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R M Roberts

Publications and source records attributed to R M Roberts.

At least 37 records · Page 2Linked to original sources
PubMed2001

The place of farm animal species in the new genomics world of reproductive biology.

Reproductive studies on farm animals have been part of the underpinnings that have led to the ready availability of low cost, safe, and nutritious food in the developed world. They have also made a significant contribution to reproductive medicine. Yet at a time when world demand for food is increasing and the National Institutes of Health budget is set to double between 1998 and 2003, funding for animal agriculture remains low, erratic, and politically vulnerable. There are also those who question whether the food animals have value any longer as comparative models for studying reproduction as it related to human health and well being. In this paper I describe how such research is presently funded at the federal level and discuss why support for agricultural science is in decline, despite many unmet needs. I then suggest that the human genome project and the developing areas of comparative gene mapping and functional genomics are beginning to provide new impetus to studies on farm animal species. Finally I argue that although rodents and, above all, the mouse, with all its genetic advantages, occupy lofty positions as models for studying reproductive processes and their abnormalities in the human, there will continue to be a need to take a broader comparative approach that will inevitably involve farm animals.

Agriculture↗
PubMed2000

Adaptive diversification within a large family of recently duplicated, placentally expressed genes.

The pregnancy-associated glycoproteins (PAG) are putative peptide-binding proteins and products of a large family of genes whose expression is localized to the placental surface epithelium of artiodactyl species. We have tested the hypothesis that natural selection has favored diversification of these genes by examining patterns of nucleotide substitution in a sample of 28 closely related bovine, caprine, and ovine family members that are expressed only in trophoblast binucleate cells. Three observations were made. First, in codons encoding highly variable domains of the proteins, there was a greater accumulation of both synonymous and nonsynonymous mutations than in the more conserved regions of the genes. Second, in the variable regions, the mean number of nonsynonymous nucleotide substitutions per site was significantly greater than the mean number of synonymous substitutions per site. Third, nonsynonymous changes affecting amino acid charge occurred more frequently than expected under random substitution. This unusual pattern of nucleotide substitution implies that natural selection has acted to diversify these PAG molecules at the amino acid level, which in turn suggests that these molecules have undergone functional diversification. We estimate that the binucleate cell-expressed PAG originated 52 +/- 6 million years ago, soon after the divergence of the ruminant lineage. Thus, rapid functional diversification of PAG expressed in trophoblast binucleate cells seems to have been associated with the origin of this unique placental adaptation.

Amino Acid Sequence↗
PubMed2000

Caprine pregnancy-associated glycoproteins (PAG): their cloning, expression, and evolutionary relationship to other PAG.

Pregnancy-associated glycoproteins (PAG) are structurally related to aspartic proteinases and belong to an extensive, rapidly evolving family of recently duplicated genes expressed in the placentas of artiodactyl species. The aim of the present study was to clone PAG from the goat, study their temporal and cell-specific expression, and determine their phylogenetic relationship to PAG from other species. RT-PCR was used to generate PAG cDNA from pooled placental RNA obtained between days 45 and 115 of pregnancy. A total of 11 cDNA, which differed by > 5% from each other, were selected for complete bidirectional sequencing from 60 clones analyzed. A group of nine (caPAG1, caPAG3-7(var), caPAG9-11), which displayed > 80% sequence identity with each other, were expressed after day 45 of pregnancy and were localized to trophoblast binucleate cells. These PAG demonstrated an unusually high ratio of nonsynonymous (amino acid changing) to synonymous nucleotide differences. CaPAG2, by contrast, was detectable only in early pregnancy (days 18 and 19) and expressed throughout trophectoderm. It was of more ancient origin than the PAG1 group, but more recent than caPAG8. The latter was expressed at all stages examined (days 18 to 115). The data confirm that many PAG genes, with different patterns of temporal and spatial expression, are transcribed in the placenta of the goat. The data also suggest that the recently duplicated PAG genes are being selected for rapid diversification of function.

Amino Acid Sequence↗
PubMed2000

Independent origin of IFN-alpha and IFN-beta in birds and mammals.

Phylogenetic analysis of type I interferon (IFN) from birds and mammals strongly supported the hypothesis that the gene duplication giving rise to the alpha and beta families of mammalian IFN occurred after the divergence of birds from mammals, whereas the bird IFN that have been designated alpha and beta duplicated independently in the avian lineage. Therefore, IFN designated alpha and beta in birds are not orthologous to those similarly designated in mammals.

