Development of a 19-plex short tandem repeat typing system for individual identification and parentage testing of horses (Equus caballus).

Individual identification of horses for pedigree verification and registration is important for the sustainable development of the horse industry. Horse individual identification and parentage tests commonly use the 17 short tandem repeats (STRs) recommended by the International Society for Animal Genetics (ISAG) and the locus LEX33. While many multiplex STR typing systems have been established for the horse, a sex determining marker is usually absent, and none of them can simultaneously detect all 17 ISAG recommended loci and the locus LEX33. Here, we present a 19-plex STR typing system that contains the 17 ISAG recommend loci, the locus LEX33 and amelogenin as sex determining loci. The results of our sensitivity, species specificity, stutter analysis and population data analysis, indicate that this system is a specific, sensitive, and robust tool for the identification of individuals, parentage testing and genetic research in the horse.

A novel 13-plex STR typing system for individual identification and parentage testing of donkeys (Equus asinus).

BACKGROUND: Previous studies investigating donkey parentage and genetic diversity used horse-specific multiplex systems. However, several mis-allele and null-allele issues were found with some of the horse primers when used in donkeys. In 2017, the International Society for Animal Genetics (ISAG) recommended 13 dinucleotide short tandem repeats (STRs) (AHT4, ASB23, HMS2, HMS3, HMS6, HMS7, HMS18, HTG7, HTG10, TKY297, TKY312, TKY337 and TKY343) as a core panel that should be used to identify individuals and to test for parentage in donkeys. To date, no single multiplex STR typing system containing all 13 donkey STRs recommended by the ISAG has been reported. OBJECTIVES: To establish a novel and donkey-specific multiplex STR typing system containing all 13 recommended STRs. STUDY DESIGN: Assay development and validation in field population. METHODS: Primers for seven of the STRs were redesigned and conditions for polymerase chain reaction (PCR) were optimised. We analysed the allele sequences, sensitivity, species-specificity and stutter ratios of this new system. RESULTS: A 13-plex STR typing system for donkey was established. A full profile could be generated from a single PCR reaction using as little as 5 ng of DNA template with the 13 pairs of primers labelled with fluorescent dyes. An allele ladder, containing 101 alleles from the 13 STRs, was generated. No full genotype profile was generated with these primers if DNA from humans, or 11 other commonly encountered animals, was used. Genotypes could be generated for the horse and horse-donkey hybrids (mule and hinny). Stutter ratios and population genetic parameters were calculated based on samples from 150 donkeys. The combined probabilities of paternity exclusion for this system were 0.988907326 (CPEduo) and 0.999665018 (CPEtrio). MAIN LIMITATIONS: This system cannot detect sex. CONCLUSIONS: Our results indicate that our donkey-specific 13-plex STR typing system is sensitive, species-specific and robust for individual identification, paternity testing and population genetic analysis in donkeys, and has potential forensic applications.

NOVEL GENETIC FINDINGS IN A CHINESE FAMILY WITH EARLY-ONSET FEMALE-RELATED TYPE 2 DIABETES.

No inheritance of early-onset female-related type 2 diabetes was reported within Chinese families. In this study, we aim to describe the inheritance pattern of type 2 diabetes in a 3-generation family and identify the gene responsible for type 2 diabetes. Genome-wide multipoint parametric linkage analysis revealed a maximum multipoint logarithm of odds (lod) score of 2.1 for a locus being associated with type 2 diabetes in this family on chromosome 20p11.2-12 between 23.5~30.8cM. Type 2 diabetes may be transmitted as an autosomal dominant trait with a high female-related penetrance in this family. Here we describe the first genetic locus for type 2 diabetes at chromosome 20p11.2-12. This region contains 8 known or predicted genes (PLCB1, PLCB4, LAMP5, PAK7, ANKEF1, SNAP25, SLX4IP, and JAG1). Gene SNAP25 which linked to energy or glucose homeostasis associated phenotypes may play a role in the development of type 2 diabetes in this family.

