [6] and cDNA sequences [7-10], oTP-1 and bTP-1 (the bovine homologue) have been classified as part of the large, multi-gene type I interferon (IFN) family, which includes IFN-aII [11], also called IFN-omega [12]. Ovine trophoblast protein-1 also possesses functional characteristics of IFN, such as potent antiviral [13], antiproliferative [14, 15], antitumor [16], and immunosuppressive [14, 15] activities. In humans and in cattle, IFN-omega is encoded by multiple, functional genes [11, 17]. Several observations suggest that this may also be true for oTP-1: 1) multiple oTP-1 cDNAs have been isolated [18]; 2) cell-free translation of poly(A) + RNAs extracted from ovine and bovine conceptus have produced polypeptides with multiple isoelectric variants [19]; 3) at least five different isoforms of oTP-1 exist [20]; and 4) Southern blot analysis of high-molecular-weight ovine DNA [21, 22] has revealed numerous hybridizing bands that most likely represent multiple genes. In addition, a more than tenfold increase in oTP-1 secretion is detected as the conceptus changes morphologically from spherical to filamentous forms [23]. Magnitude of oTP-1 production (a single Day 16 sheep conceptus secretes up to 100 pug of oTP-1

A series of coordinated interactions must occur between the mother and developing embryo in order for pregnancy to continue beyond the early stages. A substance produced by the conceptus and required for the initial establishment of pregnancy in sheep is ovine trophoblast protein-1 (oTP1) [1], also designated as trophoblastin [2]. This low-molecular-weight acidic protein is the major secretory product of the peri-attachment ovine conceptus between Days 13 and 21 of pregnancy [1]. Production of oTP-1 by the conceptus prolongs ovarian progesterone production and attenuates uterine prostaglandin F2 . production [3, 4]. Potential mech-

Accepted November 25, 1992. Received March 2, 1992. 'This work was supported in part by funds from The Women's Research Institute, The Wesley Foundation, and grants to K.P.N. from The University of Kansas School of Medicine-Wichita and Wesley Medical Research Institutes, the Maizie Wilkonson Memorial Endowed Fund for Cancer Research. Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable. ZCorrespondence: Kazuhiko Imakawa, The Women's Research Institute, Department of Obstetrics and Gynecology, University of Kansas Schcol of Medicine, 2903 E. Central, Wichita, KS 67214-4716. FAX: (316) 687-5231.

768

oTP-1 AND TYPE I IFN GENE EXPRESSION TABLE 1. Oligonucleotides used to characterize 5' noncoding regions of oTP-1 and related IFN genes. a Primers 1

Sequences 5'-TAmTrCTACTTGACCTAG-3'

2

5'-TCTTACACATAGGAAT-3'

3 4

5'-CATGAACACATCTGTAGC-3' 5'-GAGCCCACAGGTGTAT-3'

5

5'-CTATAAGTCmTTCCATAC-3'

769

was isolated from these cell preparations as described by Sambrook et al. [26].

Location oTP-o2 oTP-o7 oTP-o8 oTP-olO oTP-o2 oTP-o7 oTP-o8 oTP-olO oTP-o9 oTP-o2 oTP-o8 oTP-olO oTP-o2 oTP-o7 oTP-o8 oTP-olO

(-114 to -97) (-112 to -95 (-116 to -99) -112 to -95) (-394 to -379) (-391 to -376) (-392 to -377) (-474 to -459) (-328 to -311) (-658 to -643) (-656 to -641) (-764 to -749) (-258 to -241) (-257 to -240) (-260 to -243) (-340 to -323)

aLocations of oligonucleotides (base numbers) are identical to those in Figure 3.

during a 24-h culture period in vitro) [24] may require transcription from more than one gene. The first ovine type I IFN gene isolated and characterized was oTP-1. To date, however, only two other members of this apparent multi-gene subfamily in sheep have been reported [22, 25], and there is no information regarding their pattern of expression. Therefore, the objectives of the present study were twofold. The first was to investigate differences in the 5' noncoding regions of multiple oTP-1 and other related type I IFN genes, and the second was to determine whether a developmental and/or differential pattern of type I IFN gene expression exists during pregnancy in sheep. MATERIALS AND METHODS Extraction of High-Molecular-Weight (HMW) DNA from Sheep Conceptus Crossbred ewes used for tissue collection were housed at the Roman L. Hruska U.S. Meat Animal Research Center (Clay Center, NE). Ewes were checked daily for estrous behavior through use of vasectomized rams. Ewes were bred with intact rams of proven fertility at the first sign of estrus (Day 0) and thereafter at approximately 12-h intervals until the end of estrus. Concepti (n = 3) were collected on Day 25 by hysterectomy under anesthesia and aseptic conditions. Concepti were excised from the uterus, washed, and then minced in 5 ml of Dulbecco's PBS. Minced tissue was pooled and placed in 10 ml prewarmed PBS containing 0.3% (w/v) collagenase (Sigma Chemical, St. Louis, MO), dispersed using a spinal needle and a Dubnoff shaker bath (37°C, 45 min, medium speed), and collected by centrifugation at 900 x g for 4 min, 22°C. The cell pellets were resuspended and washed three times with fresh PBS. Cells were resuspended in a final volume of 2 ml PBS, and cell density was determined via a hemacytometer. HMW DNA