Animals↗
PubMed2000

A classification for the interferon-tau.

An attempt has been made to provide a rational organization for the many interferon-tau (IFN-tau) sequences entered in GenBank based on phylogenetic analysis and common amino acid substitutions, which might form the basis for a universal nomenclature scheme. Over the 13 years since these genes were first discovered, large numbers of cDNA and gene sequences have been reported, and there is reason to suspect that representatives of all the major ovine and bovine forms have now been described. The data are consistent with the presence of many genes and also allelic variants in sheep and cattle analogous to what has been observed for the IFN-alpha in the human. Future variants should be easily accommodated into the scheme outlined here. A flexible system of nomenclature, based on that used for HuIFN, is needed to provide a common base for comparison between research done in different laboratories and to assign relative biologic potencies to these molecules.

Animals↗
PubMed2000

Pregnancy-associated bovine and ovine glycoproteins exhibit spatially and temporally distinct expression patterns during pregnancy.

The pregnancy-associated glycoproteins (PAG) constitute a large family of recently duplicated genes. They show structural resemblance to pepsin and related aspartic proteinases. A total of 21 bovine (bo) PAG and 9 ovine (ov) PAG cDNA have been identified. Phylogenetic analysis indicated that the PAG are divided into two main groupings that accurately reflect their tissue expression, as determined by in situ hybridization. In the first pattern, represented by ovPAG-2 and boPAG-2, -8, -10, and -11 (where the numbering is arbitrary and reflects order of discovery within species), expression occurred throughout the outer epithelial layer of the placenta (trophectoderm). The second pattern was predominant localization to binucleate cells. Ribonuclease protection assays, which allow discrimination between closely related transcripts, have shown that the expression of PAG varies in a temporal manner over pregnancy. Of those bovine PAG expressed predominantly in binucleate cells, boPAG-1, -6, and -7 are expressed weakly, if at all, by Day 25 placenta, but are present at the middle and end of pregnancy. Others, such as boPAG-4, -5, and -9, are expressed at Day 25 and at earlier stages. Although not among the earliest PAG produced by the trophoblast, boPAG-1 has been used for pregnancy diagnosis, particularly in dairy cows, where there is a major need for a sensitive method capable of detecting pregnancy within 1 mo of conception. It seems likely that some of the newly discovered PAG will be better candidates than PAG-1 for pregnancy diagnosis.

Animals↗
PubMed1999

Trophoblast interferons.

The mechanisms responsible for prevention of corpus luteum regression during early pregnancy are diverse and appear to have arisen in concert with the evolutionary divergence of placental structure. That used by the sub-order Ruminantia is unique and involves the production of a Type I interferon (IFN), IFN-tau (tau). Although IFN-tau resembles other Type I IFNs (such as IFN-alpha, -beta, and -omega) in structure as well as in many of its biological properties, it is not virally inducible and is instead produced constitutively by embryonic trophectoderm during the period immediately prior to implantation. The transcription factor Ets-2 is a component of the regulatory mechanism involved in transcription of IFN-tau. These genes probably arose as the result of a duplication of an IFN-omega gene, 36 million years ago, at about the time the Ruminantia sub-order emerged. They have duplicated extensively since then and there may be 10 or more genes in some present-day species. The expression of different IFN-tau is unequal and they differ in biological potency. The rapid evolution of IFN-tau genes possibly reflects the placenta as a site of considerable genetic experimentation.

Animals↗
PubMed1999

The cross-species antiviral activities of different IFN-tau subtypes on bovine, murine, and human cells: contradictory evidence for therapeutic potential.