Incretin hormones and the expanding families of glucagon-like sequences and their receptors.

Peptide hormones encoded by the proglucagon (Gcg) and glucose-dependent insulinotropic polypeptide (Gip) genes are evolutionarily related glucagon-like sequences and act through a subfamily of G-protein-coupled receptors. A better understanding of the evolutionary history of these hormones and receptors should yield insight into their biological functions. The availability of a large number of near-complete vertebrate genome sequences is a powerful resource to address questions concerning the evolution of sequences; here, we utilize these resources to examine the evolution of glucagon-like sequences and their receptors. These studies led to the discovery of novel genes for a glucagon receptor-like receptor (Grlr) and a glucagon-like sequence (exendin) in vertebrates. Both exendin and GRLR have ancient origins, early in vertebrate evolution, but have been lost on the ancestral lineage leading to extant mammals. We also show that exendin and GRLR are both expressed in the brain of the chicken and Xenopus tropicals, results that suggest that the products of these genes function in this tissue. The lack of exendin or Grlr genes in mammals suggests that other genes may have acquired the functions of exendin and Grlr during mammalian evolution.

The fish endocrine pancreas: review, new data, and future research directions in ontogeny and phylogeny.

The literature on the ontogeny and phylogeny of the endocrine pancreas of ray-finned fishes is summarized since the latest review in fish [Youson, J.H., Al-Mahrouki, A.A., 1999. Review. Ontogenetic and phylogenetic development of the endocrine pancreas (islet organ) in fishes. Gen. Comp. Endocrinol. 116, 303-335]. A basic description and a demonstration of the diversity of the fish islet organ is provided through new immunohistochemical data on islet tissue from a basal teleost, an osteoglossomorph, and a more derived teleost, a perciforme. Unlike the previous review, the present report provides a review and discussion of the utility of sequence data of insulin, somatostatin, and NPY- and glucagon-family peptides in phylogenetic analyses of jawed and jawless fishes. The present study also provides the first comparative analysis of sequences of preprohormones of endocrine peptides from closely related basal teleost species. Some nucleotide and deduced amino acid sequence data for preprosomatostatins (PPSS-I and/or -II) are compared for four species of bonytongues, Osteoglossomorpha, and with PPSSs of the white sucker, Catostomus commersoni, representing Cypriniformes, a more generalized teleost order. Phylogenetic analysis of deduced amino acid sequences of the PPSSs of these species and others from databases indicates good support for the monophyly of Osteoglossomorpha and some support for the present taxonomic grouping of the osteoglossomorphs examined, and also the white sucker. However, PPSS may have limited phylogenetic utility due to the relative short sequence, particularly in resolving relationships among lineages that diverged over a short period of time. Since in the few fish species examined we have just touched the surface in describing the diversity of structure of the islet organ, and likely the nature of the products of its cells, this report promotes the continued study of this organ.

Evolution of new hormone function: loss and gain of a receptor.

The vertebrate proglucagon gene encodes three glucagon-like sequences (glucagon, glucagon-like peptide-1 [GLP-1], and glucagon-like peptide 2 [GLP-2]) that have distinct functions in regulating metabolism in mammals. In contrast, glucagon and GLP-1 have similar physiological actions in fish, that of mammalian glucagon. We have identified sequences similar to receptors for proglucagon-derived peptides from the genomes of two fish (pufferfish and zebrafish), a frog (Xenopus tropicalis), and a bird (chicken). Phylogenetic analysis of the receptor sequences suggested an explanation for the divergent function of GLP-1 in fish and mammals. The phylogeny of our predicted and characterized receptors for proglucagon-derived peptides demonstrate that receptors for glucagon, GLP-1, and GLP-2 have an origin before the divergence of fish and mammals; however, fish have lost the gene encoding the GLP-1 class of receptors, and likely the incretin action of GLP-1. Receptors that bind GLP-1, but yield glucagon-like action, have been characterized in goldfish and zebrafish, and these sequences are most closely related to glucagon receptors. Both pufferfish and zebrafish have a second glucagon receptor-like gene that is most closely related to the characterized goldfish glucagon receptor. The phylogeny of glucagon receptor-like genes in fish indicates that a duplication of the glucagon receptor gene occurred on the ancestral fish lineage, and could explain the shared action of glucagon and GLP-1. We suggest that the binding specificity of one of the duplicated glucagon receptors has diverged, yielding receptors for GLP-1 and glucagon, but that ancestral downstream signaling has been maintained, resulting in both receptors retaining glucagon-stimulated downstream effects.