Construction, Screening, and Sequencing of Ovine Subgenomic Library A 10-apg sample of HMW DNA was digested with EcoRI restriction endonuclease (Promega, Madison, WI) for 90 min at 37°C, then electrophoresed for 8 h at 75 V in 0.8% agarose gel (Bio-Rad, Richmond, CA) containing 1 pLg/ml ethidium bromide. DNA fragments ranging from 1 to 10 kb were excised, electroeluted, concentrated, and analyzed via PCR using oTP-1-specific oligonucleotide primers. The primers were designed on the basis of an oTP-1 cDNA [7]: oTP-1 cDNA 5' primer: 5'-CCTGTCTGCAGGACAGAAAAGACTT-3' oTP-1 cDNA 3' primer: 5'-TCTGAATTCTGACGATTTCCCAGGC-3' Primer location relative to a potential cap site of the cDNA was 221-245 bases for the 5' primer (Fig. la) and 556-580 bases for the 3' primer (Fig. f). The size range of 3.0 to 4.2 kb responding to PCR analysis was cloned into gtll vector [27, 28]. Approximately 90 000 plaque-forming units (pfu) were plated with Escherichia coli Y1090 cells (Promega) on Luria broth agar plates and incubated for 18 h at 370C. Duplicate nitrocellulose facsimile filters (Micron Separations, Westborough, MA) were overlaid on the plates [26], prehybridized in the buffer described above for 2 h at 42°C, and then hybridized in the same buffer containing 20% (v/ v) formamide and 3 2P-labeled 3' oTP-1 cDNA probe [29] instead of herring sperm DNA The filters were washed twice with 5-strength SSC (single-strength SSC: 0.15 M NaCl, 0.015 M sodium citrate) and 0.1% (w/v) SDS for 5 min at 42°C, washed twice with double-strength SSC and 0.1% (w/v) SDS for 5 min at 42°C, and then exposed to x-ray film (XAR; Eastman Kodak, Rochester, NY) plus an intensifying screen for 18 h at -80°C. Plaques that hybridized to the probe were identified and analyzed by PCR with the primers described above (oTP-1 cDNA 5' and 3'). Six plaques were further screened and purified [26]. Phage DNA was isolated [30] and digested with EcoRI restriction endonuclease to excise DNA inserts. DNA fragments were then subcloned into pUC19 plasmid vector and transformed in E. coli JM101 cells (Promega). Doublestranded DNA sequencing [31] was performed on isolated plasmids using the dideoxy termination method [32] with Sequenase (U.S. Biochemical, Cleveland, OH), 35S-dATP (Dupont-NEN Research Products, Boston, MA), and oligonucleotides synthesized at the Oligonucleotide Synthesis Facility, University of Missouri (Columbia, MO). So that nucleotide strands could be sequenced completely and in both orientations, several oligonucleotides were designed and used (Table 1). Sequence comparisons [33] were made using DNASTAR software (DNASTAR Inc., Madison, WI).

770

NEPHEW ET AL.

RNA Preparationand Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Ovine concepti (Days 13, 15, 17, 20, 30, and 45), placenta (Day 75), and placentome (Day 75) were collected, snapfrozen in liquid nitrogen, and stored at -80°C until analyzed. Ovine venous blood samples were collected from mature ewes in tubes containing EDTA, and peripheral blood lymphocytes were isolated by density-gradient centrifugation on a Ficoll-Isopaque gradient (Histopaque; Sigma). Ovine peripheral blood lymphocytes appeared at an apparent gradient density of 1.077 g/ml. Total cellular RNA was prepared from each of these tissues by means of a standard procedure [34]. All tcRNA used in the present study was initially digested with RNase-free DNase (20 U; Stratagene, La Jolla, CA), 40 mM Tris-HCl (pH 7.5), 6 mM MgCl 2 , 2 mM CaCI2 , and sterile diethyl pyrocarbonate (DEPC)-treated water at 37 0C for 30 min. Total cellular RNA was precipitated at -80 0C by the addition of sodium acetate to 0.3 M and two volumes of absolute ethanol, then vacuum dried and resuspended in 10.5 1l sterile DEPC-treated water. The DNase-treated tcRNA samples were reverse transcribed at 42°C for 60 min by means of an avian myeloblastosis virus reverse transcriptase (AMV-RT) kit (Gibco BRL, Gaithersburg, MD). Briefly, reactions were carried out in a 2 5-1ul reaction volume that included 5 1 of 50 mM KCI; 5 l1of reverse transcription buffer (500 mM Tris-HCl [pH 8.3], 50 mM MgCl 2 and 50 mM dithiothreitol); 0.5 l1of RNasin (9 U; Promega); 0.5 mmol/L each of dATP, dTTP, dCTP, dGTP; 500 ng of oTP1 cDNA 3' primer, instead of an oligo(dT) primer; and 5 units of AMV-RT. After 60-min incubation at 42°C, reactions were terminated by heating at 95°C for 3 min. Products of the RT reaction then served as templates for PCR studies using specific primers (Fig. 1) [35]. The expected PCR amplification product lengths for oTP-1, o2, o1O, o9, and o12 mRNA were 361, 296, 387, 425, and 432 bp, respectively. The oTP-1 cDNA 5'-specific primer displayed 100% sequence similarity to the oTP-1 genes isolated in the present study (but not IFN 09 and o12), and thus was utilized as a positive control. PCR amplifications were carried out in a 20-,al volume consisting of 2.5 1 l RT reaction product (template), 10 pmol/L of specific primer, 10 mM TrisHCI (pH 8.3), 50 mM KCI, 1.5 mM MgCl 2, and 20 pxmol/L each of dATP, dTTP, dCTP, and dGTP. After PCR amplification for 25 cycles (each for 1 min at 94°C, 1 min at 60°C, 2 min at 720 C), products were subjected to electrophoresis in a 1.5% agarose gel with 1 ag/ml ethidium bromide added. After electrophoresis, agarose gels were photographed under UV illumination. PCR products were then either Southern blotted [36] or subcloned and sequenced as described above. For Southern blot analysis, PCR products were electrophoresed and transferred to nylon membranes (IBI, New Haven, CT). The membranes were prehybridized in 5strength SSC, 50% (v/v) formamide, 0.6% (w/v) SDS, 0.5%