It is claimed that interferon-tau (IFN-tau) has broad cross-species reactivity and less cytotoxicity than other type I IFN when used at high concentration either in vitro or in living animals. It can also amelioriate the development of experimental allergic encephalomyelitis (EAE) without the usual side effects of IFN therapy in mice autoimmunized with myelin basic protein. For these reasons, IFN-tau may have therapeutic potential in humans. Here, the antiviral (AV) activities of eight different recombinant IFN-tau were compared with those of several bovine, human, and murine type I IFN on bovine MDBK cells, murine L929 cells, and human WISH cells. The data show that only one of the IFN-tau, OvIFN-tau4, has broad cross-species reactivity. It was comparable in this respect to HuIFN-omega1 and HuIFN-alpha1. The other IFN-tau, including the variant form (OvIFN-tau1mod) tested by others in cytotoxicity experiments and for its ability to protect mice against EAE, had relatively weak AV activity on mouse and human cells. It is possibly because this particular bioengineered form of IFN-tau binds the common type I receptor of these two species with such low affinity that it lacks cytotoxic effects. The basis for its potent anti-EAE activity is unclear, but it seems possible that it does not involve the type I IFN receptor.

Amino Acid Sequence↗
PubMed1999

Identification of a new aspartic proteinase expressed by the outer chorionic cell layer of the equine placenta.

The pregnancy-associated glycoproteins (PAGs) are placental antigens that were initially characterized as pregnancy markers in the maternal circulation of domestic ruminant species. They are members of the aspartic proteinase gene family, having greatest sequence identity with pepsinogens. However, some are not capable of functioning as enzymes. The PAGs are associated with a large gene family within the Artiodactyla order (cattle, camels, pigs). So far, no members of this family have been characterized in species outside this order. This report describes the cloning and initial characterization of a PAG-like protein (equine PAG or ePAG) expressed in the placenta of the horse and zebra (order Perrisodactyla). Equine PAG is a proteinase capable of degrading 14C-hemoglobin and catalyzing the removal of its own pro-peptide. The ePAG mRNA is restricted to the chorion both prior to implantation and in the term placenta. Equine PAG is secreted from cultured placental tissue as both a processed (mature) and unprocessed (zymogen) form. Equine PAG shares similar identity with the PAGs and pepsinogens and probably arose from a pepsinogen-like precursor that gained the ability to be expressed in the placenta. The promoter of the ePAG gene shares sequence identity with the promoter from a bovine PAG gene but not with promoters of other aspartic proteinases. Therefore, we hypothesize that ePAG is a remnant of the pepsinogen-like progenitor gene that was expanded within the Artiodactyla to create the large and highly diverse PAG family.

Amino Acid Sequence↗
PubMed1999

Identification of the expressed forms of ovine interferon-tau in the periimplantation conceptus: sequence relationships and comparative biological activities.

Interferon-tau (IFN-tau) is secreted from trophectoderm of periimplantation ruminant conceptuses and is a critical component of pregnancy recognition. Multiple genes encode IFN-tau. The objectives of this study were to identify expressed forms of ovine IFN-tau and to compare their biological activities. Sequences analyzed after cloning 36 reverse transcription-polymerase chain reaction products of ovine conceptus RNA provided seven new cDNA that were similar in sequence to previously cloned forms (p3, p6, and p8 cDNA). Phylogenetic analysis of amino acid sequence for all new and previously reported forms showed that ovine IFN-tau forms can be divided into three main groups. Equivalent amounts of mRNA for p3, p6, and p8 forms were detected in conceptuses following RNase protection. Recombinant p3 and p8 protein had similar antiviral activity on ovine and bovine cells whereas p6 protein was less active. The p3 form was the most potent of the three in its ability to extend estrous cycle length in nonpregnant ewes. In summary, there appeared to be three main groups of ovine IFN-tau, each containing several variant forms. Antiviral activity was not particularly well correlated with ability to prevent luteolysis, suggesting that distinct intracellular mechanisms are used to exert the various actions of IFN-tau.

Amino Acid Sequence↗
PubMed1998

Patterns of prehistoric human mobility in polynesia indicated by mtDNA from the Pacific rat.

Human settlement of Polynesia was a major event in world prehistory. Despite the vastness of the distances covered, research suggests that prehistoric Polynesian populations maintained spheres of continuing interaction for at least some period of time in some regions. A low level of genetic variation in ancestral Polynesian populations, genetic admixture (both prehistoric and post-European contact), and severe population crashes resulting from introduction of European diseases make it difficult to trace prehistoric human mobility in the region by using only human genetic and morphological markers. We focus instead on an animal that accompanied the ancestral Polynesians on their voyages. DNA phylogenies derived from mitochondrial control-region sequences of Pacific rats (Rattus exulans) from east Polynesia are presented. A range of specific hypotheses regarding the degree of interaction within Polynesia are tested. These include the issues of multiple contacts between central east Polynesia and the geographically distinct archipelagos of New Zealand and Hawaii. Results are inconsistent with models of Pacific settlement involving substantial isolation after colonization and confirm the value of genetic studies on commensal species for elucidating the history of human settlement.