Amphibian glucagon family peptides: potent metabolic regulators in fish hepatocytes.

Peptides analogous to glucagon-like peptide-1 (GLP-1) have been isolated from amphibian pancreas and intestine, and their amino acid sequences and cDNA structures elucidated. Just like their mammalian counterpart, these peptides are potent insulinotropins in mammalian pancreatic cells. We show here that these peptides also exert strong glycogenolytic actions when applied to dispersed fish hepatocytes. We compared the potencies of three synthetic GLP-1s from Xenopus laevis and two native GLP-1s from Bufo marinus in the activation of glycogenolysis in the hepatocytes of a marine rockfish (Sebastes caurinus) and two freshwater catfish (Ameiurus nebulosus and A. melas), and demonstrated their effectiveness in increasing the degree of phosphorylation of glycogen phosphorylase. We also compared the glycogenolytic potency of the peptides with those of human GLP-1 and glucagons from human and B. marinus. Sensitivity to these peptides is species-specific, with the rockfish responding at lower concentrations to GLP-1s and the two catfish reacting better to glucagons. However, the relative potency of the amphibian GLP-1s and glucagons is similar in the three species. Xenopus GLP-1C (xGLP-1C) is consistently more potent than xGLP-1B, while xGLP-1A displays the smallest activation of glycogenolysis. Similarly, Bufo GLP-1(32)-the peptide with the highest amino acid sequence identity to xGLP-1C-always shows a higher potency than Bufo GLP-1(37), which is closely related to xGLP-1B. The relative hierarchy of these glycogenolytic GLP-1s differs from their ranking as insulinotropins in mammalian beta-cells. In the rockfish system, Bufo glucagon-36, a C-terminally extended glucagon, is more potent than the shorter bovine glucagon and Bufo glucagon-29 in the activation of glycogenolysis; when tested in A. nebulosus hepatocytes, bovine and amphibian glucagons are equipotent. Amphibian GLP-1s and glucagons activate glycogenolysis in fish hepatocytes through increased phosphorylation of glycogen phosphorylase, implying involvement of the adenylyl cyclase/protein kinase A system in signal transduction. We conclude that the broad physiological effectiveness of GLP-1 has been retained throughout vertebrate evolution, and that both insulinotropic activity and glycogenolytic actions belong to the repertoire of GLP-1.

Molecular cloning of preproinsulin cDNAs from several osteoglossomorphs and a cyprinid.

Several preproinsulin cDNAs were isolated and characterized from four members of the Osteoglossomorpha (an ancient teleost group); Osteoglossum bicirrhosum (arawana), Pantodon buchholzi (butterfly fish), Notopterus chitala (feather fin knife fish), Hiodon alosoides (goldeye) and Gnathonemus petersii (elephantnose). In addition, we isolated and characterized the preproinsulin cDNA from Catostomus commersoni (white sucker, as a representative of a generalized teleost). The comparative analysis of the sequences revealed conservation of the cystine residues known to be involved in the formation of the disulfide bridges, as well as residues involved in the hexamer formation, except for B-17 in the butterfly fish, the arawana and the goldeye. However, the N-terminus of the B-chain was very weakly conserved among the species studied. Residues known to be significant for maintaining receptor-binding conformation and those known to comprise the receptor-binding domain were all conserved, except for a conservative substitution at B13, aspartate substituted glutamate in the arawana, goldeye, butterfly fish and white sucker, and at B16, phenylalanine substituted tyrosine in the elephantnose. Phylogenetic analysis of the sequences revealed a monophyletic grouping of the osteoglossomorphs, and showed that they were not the most basal living teleost. Comparative sequence analysis of preproinsulins among the osteoglossomorphs was useful in assessment of intergroup relationship, relating elephantnose with the feather fin knife fish and the arawana, butterfly fish, and goldeye. This arrangement of species is consistent with relationships based on other more classical parameters, except for the goldeye which was assessed as being sister to all the osteoglossomorphs. The white sucker was grouped with the common carp and both are cyprinids.