(w/v) nonfat dry milk, 20 mM Tris-HCl (pH 7.5), 4 mM EDTA, and 0.2 mg/ml single-stranded herring sperm DNA (Promega) for 4 h at 42°C; they were then hybridized in the buffer described above with IFN (o9 or o012) cDNA probe. The probe was labeled with 32 P-dATP (sp. act. 5 x 108 cpm/ pxg DNA; Dupont-NEN Research Products) through use of a random-prime labeling kit (Boehringer-Mannheim, Indianapolis, IN). Blots were washed twice for 10 min at 42°C with 5-strength SSC and 0.1% SDS (w/v) and twice for 5 min with double-strength SSC and 0.1% SDS, then exposed to x-ray film (XRP; Eastman Kodak) for 24 to 48 h at -80°C in the presence of an intensifying screen. The probes used were a 652-bp NCOI to EcoRI DNA fragment representing the coding region of IFN 09 (69 to 720), and a 564-bp Pst I/EcoRI DNA fragment representing the region 325 to 889 of IFN o12. Complementary RNA Preparationand Quantitative RT-PCR In order to prepare cRNA corresponding to various oTP1 and related IFN genes, ranges of nucleotide residues that would contain primer binding sites were excised by means of various restriction endonucleases (base numbers are identical to those in Fig. 1): oTP-1, 1-640 (EcoRI-Bgl II); oTP-o2, 69-725 (NCOI-EcoRI); oTP-olO, 69-640 (NCOI-Bgl II); oTP-o9, 69-720 (NCOI-EcoRI); oTP-ol2, 128-889 (BIN1EcoRI). These fragments were subcloned into pBS M13 vector (Stratagene), and their orientation and nucleotide residues were determined by dideoxy termination sequencing method as described previously. Upon linearization, sensestrand cRNAs were prepared from these constructs by incorporating 3 H-CTP (Dupont-NEN). Amounts of cRNA were determined from trichloroacetic acid (TCA)-precipitable counts of 3 H-CTP, and integrity of cRNA was determined by Northern blot analysis. Standard curves, ranging from 1 fg to 1.25 ng, were constructed from each of the cRNAs corresponding to various oTP-1 genes. Although quantitative RT-PCR reactions for oTP-1, o2, olO, o9, and o12 mRNA were performed separately, titrated amounts of cRNA were used as an internal standard in the same RT-PCR reaction through which the unknown samples were examined [35]. Products of the RT reactions were PCR amplified using the 32 P-yATP (Dupont-NEN)-end-labeled various oTP-1 primers. PCR conditions were as described above. Amounts of various oTP-1 mRNAs were determined after 25 cycles of PCR amplification, the point at which ex-

FIG. 1. Comparison of nucleotide sequences of oTP-1 and related type I IFN genes. Alignment of nucleotides represents the open-reading frame only. Nucleotide residues of various oTP-1 and related type I IFN genes differing from that of oTP-1 [7] are shown. Nucleotide sequences for specific primers used in RT-PCR analyses are underlined and labeled. oTP-1 cDNA 5' primer (a), oTP-o2 5' primer (b), oTP-olO 5' primer (c), oTP-o9 5' primer (d), oTP-o12 5' primer (e), and oTP-1 cDNA 3' primer (f). Actual nucleotide sequence for oTP-1 cDNA 3' primer is in the text.

oTP-1 AND TYPE I IFN GENE EXPRESSION

771

CTGGGTC, CATGGCCCAGGGGAiTCT

ATGGCTTCGTTATTCATCGCCTCGT

GTTACCTATC

oTP 07 010

...........

C

G.................. 09

GC.CG

+149

+70

(a)

TGAGAGACTCATGCTGGATGCCAGGGAGAACCTCAAGCTCCTGGACCGAATGAACAGACTCTCCCCTCATTCCTGTCTGC .C ................................. G........................ .CG ............................... ............................................ G

C ....

G ........

....................

AC. A..A .G....

(eC.C.ACTCTGCCCC --------

......

.G.*M)C. ---..----------.. G..A.... GG..' . ..... T..G...CC ........ G ........ A .TC..C.T.GGT...T...... AG .... GG..GG .......

c........................... T..GC.T........ GTC.TG ..... GC

G ................................................... ........................ t + .... ,

....................................................... . . ...................... T.C ........

.......................

oTP 07 08.... 02 08 09 09

... G .... .. A..T ........

+309

+230

TACGAGATGCTCCAGCAGAGCTTCAACCTCTTCTACACAGAGCACTCCTCTGCTGCCTGGGACACCACCCTCCTGGAGCA .. T .............................. C ......... G ................ A ...................

----

---------

oTP 02 07 09

..........

C

+______++310

+389

+310

+389

GCTCTGCACTG GACTCAACAGCAGCTGGACCACCTGGACACCTGCAGGGGTCAAGTGATGGGAGAGGAAGACTCTGAAC .............................. GG ..................... ................................................................ ............................. GG........ CG..C ............. T... ... CG.. + ....................... TG.... ...... --------

--

------------

+

I

-

....... ................ C ............ ...

oTP 02 07 08

CC. C. +.....0909

------

+390

+469

TGGGTAACATGGACCCCATTGTGACCGTGAAGAAGTACTTCCAGGGCATCTATGACTACCTGCAAGAGAAGGGATACAGC .... A..G.........................................C ............................. .... A..G................................C .............................

A.GA.C..G.....CCC.......

.........

................................................---....-------------....

+

....

T..

oTP 02 07 08 010 09

....

+470

+549

(f)

GACTGCGCCTGGGAAATCGTCAGAGTCGAGATGATGAGAGCCCTCACTGTATC AACCACCTTGCAAAAAAGGTTAACAAA ................ C ......... G ............ TC. ................................................ TC .............................. .... .............................. TC .................................... I................................................. ................. T ...... C.G..A..C ...... G T .G...T.... ..... C ........ A.G C.G ....... TT.CTC.A..G.AGA...... GG ....C... G..G +550

oTP 02 07 08 010 09 012

+629

GATGGGTGGAGATCTGAACTCACCT

oTP 02 02

.........................

