JOURNAL OF INTERFERON RESEARCH 12: Mary Ann Liebert, Inc., Publishers

Il (1992)

Genes for the Trophoblast Interferons in Sheep, Goat, and Musk Ox and Distribution of Related Genes Among Mammals DOUGLAS W. LEAMAN and R. MICHAEL

ROBERTS'

ABSTRACT

trophoblast interferons (IFNs) are a family of Type 1 IFN found in domestic ruminants that are most closely related to the little-studied 172-amino-acid IFN-w. They are produced in massive amounts by the preimplantation conceptus at a time coincident with maternal recognition of pregnancy, and are implicated in playing an important role in this process. Here we report the characterization of four distinct members of the ovine trophoblast IFN (oTP-1) gene family, and demonstrate that they, along with previously characterized bovine trophoblast (bTP-1) genes, possess distinctive promoter sequences when compared to ovine and bovine IFN-w genes. Genomic Southern blot analysis of numerous mammalian species (zoo blots) indicate that, whereas the IFN-w are widely distributed among mammals, genes for the trophoblast IFN appear to be limited to ruminant species within the Artiodactyla order. Further polymerase chain reaction (PCR) analysis of trophoblast IFN genes in these ruminant species has permitted isolation of genes for goat and musk ox trophoblast IFN. These data suggest that the trophoblast IFNs are a distinct family of IFN with a limited The

distribution among mammals.

INTRODUCTION

unknown if the

trophoblast IFN proteins also possess other unique biological properties that would further distinguish them from previously studied IFN. The Type I IFN are classified into three major subtypes (IFN-a, IFN-ß, and IFN-w) based primarily on divergence in primary sequence and antigenic distinctiveness. They may also differ according to the cells that produce them." " It has been proposed that all Type I IFN evolved from a common primordial progenitor gene, distinct from the one that gave rise to the structurally unrelated Type II IFN (IFN--,)."2' The divergence between INF-a and IFN-ß is believed to have occurred greater than 400 million years ago."31 Consequently, these IFN are

major Ovine protein produced unusually large period embryonic trophectoderm during by

protein-1 (oTP-1) is a concepin amounts secretory the critical of the maternal recognition of pregnancy in the sheep.",2) Recent evidence has demonstrated that oTP-1 is a Type 1 interferon (IFN) most closely related to the 172-amino-acid IFN-u) (a,,).'3,4' Ovine TP-1 is now regarded as the critical substance involved in the prevention of luteal regression in the ewe, presumably by acting to alter either the production or release of the luteolytic substance PGF2a from the uterus.151 A homologous protein, bovine trophoblast protein-1 (bTP-1), is produced by the bovine conceptus at the time of maternal recognition of pregnancy in the cow.16,7' The trophoblast IFN, oTP-1 and bTP-1, share a high degree of amino acid and nucleotide sequence identity with each other, and differ substantially from known IFN-co within the 3' noncoding region of the transcriptional unit.'8' Although themselves only minimally responsive to virus, they exhibit antiviral and antiproliferative activities "" However, it is currently characteristic of other Type I IFN.19 TROPHOBLAST

tus

found in all mammals

as

well

as

many nonmammalian verte-

brates."41 The IFN-io likely diverged from IFN-a 100-300 million years ago, still

presumably before the radiation of mam-

mals."5' Curiously, however, IFN-w appear

to be absent in mammalian species such as the dog,"6' and have perhaps been lost during the course of evolution. Genes for IFN-w, like IFN-a, are present in multiple copies within the genomes of those species known to possess them. However, this multiplicity is exaggerated in the ruminants where certain species, e.g., some

Departments of Animal Sciences and 'Biochemistry, University of Missouri—Columbia. Columbia, MO 65211. This research was supported by NIH Grant HD21896. The sequences reported in this paper have been submitted to the GenBank Data Bank with accession numbers M73241, M73242, M73243, M73244, and M73245. Received 19 June 1991/Accepted 12 September 1991 1