Evolution of receptors for proglucagon-derived peptides: isolation of frog glucagon receptors.

The mammalian proglucagon gene encodes three glucagon-like sequences, glucagon, glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 2 (GLP-2). Each of these three functionally distinct proglucagon-derived peptides has a unique, but related, receptor. To better understand the origin of the unique physiological functions of each proglucagon-derived glucagon-like sequence we have cloned glucagon-like receptors from two species of frogs, Xenopus laevis and Rana pipiens. The cloned glucagon-like receptor sequences were found to be most closely related to glucagon receptors. To determine whether the evolutionary history of the receptors for proglucagon-derived peptides was the same as that inferred for the peptide hormones, we conducted a phylogenetic analysis using both parsimony and distance methods. We show that the evolutionary history of the receptors for glucagon-like sequences differ from the history of the glucagon-like sequences. The phylogeny of receptors for proglucagon-derived peptides is not monophyletic (i.e. they are not each other's closest relatives), as the receptor for the hormone glucose-dependent insulinotropic peptide (GIP) is more closely related to the glucagon receptor than either the GLP-1 or GLP-2 receptors. In contrast to the evolutionary origin of glucagon-like sequences, where glucagon is of most ancient origin, we found that the GLP-2 receptor has the most ancient origin. These observations suggest that the diversification of the glucagon-like sequences encoded by the proglucagon gene and of the receptors for these peptides occurred independently, and that either these hormones or their receptors have been recruited for new functions.

Decreased CRH mRNA expression in the fetal guinea pig hypothalamus following maternal nutrient restriction.

The regulation of corticotropin-releasing hormone (CRH) mRNA expression following maternal nutrient restriction was examined in the fetal hypothalamus. Pregnant guinea pigs were food restricted for 48 h or fed normally during late gestation. After nutrient restriction, CRH mRNA levels in the hypothalamic paraventricular nucleus of the fetus were determined using in situ hybridization and were found to be significantly decreased (P<0.0001) compared to controls. In conclusion, we have successfully sequenced the coding sequence of the guinea pig CRH gene, and have shown that a short period (48 h) of maternal nutrient restriction inhibits CRH mRNA expression in the fetal hypothalamus.

Molecular evolution of proglucagon.

The vertebrate proglucagon gene encodes glucagon, and the two glucagon-like peptides GLP-1 and GLP-2. To better understand the origin and diversification of the distinct hormonal roles of the three glucagon-like sequences encoded by the proglucagon gene, we have examined the evolution of this gene. The structure of proglucagon has been largely maintained within vertebrates. Duplication of the proglucagon gene or duplications of sequences within the proglucagon gene are rare. All proglucagon gene duplications are likely to be the result of genome duplication events. Examination of the rates of amino acid sequence evolution of each hormone reveals that they have not evolved in a uniform manner. Each hormone has evolved in an episodic fashion, suggesting that the selective constraints acting upon the sequence vary between, and within, vertebrate classes. Changes in selection on a sequence often reflect changes in the function of the sequence, such as the change in function of GLP-1 from a glucagon-like hormone in fish to an incretin in mammals. We found that the GLP-2 sequence underwent rapid sequence evolution in the early mammal lineage, therefore we have concluded that mammalian GLP-2 has acquired a new biological function that is not found in other vertebrates. Comparisons of the hormone sequences show that many amino acid residues that are functionally important in mammalian hormones are not conserved through vertebrate evolution. This observation suggests that the sequences involved in hormone action change through evolution.

cDNA cloning of proglucagon from the stomach and pancreas of the dog.