:::::::::::::::::::::::::C~~~~~~~~

.63

010 09 012

+229

AGGACAGAAAAGACTTTGGTCTTCCCCAGGAGATGGTGGAGGGCGACCAGCTCCAGAAGGACCAGGCCTTCCCTGTGCTC .... TG........ :C. ................................................ ...................... T :..G .. .

+630

0

+___-------------------_+____,____+____,____+____,____+___

+150

....

oTP 02 07

....

C+.....

+ACG

+654

~08

07

772

NEPHEW ET AL.

MAFVLSLLMALVLVSYGPGGSLGCYLSERLMDARENLKLDJRMRLSPH oTP . . R....Q.........Q.02 R .Q R......... R. 07 .......................... .Q.....................

Q..........

08 010

R

EP.

.Q.........................

........................ D..QNHV.VGSQ..R.GQ.R... LR -23

+1

+27

SCLQDRKDFGLPQEMVEGDQLQKDQAFPVLYEMLQQSFNLFYTEHSSAAW ...................... EA...C ............. .R. ..................................................

F........AF....... ____ ___+

... ____ ___+

09

oTP 02 07

08 010 EA.. S..H......... ____ ___+ ____ ___+…_ _

+

09

+77

+28

DTTLLEQLCTGLQQQLDHLDTCRGQVMGEEDSELGNMDPIVTVKKYFQGI N ............... ED ...... P....K..... K.... K..... ........................ ..... D..........ED...................... ED ...................... ................ ..R...H .... D..A.L.E.T.....A. .RTG.LAMR.

oTP 02

07 08 010 09

___,__+_--__,___+___,_------+--_---_+____,__

--78

+127

YDYLQEKGYSDCAWEIVRVEMMRALTVSTTLQKRLTKMGGDLNSP S..........T.. H .............. T .......... .

S................

.. .....................

oTP

02 07 08

...............................

....

HV..K ............. + +128

--

L.I

010

........

S.SS...S.H ... RM.D... S..

_+--_,---+128

,_

09

_172_+ +--+ +172

FIG. 2. Comparison of amino acid sequences deduced from various oTP-1 genes. Only differences in amino acid (vs. oTP-1 171)are shown. Signal sequences (aa -23 to -1) are italicized. A single underline (clone o2) indicates potential N-glycosylation site (Asn-Thr-Thr).

ponential accumulation of PCR products plateaued (five additional cycles did not yield at least twice as much product). After gel analysis of the PCR products, bands were excised and counted in a liquid scintillation counter. The absolute yield of cDNA was determined by linear regression, correcting for primer efficiency of cRNA vs. tcRNA by TCA precipitation (according to protocol provided with the AMVRT kit; Gibco). RESULTS StructuralAnalysis of oTP-1 and Other Type I IFN Genes Of the 90 000 plaques screened from the genomic library, 30 positive clones were initially detected, and 6 (designated as o2, o7, o8, o9, o10, and ol) were chosen for sequencing on the basis of PCR analysis using oTP-1 cDNA 5'/3'-specific oligonucleotide primers. All contained DNA inserts of 3.0-4.2 kb in length representing -2.5-0.9 kb (relative to the cap site, +1) of an oTP-1 cDNA [7]. An additional clone analyzed in this study, defined as o12, had

been isolated from an ovine genomic DNA library previously prepared in our laboratory [25]. Between -1000 and +900 bp were sequenced, depending upon the individual gene. On the basis of the high degree of nucleotide similarity (> 95%) to other oTP-1 sequences [7, 8, 10, 22] in the coding region, it was concluded that clones o2, o7, o8, o10, and oll were oTP-1 genes. Additional sequence characteristics suggested that these were functional oTP-1 genes, including a TCCCCATG translation initiation sequence and two sequential TGA stop codons. No premature termination codons were present in any of the oTP or other type I IFN clones isolated in this study. The open-reading frame (ORF) of 595 nucleotides coded for a 195 aa polypeptide that contained a 23 aa signal peptide followed by a 172 aa mature protein (Fig. 1). It was

FIG. 3. Comparison of the nucleotide sequence of the 5' flanking regions of oTP-1 and related type I IFN gene. The TATA box and putative sites for various transcription factors are in bold print and underlined (see text for details).

AACTCAACCTTGCACTACTG -1040

010

-961

GAGATCACAAGTCATCTAGAGATCAGTCTTGGTATCTGAAACTACGAACTGAACTA fiMAcCTGAAAAOTAGTCTGTCTATATATCTGACTATCACCTTGAAA-MTCACAACCCAAA AAAGCTAAACTGcAATATTTG CGAGAGACAT AACACTC C TCATCCC ....+.... w.... 4. , + .... , · .... + + .... ,· .... _- +..--t ....+ .. + ....· , .... + + ....· ,....4+....· ++.... w .... + -960 481

02 07 08 010

ZATTTGGCCACCTCATGTGAAGAGTAACTATT AACTCTATGCTOAGAGGATTGGGGCAGTCAAGAAGGCCTA GTGAAGAGATATGTcTTATTGGTGGGTATTGccAGcGTAAGAGCAcCTATAGATCTCACTGGGCOOGGGo CACCTCATGTGAAGAGTAACCTATTGAAGAACTCTGATGACTGAAGGA GGGCAGGAAAAA GACTCGATGAGCTGAGTTTTGAACTCTGGAGTTGGTGATGA GTGTCA GTGTCAATACTCACTTA CAGCTCAGAA AATTCAGCTATTGAGCTGCTG +4... .. .. .... . .-+-4 -- + ... .. .... ........ -,----+-- +--- , .... ...... . -- ,----- , 480 4-01