LEAMAN AND ROBERTS

2 cattle and sheep, have been shown by genomic Southern blotting to possess upwards of 20 IFN-io-like genes. The musk ox blood was a gift of Dr. P.F. Flood (Department

of

Veterinary Anatomy, University

deer (Odocoileus virginianus) DNA was isolated from cultured fibroblasts obtained through Dr. Dan Gal lager (Texas A&M University). The Thompson's gazelle fibroblasts were originally established from skin biopsies obtained from Dr. M. Richardson, W. Trammel, and R. Evans (San Antonio Zoo). Human DNA was extracted from JAR choriocarcinoma cells also by previously published techniques.I22> Southern Blot Analysis: For genomic southern blots, DNA (5-8 |JLg) was digested to completion with restriction enzyme Eco RI (Promega, Madison, WI), loaded onto 0.85% (wt/vol) agarose gels, and separated electrophoretically (20 V overnight). The DNA was then transferred to nylon membranes (ICN, Irvine, CA) by vacuum transfer. Membranes were dried, baked, and then hybridized with the appropriate probe. Probes and Hybridization: A full-length ovine trophoblast IFN probe (cDNA oTP120, isolated from vector by Eco RI digestion) was employed to identify both TP-1 and IFN-to genes. A more specific 3' ovine trophoblast IFN probe was derived from cDNA clone oTP120 by digestion with Bgl II and Ssp I (Promega, Madison, WI) to yield a 266-bp fragment.'231 A full-length equine IFN-col probe, liberated from vector by Eco RI digestion, was also used for probing Southern blots.(24> Hybridization was carried out under the following conditions:5x SSC buffer (where 1 x SSC is0.15MNaCl, 0.015M sodium citrate); 5x Denhardt's solution'221; 0.1 M sodium phosphate, pH 6.5; 0.1% (wt/vol) SDS; 0.5 mg/ml herring sperm DNA; 50% (vol/vol) formamide; 42°C. Membranes were washed under stringent conditions, and then placed on Kodak XAR film with intensifying screens for autoradiography. PCR: Trophoblast IFN and IFN-to genes were PCR amplified from genomic DNA as described previously."7' Approximately 100 ng of genomic DNA was used in each reaction, and amplification was performed in a Perkin-Elmer Cetus (Norwalk, CT) thermocycler using Perkin-Elmer DNA polymerase from Thermus aquaticus. The oligonucleotide primers were nondegenerate and specific for bTP-1 (primers A, B, C) or bovine IFN-co (D and E). Primers A and B (Table 1) were used for amplification of oTP-1 genes oTP-p7 and oTP-p9 from sheep DNA, and primers A and C were used for cloning of sheep (oTP-p2, oTP-p4), goat (G5), and musk ox (M2) trophoblast IFN genes. The IFN-cü-specific primers D and E were used for amplification of the ovine INF-co gene. All PCR amplifications were performed with appropriate negative controls, and trophoblast IFN genes from different species were amplified in separate experiments to avoid possible sources of contamination. Controlled experiments indicated that Taq polymerase gives rise to an error rate of less than 0.2% for PCR amplified DNA inserts in our laboratory (data not shown).

of Saskatch-

Canada). Giraffe (Giraffa camelopardalis), African elephant (Loxodonta africana), hippopotamus (Hippopotamus amphibius), long-beaked dolphin (Stenella longirostris), and zebra ewan,

was generously donated by Dr. David Irwin and Dr. Allan Wilson (University of California, Berkeley). Aardvark liver samples were obtained from Dr. Oliver Ryder (San Diego Zoo). Cat (Felis domesticus) and dog (Canus familiaris) DNA was donated by Dr. Gary S. Johnson (Department of Veterinary Pathology, University of Missouri, Columbia). Thompson's gazelle (Gazella thomsoni) and white tailed

(Equus grevyi) DNA

Table 1. Primers Used for PCR Amplification of Genes Name A

B C D E

Sequence

of primer

5'-GAAATTTGTTAAGTTACAT-3' 5 ' AATACAAACATCAATATGGCC- 3 ' 5'-AGCGAATTCGACTACATTTCCTAGGTC-3• 5'-ATTCAAGAAGTTCACCTTGAC-3' -

5'-TGTGAATACATACATGAAAATC-3

TROPHOBLAST IFNs IN RUMINANT SPECIES

3

Cloning of PCR Products: PCR products of the expected sizes were excised from an agarose gel following electrophoresis, treated with T4 polynucleotide kinase (Promega, Madison, WI) and subcloned via blunt-end ligation into the Sma I site of pBS(-) plasmid (Stratagene, La Jolla, CA). All inserts were sequenced to completion on both strands by using doublestranded dideoxy chain-termination sequencing procedures.' "