In human and rat, tissue-specific proteolytic processing of identical proglucagon precursors yield tissue-specific proglucagon-derived peptides. In contrast, in many non-mammalian vertebrates alternative mRNA splicing yields different proglucagon precursors in different tissues. Thus alternative mRNA splicing, in part, limits the choices of proglucagon-derived peptides that can be produced by proteolytic processing. Stomach proglucagon mRNAs from the rainbow trout and Xenopus laevis were found not to encode the proglucagon-derived peptide glucagon-like peptide 2 (GLP-2). To determine if the absence of GLP-2 was a general feature of stomach proglucagons we isolated and characterized proglucagon cDNAs from the stomach and the pancreas of the dog, a mammal that expresses the proglucagon gene in the stomach. A major proglucagon transcript of about 1100 bases and a minor transcript of about 800 bases were identified in both stomach and pancreas. The coding sequences of both the stomach and pancreatic proglucagon transcripts were identical. Therefore, tissue-specific proteolytic processing, and not alternative mRNA splicing, must regulate the production of tissue-specific proglucagon-derived peptides from the stomach of the dog.

Proglucagon cDNAs from the leopard frog, Rana pipiens, encode two GLP-1-like peptides.

We have isolated and characterized proglucagon cDNAs from the intestine and pancreas of the leopard frog Rana pipiens. R. pipiens proglucagon encodes glucagon, glucagon-like peptides 2 (GLP-2), and two glucagon-like peptide 1 (GLP-1) like sequences. The pancreatic and intestinal cDNAs were of identical structure and sequence suggesting that, unlike many other non-mammalian vertebrates, there is little or no alternative splicing of the proglucagon mRNA in this species. A phylogenetic analysis of the GLP-1 encoding sequences implies that the exon encoding GLP-1 was triplicated early in frog evolution, more than 150 million years ago, before the divergence of modern frogs.

Proinsulin cDNAs from the leopard frog, Rana pipiens: evolution of proinsulin processing.

We have isolated a proinsulin cDNA from the Amphibian Rana pipiens. The predicted R. pipiens insulin A- and B-chain amino acid sequences differ from that deduced from the closely related Rana catesbeiana at one residue (Asp for Pro at B2). The R. pipiens and Xenopus laevis proinsulin precursor sequences are of identical length, with the amino acid sequences of the mature A- and B-chains being well conserved. The proinsulin C-peptide amino acid sequence is less well conserved between R. pipiens and X. laevis and also differs in length. The R. pipiens C-peptide is shorter than the homologous X. laevis sequence due to a two amino acid residue truncation. The truncation of the R. pipiens C-peptide compensates for a two amino acid residue extension observed at the N-terminal of the A-chains of insulins from Ranid frogs. A change in the site of proinsulin processing can explain both the C-peptide and A-chain length differences. The evolution of the new proinsulin processing site required two amino acid substitutions.

Lamprey proglucagon and the origin of glucagon-like peptides.

We characterized two proglucagon cDNAs from the intestine of the sea lamprey Petromyzon marinus. As in other vertebrates, sea lamprey proglucagon genes encode three glucagon-like sequences, glucagon, and glucagon-like peptides 1 and 2 (GLP-1 and GLP-2). This observation indicates that all three glucagon-like sequences encoded by the proglucagon gene originated prior to the divergence of jawed and jawless vertebrates. Estimates of the rates of evolution for the glucagon-like sequences suggest that glucagon originated first, about 1 billion years ago, while GLP-1 and GLP-2 diverged from each other about 700 MYA. The two sea lamprey intestinal proglucagon cDNAs have differing coding potential. Proglucagon I cDNA encodes the previously characterized glucagon and the glucagon-like peptide GLP-1, while proglucagon II cDNA encodes a predicted GLP-2 and, possibly, a glucagon. The existence of two proglucagon cDNAs which differ with regard to their potential to encode glucagon-like peptides suggests that the lamprey may use differential gene expression as a third mechanism, in addition to alternative proteolytic processing and mRNA splicing, to regulate the production of proglucagon-derived peptides.