02 07 08 010 09

CAGAGATGAGATGGCTATCATCATcACTCGATGACGTGATTGGTGAACTCTGGAGTTGGTAAT0ACAGGGAGGC G AAGGGAAT ATGTCTAGTGTAT TCTTcCTGA GTGTGACTTGCAAACCTA AAAT ATAGAAGATGGcTGATGGCATcATAATcGATGGAcGTGAGTTTGGGTGAACTTCGGATTGGTAATGGACAGGGAW AAAGAGTCAGACACCACTGCAA CCCCAGTGTA _ C AGCACCTGAGGCG TCTAGAGATCACAGTAGAGGAQ TCACrAAGAAGGCTGCCATGTGACTCCGTTATAGGCTGAATC + ... .. ...-.... . 4+--- -+ .... -, .... .... . . ,.... ... .+.... ..-. -- 4+----, . .. . .-,----+4 400 -721

02 07 08 010 09

TTGGATCGTGCAATTCOTGGGGTCCAAACAGTCAACATGACTGAGCGACTGAACTAACTGATA C TGAGAGTTATGTTTTATTTGGTAGGAACTCAGAGACTGTATTTCAAGTCACCCTGAGAGAAGTGCTCCAAGGAGTGG, CTTC C CG TCAGACTGACTGAGCGACT GAACTGAACTGAGCCACGGTTAT GAACGAT GAA TGTGTCATCCTGGTT TACTGATA TGGCAGGAGATA CTTCACTCACATGACCAGGCAGGAGATACT AGTCATCCAG ATGTAGAGTCTCCTCACCCATTTC GAG AAAA ATaTGGATATTGCCAGTCTGGA +~~---- .... + ,----+ ... .. .... ...-. 4+--- -+... .... .. , .... . 4+----- + ... .... ,- --+ ... .... , --720 -641

02 07 08 010 09

CCCCTTC TGAATGTCATCC T GTTTACTATAT GGCAGGAGATACTTC GGGTAGAGGCCACGTTATATAGCGT TTG C ]AGNm2AGG TAGTTTGAACCTCAAAGGTATTTTTTGAAGTAGAG TCCCCCTTCCTGAGCACCTGGAGGGC~ CTCTTGATGAATGTGTCATCCTGGTTTACTGATATGGCrGGAGATACT TCACTCACAA TGCAACGTAATCTCAGAACCTG A T CA AACACT GAAAAGGTATCCAGAGACAA TGATGTCTG TAATAGCCGATTGTTCAAC

02 07 08 010 09

~~~

+____,____+____,____+____,____+____,____+____,____+____,____+____,____+____,____

-640

-561

ACTCACGTGACCTGGTGcCCAaOAAATOAATCTCAGGAACCCGAA.ATGTTCTCAAAAGAACAaAAACACCCAA ACCAATATCTCQAAGTTAAGAATTTAGCACTTGCTATATATGGGAAGATGCAAGAGGCTGAGCTCCTgA~AAT TCACTTCACTGACCTGGTGGCCCCAGaAAATTAATcCTAACCCGAATGAATTTCTCAAAAG AACAOAACC GAGTAGTTCATTCACCCTTAATTACAAGCTTATcTCTGTGAGACATAACAATAAAAAAATCAATATGGCCGAGTGA ACAACTACAGGACCTCATGGACTTGTAACAATrTGAC + .... +...-, --+ . .... .. ,- --+ 4..... ... ....--- +... ... .. .. ... ..... 4. ..-,----+ .. . 4 . --- 4+---560 -481

02 07 08 010 09

GTAGTTCACACCCTTAATTACAAGCTTATCCTCTGTGAGACA MM AAAAATCAACATGGCCTGAGTGA TTTTTCATGCATCTCACCTATCCGGGACACGCACTTG GTCATATCCTGAGCACTCAATATATGGCCTGAG CAATAGTTCATTCCCTTAATTACAAGCTTATCC TCTTAG ACA AAATCAACATGCCGAGT CTTGCATTCCTATTGTAAGA TAAGGAGGA-TGCAGTTAT CC AAA A TTTTCTAATCAAACTAAAGATGAACCATCTTCTGTTTCAAAGACCATAATTGCCAGAGTAACTGTAAATCACTTATA + ...--... -....--....+-........--.-....- --....-- ....------ .. ...-------------480 401

02 07 08 010 09

CTCTGCATTCCTATGTG TAAA AATAGTTAATAATTATAGACAACCAT 02 TGACTGTGCATTCCTATGTGTAAGATAAGGAGGAAATGCGTTAAGAATCAAGGAANATATTCCTGACTAAG 07 GACTCT.CATTCCTATGTGT&A A9AGO TGCAGTTAAGAATCGAGgGAATA TATTCCTGACATACGAT 08 AACAAAGfAATGCTTATATGTATTATACCTAAATTGTGCCTAATAACTATGTACACTCTATAAGTCQTTCCATAT 010 AAA ATGGCTCCTATGTAAAGAGGSAN__TGGAGCCTAAGATCAGGGCTACAGA 09 ++ ...., .... ++...., .... ++..... ,...+ +.. ..--,....4. +....-, .... 4+' ..... ,...+ +. . ,. .. · ... + +.. --, _... .. -400 -321 CO -321

~~~~~~~~~~~~ ~~~~~~~~070

AAAcAAAA~AATGTTTATATATATTATAccTAAATTTGTACATATGATATGTAcAGGTcTATAAG~2cTTTCCAThc ATAAAcAAAAAGAATGATATATATTATAccTAAATTTTGCCTAATAACTATGTACACGTCTTA&TTCTTTCCATA AACAAAA GTTATATATATTATACCTAAATTTGTACATTAATGACTATGTACAGGTCTATAAGTCTTTCCATACTT CACTTCAG CATTTAGCc AATTAT ATA ATATTGTCAAATAAATTTTTGAAAAAA TCTGTTCATGGACAAAGGAGAGCAAA CATGAGAGACAAAAG AATAATTGTAACTACACKTACAC +----....--------....------..---.. 4 .... .. ..- .-....--....--....-....--....--... -320 -241