RESULTS Isolation

of genes representing different oTP subtypes

Initial attempts to isolate oTP-1 gene clones from an EMBL3 sheep genomic library failed. However, we were successful in obtaining oTP genomic clones when we used the PCR in conjunction with highly specific oligonucleotide primers derived from upstream sequences of an already cloned bTP-1 gene"71 (5' primers A and B; Table 1) and from highly conserved regions within the 3' untranslated regions of oTP and bTP cDNA (3' primer C). A total of 11 genomic clones were fully sequenced. They fell into three major subtypes (B, C, D) which are illustrated by the three clones shown in Fig. 1. Numbering is as described previously, with + 1 arbitrarily assigned to the adenosine residue 67 bases upstream of the initiation codon, which Capon et a/."51 designated as the transcription start site for bovine IFN-w (but which, it should be noted, may only be a minor transcription start site for TP genes)."7,261 It seems likely (but not proven) that these genomic clones represent three distinct, nonallelic oTP gene subtypes on the basis of nucleotide or amino acid sequence differences as described in more detail below. Also included in Fig. 1 is a putative fourth subtype of oTP gene for which several cDNA'23' but no genomic clones have been isolated (represented here by cDNA clone oTP120). All other oTP-1 genes and cDNA isolated in our laboratory during the course of this and earlier studies appear to fit into one of these four A to D subtypes. The gene subtype A is represented by cDNA clone oTP120, isolated by Kiemann et a/.'23' Subtypes A and B differ by only 3% in nucleotide sequence within the transcriptional unit, but cDNA clones classified under subtype A possess a potential AMinked glycosylation site at Asn78 of the mature protein, and 11-13 conserved amino acid substitutions when compared to members of subtype B.'8' To date, we have not isolated any genomic clones representing subtype A by PCR methods, although genes classified as subtypes C and D (below) have very similar coding regions, including many of the amino acid substitutions within the deduced mature protein sequence. Clone oTP-p7 represents gene subtype B. It has an open reading frame that is identical to a cDNA clone (oTP-1 p3), which we described previously.'8' It is also very similar to cDNA clones reported earlier by a number of groups.'27,28' Another B-subtype genomic clone, oTP-pll (sequence not shown), is identical within its putative transcription unit to a cDNA clone (oTP-lp8) also reported previously.'23' The proteins encoded by these various cDNA differ from the one represented by oTP-p7 at only two amino acid positions, and most likely represent allelic forms of the same gene. Recently, Charlier et al.a6) have also cloned an oTP-1 gene of this subtype.

Their clone shares greater than 99% sequence identity with ours, including upstream promoter sequence similarity through base position -400, and is a good indication of the fidelity and usefulness of these PCR methods in the identification of additional oTP-1 gene subtypes. Genomic clone oTP-p4 represents the most novel type of oTP genes isolated in this study. Although the coding regions of this and two other similar genomic clones (not shown) share 9598% identity with other oTP-1 gene subtypes, the 3' untranslated regions display at best only 90% sequence conservation. In addition, the nucleotide and deduced amino acid sequences within the coding regions exhibit characteristics of oTP subtypes A and B, as well as some substitutions previously found only in bTP-1 cDNA.'7'7' Thus, we feel that oTP-p4 represents a distinct subtype, which we have designated C. Clone oTP-p9 represents the fourth subtype (subtype D) of oTP gene isolated in this study. This group is distinct from the other subtypes by virtue of a one-base deletion found at nucleotide 611 (codon 182 of the coding region) that results in a frame shift and premature termination at an in-frame ochre stop codon generated at position 185. We believe that this singlebase deletion is real and not a PCR artifact, since it was conserved between this clone and another (oTP-p2, not shown) that we separately amplified from the DNA of a different animal using an alternative PCR primer set (see Methods). Additionally, Charlier el a/.'26' have identified an oTP-1 gene with the same base deletion at position 611. However, the gene they reported also had an in-frame termination codon at base position 194 (codon 20 of the mature protein), and thus they concluded that it represented a pseudogene.'2'" None of the subtype D genes that we isolated during the course of this study contained this termination codon, and all possessed full open reading frames of a predicted 185 amino acids in length. To determine if the truncated proteins encoded by these genes were functional interferons, we subcloned subtype D genomic clone oTP-p9 into the E. coli expression vector pTrp2.'29' Lysates of E. coli strain Dl 12 expressing this gene construct under indoacetic acid induction exhibited antiviral activity (data not shown). Also, Western blots of bacterial cellular proteins from these lysates indicated the presence of an immunoreactive oTP-1 band with an apparent Mr of about 17,000 (S. Klemann and R.M. Roberts, unpublished results), which is the expected size of a mature oTP-1 protein truncated by 10 amino acids at the carboxyl terminus. While there are no obvious promoter differences between this and other gene subtypes, no cDNA possessing this one-base deletion have been isolated. Therefore, it is unknown if this gene is transcribed, but it clearly has the potential to encode a functional, albeit slightly shorter protein.