The complete consensus sequence of coxsackievirus B6 and generation of infectious clones by long RT-PCR.

The full length sequence for the human pathogen coxsackievirus B6 (CVB6, Schmitt strain) has been determined. We used long RT-PCR to generate full length DNA amplicon of CVB6, and then directly sequenced the amplicons. One-step cloning of the full length amplicon enabled us to obtain an infectious clone of CVB6. RNA generated from CVB6 amplicon DNA or CVB6 clones, by transcription with T7 RNA polymerase, was demonstrated to be infectious upon transfection into HeLa cells in vitro. The CVB6 genome is characteristic of enteroviruses, with a 5'-non-translated region (743 nucleotides) followed by an open reading frame (encoding a 2184 amino acid polyprotein) and a 3'-non-translated region (100 nucleotides) and polyadenylated tail. The predicted amino acid sequence of CVB6 clustered with the other CVB serotypes and swine vesicular disease virus (SVDV).

Glucagon-like peptide 1 increases insulin sensitivity in depancreatized dogs.

To determine whether glucagon-like peptide (GLP)-1 increases insulin sensitivity in addition to stimulating insulin secretion, we studied totally depancreatized dogs to eliminate GLP-1's incretin effect. Somatostatin was infused (0.8 microg x kg(-1) x min(-1)) to inhibit extrapancreatic glucagon in dogs, and basal glucagon was restored by intraportal infusion (0.65 ng x kg(-1) x min(-1)). To simulate the residual intraportal insulin secretion in type 2 diabetes, basal intraportal insulin infusion was given to obtain plasma glucose concentrations of approximately 10 mmol/l. Glucose was clamped at this level for the remainder of the experiment, which included peripheral insulin infusion (high dose, 5.4 pmol x kg(-1) x min(-1), or low dose, 0.75 pmol x kg(-1) x min(-1)) with or without GLP-1(7-36) amide (1.5 pmol x kg(-1) x min(-1)). Glucose production and utilization were measured with 3-[3H]glucose, using radiolabeled glucose infusates. In 12 paired experiments with six dogs at the high insulin dose, GLP-1 infusion resulted in higher glucose requirements than saline (60.9+/-11.0 vs. 43.6+/-8.3 micromol x kg(-1) x min(-1), P< 0.001), because of greater glucose utilization (72.6+/-11.0 vs. 56.8+/-9.7 micromol x kg(-1) x min(-1), P<0.001), whereas the suppression of glucose production was not affected by GLP-1. Free fatty acids (FFAs) were significantly lower with GLP-1 than saline (375.3+/-103.0 vs. 524.4+/-101.1 micromol/l, P<0.01), as was glycerol (77.9+/-17.5 vs. 125.6+/-51.8 micromol/l, P<0.05). GLP-1 receptor gene expression was found using reverse transcriptase-polymerase chain reaction of poly(A)-selected RNA in muscle and adipose tissue, but not in liver. Low levels of GLP-1 receptor gene expression were also found in adipose tissue using Northern blotting. In 10 paired experiments with five dogs at the low insulin dose, GLP-1 infusion did not affect glucose utilization or FFA and glycerol suppression when compared with saline, suggesting that GLP-1's effect on insulin action was dependent on the insulin dose. In conclusion, in depancreatized dogs, GLP-1 potentiates insulin-stimulated glucose utilization, an effect that might be contributed in part by GLP-1 potentiation of insulin's antilipolytic action.

Mosaic evolution of ruminant stomach lysozyme genes.