02 07 08 010 09

TTACATAACCTCAGCCTTATTTAG TT CT AATTGATATATATTGACAAACAAA TACATATCTTGAaCCTTTATTT CT AAATTATTAAATATGACAATCCAAA

A AA 02 TATTGG 07 TATACAATTTCAATCMAA-AT FGGGMNUAT 08 CTTCTACTATAAAACTAAGATTTAGTTTCTEmATTGATATAC.m TATTGACAAACCAAA T-ATTGGMI 010 ACTCCTGAGTTAACAAGAAaT-C.TCATTGATTGATAAATA IGATGGGCACTAGGAAG! .TATTGL 09 + ... · ... +....--... ---__... ,-....-4.-------....-+-....---...-+ ....., + ·-....4-240 -161 ACATAA

-TAAATTTCCTCTGTAAAATTAAAGTTCAGATTACTAA CTAIA%~TATCTAAATGAAUAAC ... LTAATATT T TANAAAATCTAAAGAAA AAATTCCTCTGTAACA TT CATC.AAC ~ _ A~AACTTcTACTATA AAATAAATTTATGTAAW=-xTAG~A~lAJSTATcTAaAAA~ ti 1129MM 1ACACAAAATTATATAGATTGATGATCTGATCATATTTG.ATACATAAATGA

~~~

-160

02 07 08 010 09 09

~~~~~~~~~~~~~~ ~010

41

AAATGAAO5TAGAAMGGATAATGAGTACCOTCTCC CCTTGCTTAGAACAATCTTCAT 02 AACAGGAAGTGAGGAGGAATTTTCGGATGATGAGTACCOTCTTCCTTAMAGCCTTGCTAGAACCATCGTCAT 07 ATOAJ2.OTAGAlATTTCGGATATATGAGTACCG AACATCTTAT 08 AAC&GGAAOTGGGGAGAATGTTTGGATAATGAGTACCGTCTTCCCTATITACCGTTAGAACCATGCAT_ 010 ?TAAAGATTAGAgLATATTAAAAM2T9-t'a'TACCATGTTCCTACAlATY-!GGLCTAGAAAGWCTGCATS 09 ---------- ---------------- ------ ------ ----- ------ ------ ------ ---- 1 40

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B 800

zZ

M

600

z a. E 0. 0

400

200

0 10

20

30

40

50

Days of Gestation FIG. 4. Quantitative RT-PCR analyses of oTP-1 mRNAs during conceptus development. Total cellular RNA was isolated from ovine concepti of various gestational ages (Days 13-45), reverse transcribed using oTP-1 cDNA 3' primer (Fig. 1, primer f), and PCR amplified using y-32P-ATP-labeled primers specific for each oTP-1 gene. Amounts of oTP-1 mRNAs were determined by comparing radioactive counts of PCR products to radioactive counts generated from cRNA standard (see text for detail). Panel A represents an ethidium bromide-stained gel of the amplified products, and panel B presents a summary of quantitative RT-PCR determining amounts of various oTP-1 mRNAs.

determined on the basis of high homology (> 99%) in nucleotide sequence that clones 07 and oil represented the same gene. Nucleotide sequence analysis of clones o9 and o12 also revealed a 595-bp ORF having only 85% and 71% homology, respectively, with the translated region of oTP-1 (Fig. 1). The deduced aa sequence of both clones translated into a 195 aa polypeptide containing a 23 aa signal peptide completely conserved with oTP-1. Cleavage of this presumptive signal peptide also resulted in a mature protein of 172 aa, characteristic of the IFN-odI family [11]. The mature protein contained four Cys residues at positions 1, 29, 99, and 139 that are highly conserved among oTP-1 and other type I IFNs (Fig. 2) and considered essential in maintaining struc-

tural integrity and biological activity [37]. The coding region of clone o9 had 53 aa substitutions compared to oTP-1, resulting in aa sequence homology of only 71%. Clone o9 showed > 90% identity in nucleotide and aa sequences to bovine IFN-xII isolated by Capon et al. [11] and to ovine IFN-odII10 characterized by Charlier et al. [22], and 98% identity to an IFN-omega reported by Leaman and Roberts [38]. Clone o12 displayed only 60% similarity in deduced aa sequence to any oTP-1 or to other ovine or bovine IFNs isolated to date. Thus, on the basis of these characteristics it appears that clone o9 and o12 encode functional ovine type I IFNs that are different from oTP-1. The nucleotide sequence of clone o12 has been published previously [25]. Differences in the inferred aa sequences for the oTP-1 genes are illustrated in Figure 2. Clone o2 displayed a total of 26 nucleotide changes accounting for 17 aa substitutions. The most intriguing of these were the substitutions of T for C and G for C at nucleotide positions 301 and 302 (Fig. 1), respectively, resulting in a sixth Cys residue instead of a Pro at position 55 of the mature protein. Also in clone 02, the substitution of the nucleotide A for G at position 370, resulting in Asn instead of Asp at aa position 78, provides a potential site for N-glycosylation (Asn-x-Thr). As evidenced by the various aa substitutions, the polypeptides encoded by different oTP-1 genes differ in charge (-4.6 to -11.9) and isoelectric point (4.53-5.03). A modified TATA box (TATTTAA) starting 24-26 bases upstream from the transcription start site (the position +1 adenine, 69 bases up from the ATG start codon; the consensus start site for transcription of IFN-x genes [11]) was present in all clones except o2, since clone o2 contained a tandem repeat of the 24 nucleotide sequence AATCTTCATCAGAGAACCTACCTG between -29 and + 19. Thus, the TATA box began 51 bases upstream from the transcription site in clone o2. The sequence significance of these extra bases and the relevance of their location, if any, is unknown. Perhaps these nucleotides play some modulatory role in expression of this gene. The 5' flanking regions of the oTP-1 clones are aligned in Figure 3. Comparisons performed on the region 5' to position -300 revealed a wide range of disparity, from 53.4% (clone o2 vs. 07) to 95.3% (clone o2 vs. o8), suggesting that the oTP-1 genes have diverged in this region. Unique to clone olO was a 37-bp sequence (-261 to -225) that was repeated 88 bases downstream (-174 to -138). The significance of this A/T-rich (81%) sequence is unknown. The 5' flanking region of clone o9 or o12 displayed very little homology to bTP-1 or oTP-1 (< 54%). Detection of Distinct mRNAs for oTP-1 and Related Type I IFNs To identify and distinguish among related sheep type I IFN mRNAs, specific oligonucleotide primers (Fig. 1, a-f) were constructed for RT-PCR amplification. Because the oTP1 clones isolated in the present study were so highly re-