Isolation

of ovine IFN-u>

genes

On the basis of extensive sequence dissimilarities within their cDNA and the very different organization of the promoter regions of their genes, we have suggested that in cattle the trophoblast IFN and the IFN-ü> constitute two distinct groupings within the Type I IFN family. To obtain further evidence supporting this hypothesis, we have now cloned an ovine IFN-w gene by PCR techniques similar to those described in the previous section foroTP genes. Specific primers (Table 1, primers D

LEAMAN AND ROBERTS

oTP-p7 OTP-JJ9

-30* -U» 6A6TGACT6T«*ATT*XTAT6T6TAA«TAACGA6G6AAAAATC(*ACTTAAGAAT(*AAG*'^^ .C....A.G.A.C.C.

oTP-p7 oTP-p9

-193 TTATATGTATTATACCTAAATTTGTrcCTAATAACTATGTACAt-CTCTATAACTCTTTfXATAtt^^ .A.A.A_G.G.G.A..C.

-82

oTP-p7 .TTTATATTGAI-AAATCCJUUTTTTATTGGGAAAAGTAAACTT-nACTATAAAM^ 0TP-fj9 .T_T...CT..G.C.A.T. .C.A.C.T. oTP-p* 28

1

oTP-p7 oTP-p» oTP-p*

CA AACAGGAA6TGAGGGAGGAATTTTT6CATAAT6ACTACCCTm*-g-TATTTAAA GO*TTGCTTAGAA(XATC|-T(*ATC*AGAaUtfrTAa*TGAAGATT(XCCCT6A

.GC..AT.A.A...A.C.A.A...T.G.G.. .GC..AT.A.A...A.C.A.A...T.G.G.. 121

OTP120

oTP-p7 oTP-p9 oTP-p*

OTP120

.

Ca*«TCTaU¡CCAG*XCAGCAGCAGCTGCATCTTaXC ATG GCC TTC GTG CTC TCT CTA CTG ATG GCC CTG GTG CTG GTC AGC TAT GGC CCA

.G.C.

.G.C.,. 205 .G. .A.G.

oTP-p7 oTP-p9 oTP-p*

GGA GGA TCT CTG GGT TGT TAC CTA TCT CAG AGA CTC ATG CTG GAT GCC AGG GAG AAC CTC AAG CTC CTG GAC CGA ATG AAC AGA

OTP120

..G ..A

oTP-p7 oTP-f* oTP-p*

CTC TCC CCT CAT TCC TGT CTG CAG GAC AGA AAA GAC TTT GGT CTT CCC CAG GAG ATG GTG GAG GGC GAC CAG CTC CAG AAG GAC ..G ..A.T.. G.. .C. ..G ..A.T.. G.. .C.

.G. .G. 289

OTP120

oTP-p7 oTP-p9 oTP-pt

.

373 .T.G.C.G.A. CTC TCC TCT GCT GCC TGG GAC ACC TTC TAC ACA GAG CAC CAG GCC TTC CCT GTG CTC TAC GAG ATG CTC CAG CAG AGC TTC AAC .TG.T.C.G.A. .TG.T.C.G.A. *57

OTP120

oTP-p7 0TP-p9 oTP-p*

.CC. ACC CTC CTG GAC CAG CTC TGC ACT GGA CTC CAA CAG CAG CTG GAC CAC CTG GAC ACC TGC AGG GGT CAA GTG ATG GGA GAG AAA

.G.G G.A

...