The genomes of ruminant artiodactyls, such as cow and sheep, have approximately 10 lysozyme genes, 4 of which are expressed in the stomach. Most of the duplications of the lysozyme genes occurred 40-50 million years ago, before the divergence of cow and sheep. Despite this, the coding regions of stomach lysozyme genes within a species (e.g., cow, sheep, or deer) are more similar to each other than to lysozyme genes in other ruminants. This observation suggests that the coding regions of the stomach lysozyme genes have evolved in a concerted fashion. Our previous characterization of 3 cow stomach lysozyme genes suggested that it was only the coding exons that had participated in concerted evolution. To determine whether the introns and flanking regions of ruminant stomach lysozyme genes are evolving in a concerted or a divergent fashion, we have isolated and characterized 2 sheep stomach lysozyme genes. Comparison of the sequences of the sheep and cow stomach lysozyme genes clearly shows that the introns and flanking regions have evolved, like the 3' untranslated region of the mRNAs, in a divergent manner. Thus, if the four coding exons are evolving by concerted evolution, then a mosaic pattern of concerted and divergent evolution is occurring in these genes. The independent concerted evolution of coding exons of the ruminant stomach lysozyme gene may have assisted in the accelerated adaptive evolution of the lysozyme to new function in the early ruminant.

The Xenopus proglucagon gene encodes novel GLP-1-like peptides with insulinotropic properties.

The proglucagon gene encodes several hormones that have key roles in the regulation of metabolism. In particular, glucagon-like peptide (GLP-1), a potent stimulus of insulin secretion, is being developed as a therapy for the treatment of non-insulin-dependent diabetes mellitus. To define structural moieties of the molecule that convey its insulinotropic activity, we have cloned and characterized the proglucagon gene from the amphibian, Xenopus laevis. Unexpectedly, these cDNAs were found to encode three unique glucagon-like-1 peptides, termed xenGLP-1A, xenGLP-1B, and xenGLP-1C in addition to the typical proglucagon-derived hormones glucagon and GLP-2. xenGLP-1A, -1B, and -1C were synthesized and tested for their ability to bind and activate the human GLP-1 receptor (hGLP-1R), and to stimulate insulin release from rat pancreas. All three Xenopus GLP-1-like peptides bind effectively to the hGLP-1R and stimulate cAMP production. Surprisingly, xenGLP-1B(1-30) demonstrated higher affinity for the hGLP-1R than hGLP-1 (IC50 of 1.1 +/- 0.4 nM vs. 4.4 +/- 1.0 nM, respectively, P < 0.02) and was equipotent to hGLP-1 in stimulating cAMP production (EC50 of 0.17 +/- 0.02 nM vs. 0.6 +/- 0. 2 nM, respectively, P > 0.05). Further studies demonstrated that hGLP-1, xenGLP-1A, -1B, and -1C stimulate comparable insulin release from the pancreas. These results demonstrate that despite an average of nine amino acid differences between the predicted Xenopus GLPs and hGLP-1, all act as hGLP-1R agonists.

Selective constraints on the activation domain of transcription factor Pit-1.

The POU transcription factor Pit-1 activates members of the prolactin/growth hormone gene family in specific endocrine cell types of the pituitary gland. Although Pit-1 is structurally conserved among vertebrate species, evolutionary changes in the pattern of Pit-1 RNA splicing have led to a notable "contraction" of the transactivation domain in the mammalian lineage, relative to Pit-1 in salmonid fish. By site-directed mutagenesis we demonstrate that two splice insertions in salmon Pit-1, called beta (29 aa) and gamma (33 aa), are critical for cooperative activation of the salmon prolactin gene. Paradoxically, Pit-1-dependent activation of the prolactin gene in rat is enhanced in the absence of the homologous beta-insert sequence. This apparent divergence in the mechanism of activation of prolactin genes by Pit-1 is target gene specific, as activation of rat and salmon growth hormone genes by Pit-1 splice variants is entirely conserved. Our data suggest that efficient activation of the prolactin gene in the vertebrate pituitary has significantly constrained the pattern of splicing within the Pit-1 transactivation domain. Rapid evolutionary divergence of prolactin gene function may have demanded changes in Pit-1/protein interactions to accommodate new patterns of transcriptional control by developmental or physiological factors.