Animals↗
PubMed1998

Control of interferon-tau gene expression by Ets-2.

Expression of the multiple interferon-tau (IFN-tau) genes is restricted to embryonic trophectoderm of ruminant ungulate species for a few days in early pregnancy. The promoter regions of these genes are highly conserved. A proximal (bp -91 to -69) sequence has been implicated in controlling trophoblast-specific expression. Here it was used as a target for yeast one-hybrid screening of a day 13 conceptus cDNA library. Two transcription factors of the Ets family, Ets-2 and GABPalpha, were identified, consistent with the observation that active ovine IFN-tau genes contain a single 10-bp Ets motif (core: GGAA) in the proximal segment, whereas three known inactive ovine genes contain a mutated core motif (TGAA). Cotransfection of a promoter- (-126 to +50) luciferase reporter construct from an active gene (bovineIFN-tau1; boIFNT1) and an Ets-2 expression plasmid in human JAr cells provided up to a 30-fold increase in reporter expression, whereas promoters from inactive genes were not transactivated. GABPalpha alone was ineffective and had only a approximately 2-fold positive effect when coexpressed with its partner GABPbeta. Other Ets-related transcription factors, which were not detected in the genetic screen, also provided a range of lesser transactivation effects. Coexpression of Ets-2 and activated Ras failed to transactivate the IFNT promoter greater than Ets-2 alone in JAr cells. The presence of Ets-2 in nuclei of embryonic trophectoderm was confirmed immunocytochemically. Together, these data suggest that Ets-2 plays a role in the transient expression of the nonvirally inducible IFNT genes.

Amino Acid Sequence↗
PubMed1998

Relationship between age of blastocyst formation and interferon-tau secretion by in vitro-derived bovine embryos.

This study was designed to examine the relationship between the speed at which bovine embryos reach the blastocyst stage, their cell number, and interferon-tau production. A total of 800 oocytes were fertilized by frozen-thawed semen. On day 2, 44 hr after exposure to sperm, 78, 320, and 296 embryos were at the two-, four-, and eight-cell stages, respectively, with an overall cleavage rate of 86.8%. Within these three groups 15 (19.2%), 106 (33.1%), and 158 (53.4%) embryos proceeded to the blastocyst stage. Of these 46.7%, 65.1%, and 63.3% hatched in the three groups, respectively. Blastocysts began to appear at day 7, but a few did not form until as late as day 13. Expanded blastocysts (n = 279) were cultured individually for 48 hr in 50-microliter droplets of medium, fixed for cell counts, and the concentration of interferon-tau in the medium was determined. Blastocysts originating from two-cell embryos had significantly fewer cells (46.5 +/- 23.3) than either four-cell- (97.2 +/- 13.5) or eight-cell-derived embryos (113.8 +/- 13.6; P < 0.05). Hatching was accompanied by an increase in cell number (129.8 +/- 15.5 versus 41.9 +/- 14.4; P < 0.01). Blastocysts derived from embryos that had reached the eight- or four-cell stage 44 hr after insemination produced significantly more interferon than embryos derived from two-cell embryos (941.7 +/- 92.1, 930.1 +/- 163.1, versus 232.8 +/- 70.1 pM). In contrast, hatching, ovary batch, the speed of early cleavage, cell number, and quality grade had no effect on interferon-tau secretion. The embryo's age at blastocyst formation was not related to the number of its cells but did have a significant effect (P < 0.001) on interferon-tau production, with mean concentrations in the medium of 294.8 +/- 57.9, 563.3 +/- 82.0, 1126.3 +/- 133.6, 1778.5 +/- 297.2, 512.9 +/- 82.0, 315.0 +/- 157.5, and 157.5 pM among blastocysts appearing from days 7 to 13, respectively. These data suggest that blastocysts that form at days 7 and 8 produce less interferon-tau than those that form on days 9 or 10. Since early-forming blastocysts are generally considered more developmentally competent than those which form late, there may be a negative relationship between early interferon-tau production and competence.