.G.G G.CC. .G.G G.CC. 541

OTP120

0TP-p7 oTP-p9 oTP-p*

.A ..G.C. GAC TCT GAA CTG GGT AAC ATG GAC CCC ATT GTG ACC GTG AAG AAG TAC TTC CAG GGC ATC TAT GAC TAC CTG CAA GAG AAG GGA .A ..G.C.

.A ..G.C. TT.A.A. 625

OTP120

0TP-p7 OTP-p-?

oTP-p«.

.TC.

TAC AGC GAC TGC GCC TGG GAA ATC GTC AGA GTC GAG ATG ATG AGA GCC CTC ACT GTA TCA ACC ACC TTC CAA AAA AGG TTA ACA

.T.C.G.TC.*. .T.G.T.. T.G.G.G.

726 OTP120

oTP-p7 oTP-p9 oTP-pl

...

.C.C-G.

AAG ATG GGT GGA GAT CTG AAC TCA CCT TGA

TGACTCTT*»*«rrAAGATGCaU-ATCAGCCr-XTACACCCeanGTGTT(-ATTTCAGAAGACT

.C.C.C-A..T..TT. .GA.CA..A.C_G.T.A. ...

837 OTP120

.

oTP-p7

TTCTGCTCXAGCCACCAAATTCATTGAATTACTTTACTGGATACTTTGTCAG

oTP-p9 oTP-p4

OTP120

T AGTAAAAAGCAAGTAGATATAAAAGTATTCAGCTGTAGGGGCATGAGTCCTGAAA .G.G.A.G.

.C.CA.T.A...T.T.C.C.T.TG

.

0TP-p7 TGATtïCTTCCCTMTGTTATCTGTTGCTGAnTATTTATAtXTTCTTGCATTTAACATACTTAAAATATTA

oTP-**TP-p7 OTP-p9 ...GG.C.CT.C. -120 bTP-1

-100

-60

-80

-40

-20

ACTACATTTCCTAGGTCAAACAGAAAATATCTAACTGAAAACACAAACAGGAAGTGAGAGAGAAATTTTCGGATAATGAGTACCGTCTTCCCTATTTAAAAGCCTTGCTTAGAACGATCATC

0TP-p7 G.T.GT.G_A.G...G.T.C...G.. 0TP-p9 .T.GT.A.GC... T.A.A...T.. oTP-p4 G5

M2

Cons.

Fig. 5.

.GT.A.GC... T.A.A...T.. .T.GT.G...A. T.G...G.G.C.G..

.G.GT.G...A.A.A_A.A.T.G. aCTACaTTT CTAGGTAaAgtAGAAAATAt TAAaTGAAAAC

Promoter sequence

AAa

G AAGTGAG GAG AAtTTTcgGATaATGAGTACCqTcTTCCCTATTTAAA GCCTTGcTTATAAC ATC TC

alignment of the bTP-1

gene isolated

previously,"7' three oTP-1 genes isolated during this study, and

goat and musk ox trophoblast IFN genes also isolated in this study. The bases that are conserved between all promoters within the first 120 bp upstream of the putative transciption start site are indicated in capital letters of the consensus sequence (Cons.). Those bases that are conserved in all but one gene promoter are indicated as lowercase letters, and blank spaces within the consensus sequence are positions where there is no clearly preferred nucleotide. Regions that resemble elements or IRF-1 binding sites are indicated with underlines or overlines.