oTP-1 AND TYPE I IFN GENE EXPRESSION

775

45 (Fig. 4). The oTP-1 gene designated as o10 was highly expressed on Days 13-20. Thereafter, PCR products for o10 declined markedly but were still detectable on Days 30 and 45. In contrast to the pattern of oTP-1 and o10 transcripts, o2 mRNA was only weakly detectable at all stages studied (Fig. 4). No PCR product for oTP-1 was obtained with Day 75 placenta, placentome, adult liver, or lymphocyte RNA (data not shown). The patterns of expression for the related type I IFN genes are illustrated in Figure 5. Co-expression of IFN o9 mRNA (5-10 pg/V1g tcRNA) with oTP-1 transcripts occurred in Day 15 and Day 20 conceptus. Densitometric analysis revealed that hybridization signal for IFN o9 mRNA was relatively strong in Day 75 placenta (fetal chorionic tissue) but only weakly detectable in placentome (fetal and maternal cotyledons) and lymphocytes. A strong hybridization signal for IFN o12 mRNA was detected in lymphocytes, and o12 mRNA was detected in Day 75 placental tissues, but to a lesser extent. No signal for IFN o12 mRNA was detected in conceptus tissue (data not shown). DISCUSSION

FIG. 5. RT-PCR, Southern blot, and densitometric analyses of Day 75 ovine placenta (lanes 1 and 4), Day 75 ovine placentome (lanes 2 and 5), and ovine lymphocytes (lanes 3 and 6). Total cellular RNA isolated from these tissues was reverse transcribed and PCR amplified using primers specific for IFN 09 (lanes 1-3) or IFN o12 (lanes 4-6). The top panel (A) represents an ethidium bromide-stained gel of the amplified products; panel B is an autoradiograph of these products hybridized to probes specific for IFN o9 (lanes 1-3) or o12 (lanes 4-6); and panel C represents densitometric scan of autoradiographic signals.

lated, the oTP-1 cDNA 5' primer detected transcripts from all four oTP-1 genes; nevertheless, mRNA from two of the four could be distinguished by means of specific primers. The expected lengths of the PCR amplification products were confirmed by agarose gel electrophoresis. Cloning and sequencing of the oTP-1 and related Type I IFN PCR products further confirmed the specificity of the primers. The patterns of oTP-1 and related type I IFN RT-PCR amplification products, and thus oTP-1 and IFN mRNAs, are illustrated in Figures 4 and 5. As expected, samples that had no template or no RT added did not produce any PCR products; thus, the results reported here are not due to DNA contamination. Amounts of oTP-1 mRNA were high on Days 13-20 and then declined, but were still detectable on Days 30 and

The data from the present study demonstrate that oTP1 is encoded by multiple, distinct genes in sheep. To date, none of the nine full-length oTP-1 genes isolated (four in this study, one by Charlier et al. [22], two by Hansen et al. [17], and three by Leaman and Roberts [38]) have the same nucleotide sequence. Such differences are consistent with observations that multiple, distinct oTP-1 cDNAs have also been isolated [18], that in vitro translation of ovine conceptus mRNA yielded several isoelectric variants [19], and that several isoforms of oTP-1 were identified by 2-D PAGE [1] and N-terminal amino acid sequencing [6,20]. At least two of the genes isolated in the current study appear to encode isoforms of oTP-1 secreted by the ovine conceptus [20]. The functional significance of multiple genes and proteins for oTP-1 remains to be elucidated. As proposed by Gillespie et al. [39], multiple genes may produce slightly different products that differ in biological activity and possibly play different roles at the level of the feto-maternal interface. There are well-established differences in functional properties among other proteins also produced in large amounts and encoded by multiple genes, such as actin [40, 41] and human IFN-a [42]. Structure-function studies by Cheethman et al. [42] demonstrated that single aa substitutions can dramatically alter the biological activity of human IFN-oa4 and that highly conserved residues were of critical importance in biological function. The studies of Pontzer et al. [43] and Ott et al. [44] determined that the Cterminal of oTP-1, which has a structure common to other IFN-as, is involved primarily in antiviral activity; those studies also indicated that the unique reproductive properties of oTP-1 presumably are attributable to another part of the