Animals↗
PubMed1998

An aspartic proteinase expressed in the equine placenta.

This manuscript describes the cloning of a novel aspartic proteinase expressed in the placenta of the horse (order Perrisodactyla). Evidence for similar genes in the cat (Carnivora) and ruminants (Artiodactyla), indicates that these molecules have been conserved within widely divergent species with distinct types of placentation. Since ePAG is produced by the outer cell layer (trophoblast) of the placenta, it can tentatively be grouped with the pregnancy-associated glycoproteins (PAG) of cattle, sheep, and pig. The high sequence identity that ePAG shares with pepsinogens as well as the PAG, indicates that ePAG may be the evolutionary bridge that links these two groups of aspartic proteinases.

Animals↗
PubMed1998

The evolution of the type I interferons.

There are five recognized subtypes within the type I interferons (IFN), IFN-alpha, IFN-beta, IFN-delta, IFN-omega, and IFN-tau, although others may remain to be described, and the IFN-omega may have to be subdivided further because of their evident structural complexity. Together, they constitute an ancient family of intronless genes, possibly present in all vertebrates. THe IFNA/IFNB genes originated by duplication of a progenitor after the divergence of birds, most probably about 250 million years ago (MYA). The avian gene itself proceeded to duplicate to form a series of independent subtypes. The IFND, to date described only in the pig, arose from the IFNA lineage before the emergence of mammals about 180 MYA and might, therefore, be generally distributed in present day species. The IFNB, which occurs as a single gene in primates and rodents, have been duplicated in some other orders. Recent events have produced 10 or more genes in bovid species. The IFNA, which are clustered with the IFNW in humans and cattle, exist as multiple genes in all mammals so far examined as a result of a series of duplication events, some of which occurred recently and, therefore, independently in separate mammalian lineages. The IFNW diverged from the IFNA approximately 130 MYA, just prior to the emergence of mammals, and have continued to duplicate since then. The IFNT, which play a role in reproduction of ruminants, arose from an IFNW within the Artiodactyla suborder about 36 MYA and are found only in the suborder Ruminantia. These genes have also continued to duplicate to form an extensive family. Consequently, their involvement in early pregnancy is a feature of ruminants and not of other mammalian species.

Animals↗
PubMed1998

Different ovine interferon-tau genes are not expressed identically and their protein products display different activities.

Interferon tau (IFN-tau) proteins are secreted by the ovine conceptus for a few days before definitive attachment of the trophoblast to the uterine epithelium and act to prolong luteal life span. Multiple genes encode for IFN-tau in sheep, but it remains unclear which genes are expressed during early pregnancy and whether the proteins encoded by these genes are equipotent. Three distinct ovine (ov) IFN-tau gene variants, p3, p6, and s4, were examined to determine whether they differed in gene expression and whether the proteins displayed different biological activities. By using RNase protection assays, full-length protected fragments were detected for p3 and p6 in approximately similar proportions in conceptuses flushed from the uterus at Days 12-13, Days 15-16, and Days 18-19 of pregnancy, but the amount of full-length protected s4 transcripts was 10% to 20% of that for p3 and p6. Partially protected probe fragments were also evident, presumably from probe hybridization to related ovIFN-tau transcripts. Recombinant proteins were generated and exhibited 34.2 (p3), 8.4 (p6), and 11.9 (s4) units (x 10(-7)) of activity per milligram of protein when tested on Madin-Darby bovine kidney cells. Antiproliferative activity on human Daudi cells varied considerably between the interferons, with p3 being 2000-fold more potent than s4. The interferons were injected into the uterine lumen of ewes from 10 to 18 days postestrus. The functional life span of the corpus luteum (CL) was increased (p = 0.02) by either 300 microg/day p3 (31.7 +/- 7.8 days) or 300 microg/day p6 (25.5 +/- 4.2 days), but not by 300 microg/day s4 (19.2 +/- 2.9 days), when compared to controls (15.8 +/- 0.6 days). Injection of 1 mg/day s4 did, however, increase (p = 0.02) CL life span (23.5 +/- 4.1 days). These data suggest that IFN-tau genes are not equally expressed in trophectoderm and that ovIFN-tau genes encode for proteins with significantly different biological potency.

Animals↗