more degenerate oligonucleotides had been employed, all of the oTP-1 subtype genes would have been successfully identified. The more flexible approach might also have allowed us to isolate trophoblast IFN genes in closely related species, such as the giraffe and deer (where we failed). Both of these ruminant species likely possess such genes, as indicated by genomic Southern blots. The successful cloning of genes encoding putative trophoblast IFN from goat and musk ox DNA also provides evidence that such genes are common to a range of ruminant species. The goat conceptus is known to produce an immunologically related IFN at approximately the same stage of development as the conceptus in sheep and cattle,'43,44' but comparable data are lacking for other species, including musk ox. The apparent absence of genes similar in structure to the trophoblast IFN in species other than the large ruminants is of special interest in terms of the evolution of these genes and the acquisition of their highly specialized function. Genes for the IFN-a and IFN-ß are found in all the mammalian species where they have been sought, but the number for both types varies widely from species to species.'45' The IFN-to, on the other hand, are represented by multiple genes in most species examined, but are completely absent in others. In the dog, for example, IFN-to genes appear to have once existed but subsequently became lost since remnants of such genes have been detected. The ruminant species are unusual in that they not only possess extremely high numbers of IFN-to-like genes (including those for the trophoblast IFN), but also have multiple copies of IFN-ß genes. The latter are generally found in only one or two copies in most mammalian species.'45' The explanation for this seemingly dynamic state of IFN gene copy number in ruminants is unclear, but may have involved duplication of chromosomal regions containing multiple IFN loci.'46' Certain species examined in this study are closely related to ruminants evolutionarily but apparently lack trophoblast IFN genes. For example, the pig and hippopotamus (Suina suborder) and the llama (Tylopoda suborder) are within the order Artiodatcyla and diverged from the Ruminantia suborder between 55 and 60 million years ago.'47' Similarly, the horse and

previously

identified viral response

zebra are members of the order Perissodactyla, which is estimated to have diverged from the Artiodactyla rather more than 65 million years ago.'47' The absence of genes recognized by the trophoblast IFN probe in all of these species suggests that the trophoblast IFN diverged from the IFN-to within the last 55 million years. Furthermore, because the cow and sheep each contain approximately the same numbers of TP-1 genes, it is likely (but not certain) that these genes became duplicated prior to the divergence of the two species, approximately 20 million years ago. Southern blots of DNA from the giraffe (Giraffidae family), which diverged from cattle and sheep (Bovidae family) an estimated 30 million years ago, gave a single, but strongly hybridizing band when probed for trophoblast IFN genes (see Fig. 5). Conceivably more than one gene is present but they have not diverged significantly. These data together suggest that the trophoblast IFN genes evolved from a single IFN-to gene some 30-65 million years ago, and duplicated to their present numbers in domestic ruminants more recently. Direct sequence comparisons to determine more precisely when this divergence occurred and from which member of the IFN-to subfamily the trophoblast IFN evolved will have to await detailed analysis of all potential trophoblast IFN and IFN-to genes within a single species. Apparent constitutive production of IFN (usually detected as antiviral activity) has been reported in a range of nonruminant species (for a discussion see Ref. 48). In general, amounts have been small and the nature of the IFN produced obscure. Human cytotrophoblast can, however, be induced to produce IFN-ß by exposure to poly(I) poly(C),149' and the pig trophoblast constitutively secretes both Type II IFN (IFN-7) and a poorly characterized Type I IFN after about day 11 of pregnancy.'50,5" Thus, while the "true" trophoblast IFN may be limited to ruminant species within the order Artiodactyla, where they have a demonstrable role in delaying regression of the corpus luteum,'52' IFN production during pregnancy may be a fairly general phenomenon even though its significance outside the ruminant species is unclear. •

10

LEAMAN AND ROBERTS

ACKNOWLEDGMENTS 11.

We are grateful to Dr. Jay Cross, Dr. Tod Hansen, and Dr. Steve Klemann for invaluable discussion and input throughout this study. We also thank Jim Bixby for technical services and computer analysis of various oTP-1 genomic clones, and Jude Alexander for work with the expression of oTP-p9 in E. coli. For providing genomic DNA samples, we thank Dr. P.F. Flood, Dr. G.S. Johnson, Dr. Oliver Ryder, Dr. Dan Gallager (and Drs. Richardson, Trammel, and Evans), and especially Dr. David Irwin and Dr. Allan Wilson for initial sources of DNA for zoo blots. The equine IFN-co probe was provided by courtesy of Drs. R. Hauptmann and G.R. Adolf, Boehringer Ingelheim. A special thanks to Gail Foristal for preparing the manuscript. This paper is #11,511 from the Missouri Agricultural Experiment Station.

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Dr. R.M. Roberts 158 Animal Science Research Center University of Missouri Columbia, MO 65211 Received 19 June

1991/Accepted

12

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1991