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molecule. It is tempting to speculate, then, that the various aa substitutions encoded by distinct oTP-1 genes in the present study could alter the biologically active domains as well as other regions of oTP-1, resulting in proteins that differ subtly in biological function. Such may be particularly true for clone o2, which encodes a protein containing a sixth Cys at position 55 instead of a highly conserved Pro. This would give the potential for three disulfide bonds, instead of two, and potentially eliminate a 3 turn, a component frequently indicated by Pro in sequences [45]. Such structural alterations could affect the biologically active conformation of the molecule. Furthermore, it could be biologically advantageous to activate multiple oTP-1 genes, perhaps at different times during early pregnancy, in order to achieve the magnitude of protein production necessary for maternal recognition of pregnancy. It is intriguing that intramuscular injection of recombinant IFN during early pregnancy significantly increased pregnancy rates in sheep [46,47], perhaps by ensuring that a proposed threshold level of oTP-1 [23] was attained before luteal regression could be initiated fully. On the basis of the amount of mRNA corresponding to the oTP-1 genes examined, it appears that these genes are differentially transcribed during pregnancy establishment (Fig. 4). Transcripts for o10 predominated, compared to those for o2, during the period of maternal recognition of pregnancy (Days 13-20). Furthermore, co-expression of the related IFN o9, but not of o12, with oTP-1 mRNA was evident. In contrast to o9 and oTP-1 mRNAs, IFN o12 mRNA was not detected in conceptus, but was highly expressed in tissues from a later stage of pregnancy and in adult lymphocytes. Collectively, perhaps more interestingly, a somewhat tissuespecific and temporal pattern of gene expression was observed in this study. More specifically, there appears to be a transition from trophoblastic and fetal IFN gene activation (oTP-1 and IFN o9, respectively) to an adult IFN gene activation (12). This kind of gene regulation during development has been documented for a-fetoprotein (AFP), the major serum protein of the mammalian fetus. The AFP gene is expressed at high levels in fetal liver and in yolk sac [48]. Its expression is turned off after birth, and production of an adult form of serum albumin is established [48]. Coexpression of IFN-a and IFN-omega subtype mRNA in ovine [49] and bovine [9, 50] conceptus has been demonstrated. The observed pattern of transcripts further suggests that control of activation of the IFN o12 gene is most likely to differ from that of oTP-1 or IFN o9. IFN o12 might be constitutively expressed, because the lymphocytes used in the current experiments were collected from apparently healthy ewes and there is no reason to suspect that this IFN was induced by viral infection. Constitutive expression of other types of IFN-ats has been documented (see review by Chard [51]). The functional significance of IFN o9 and o12 has yet to be identified.

It is conceivable that the rapidly changing uterine milieu during early pregnancy might differentially activate oTP-1 genes, i.e., that different oTP-1 genes respond to different uterine stimulatory factors that could in turn counteract the short half-life of oTP-1 transcripts [52] and perhaps ensure that adequate amounts of this antiluteolytic signal are present at the time of pregnancy recognition. Differential expression of other types of IFNs has been reported and could be attributed to subtle differences in the promoter region. For example, 6 nucleotides out of 35 were thought to account for the differences in activity between murine IFN-a4 and IFN-o6 genes [53]; and just one-point mutation resulted in a 50% reduction in inducibility of IFN-all as compared to IFN-a4 [54,55]. Differences in the number and location of cis-acting elements in the 5' flanking region (Fig. 3) were observed among the oTP-1 genes, and perhaps subtle differences in the organization of this region contributed to differences in oTP-1 gene expression. Present within the 5' region of clones o2, 07, o8, and o10 were 14, 13, 13, and 8 GAAANN motifs (where N signifies any base), respectively. This motif has been implicated in IFN gene induction [56,57]. The overlaps of GAAANN with AAATGA and/ or AAAGGA sequences between -115 and the cap sitepotential binding sites for IFN regulatory factor-1 (IRF-1) [58, 59]-are conserved in all trophoblast IFN genes reported to date, as well as in related ovine (this study and [38]) and bovine [17] type I IFN genes. Clones o2 and o10 contained additional repeats of the IRF-1 hexamer motif (AACTGA; -778 to -763). Unique to clones o2 (-535 to -530) and 08 (-532 to -527) was a GAAATG, a motif not previously thought to be present in trophoblast IFN genes [17]. This motif binds a protein distinct from IRF-1 that may be involved in mediating IFN inducibility [57]. In both genes, the GAAATG sequence shares an AGTGAA and is 16 bp upstream from another AGTGAA, a motif implicated in murine IFN-ao gene expression [60]. Unique to clone o10 was an AP-1-like sequence located at -929 to -920. The AP-1 regulatory element is activated by the proto-oncogenes c-jun and c-fos [61]. Increases in levels of c-jun and c-fos mRNA and in JUN protein were induced by the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) [61]. Expression of GM-CSF transcripts in murine placenta has been demonstrated [62], and GM-CSF stimulated proliferation [63] and secretory activity [62] of mouse trophoblast cells in vitro. Both c-fos ([64] and K. Imakawa, RK. Christenson, unpublished data) and GM-CSF (K. Imakawa, RK. Christenson, unpublished data) mRNA have been detected in ovine conceptus; furthermore, GM-CSF induced increases in both oTP-1 and oTP-1 mRNA production by conceptus in vitro (K.P. Nephew, R.K. Christenson, K. Imakawa, unpublished results). Previous studies have shown that oTP-1 is secreted by conceptus principally from Day 13 to Day 21 [1], and oTP1 mRNA levels reflect the transient nature of oTP-1 production [65]. Data obtained by Northern [65], dot-blot [65],

oTP-1 AND TYPE I IFN GENE EXPRESSION and in situ [29, 66] analysis demonstrated that oTP-1 mRNA declined to very low levels by Day 23 of pregnancy, although later stages of gestation were not examined in those studies. Our results are consistent with those observations and further demonstrate that oTP-1 mRNA can be detected at least to Day 45 of pregnancy by means of RT-PCR, an extraordinarily sensitive method that can detect as few as 1 to 100 copies of specific RNA [67, 68]. Ott et al. [69] showed by Western blotting that oTP-1 was present in allantoic fluid from Days 25 to 40 of gestation. Our results and those of Ott et al. [69] support the possibility that oTP-1 production is not confined to the first three weeks of pregnancy; however, the biological significance of a second oTP-1 signal remains to be elucidated. Furthermore, our results suggest that the second period of oTP-1 secretion originates from the embryo proper as well as the chorion [69]. In summary, we have documented the patterns of expression of mRNA corresponding to two distinct oTP-1 and two related type I IFN genes. The temporal change in abundance of mRNA, coupled with the somewhat tissuespecific pattern of expression, suggests that type I IFN gene expression in sheep is developmentally regulated. ACKNOWLEDGMENTS The authors extend thanks to Dr. Rodney Geisert of Oklahoma State University, Department of Animal Science, for providing the sheep blood from which monocytes were isolated; to Dr. Joe Forrester, University of Missouri, for oligonucleotide synthesis; and to Dr. Bruce D. Schanbacher for initial collaboration. We are grateful to Ms. Arleene Moore for manuscript preparation and to Rick Carroll for data analyses.

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