Timothy E. Adams

The sheep growth hormone receptor : molecular cloning and ontogeny of mRNA expression in the liver

Two overlapping cDNA clones encoding the sheep growth hormone (GH) receptor were isolated from a sheep liver cDNA library. The translated amino acid sequence predicts a polypeptide precursor of 634 amino acids with a calculated molecular weight of 70,799. The mature GH receptor comprises an extracellular domain of 242 amino acids, a hydrophobic transmembrane region of 24 amino acids, and a cytoplasmic domain of 350 amino acids. The nucleotide and translated amino acid sequences display extensive similarity with sequences established for GH receptors from a number of other mammalian species. A prominent transcript of 4.5 kb and a minor transcript of 1.9 kb are detected following Northern blot hybridization of poly(A)+ RNA isolated from sheep liver. The onset of GH receptor mRNA expression in the liver is developmentally regulated: GH receptor transcripts are first detected by Northern blot hybridization in liver taken from a term (145 days of gestation) fetus and reach maximum levels within one week following birth. Ribonuclease protection assays reveal heterogeneity within the 5 untranslated region of GH receptor mRNA transcripts detected in liver and a number of other tissues. At least one transcript appears to be expressed in a liver-specific fashion, supporting a role for alternative RNA splicing in the tissue-specific regulation of sheep GH receptor expression.

Identification of a liver-specific promoter for the ovine growth hormone receptor

Growth hormone (GH) receptor cDNA clones from several species are characterized by heterogeneity in the 5 untranslated region (5UT). This has been attributed to different promoters directing the expression of the gene from exons encoding 5UTs which are alternatively spliced onto a common splice acceptor 11 basepairs (bp) upstream of the initiating AUG on exon 2. The following study identifies exon 1A of the ovine (o) GH receptor gene, corresponding to the 5UT of a developmentally regulated, liver-specific transcript. Exon 1A spans 206 bp at a position 17 kilobases (kb) upstream of exon 2. Sequencing of the 669 bp region 5 to the transcription initiation site (+1) reveals a TATA box at -31, a CCAAT box at -88, and putative binding sites for several transcription factors involved in liver-specific gene expression. Two repetitive sequence elements are located in the 5 and 3 flanking regions of exon 1A. Functional analysis of the 4.5 kb region upstream of exon 1A was performed by transfecting the human hepatoma cell line HuH7 with luciferase reporter gene constructs. Positive and negative regulatory regions are identified, with basal promoter activity within 473 bp of the transcription initiation site. A 47 bp region containing putative binding sites for the activated glucocorticoid receptor and C/EBP-like proteins, between -180 and -133, is essential for transcriptional activation.

Nucleotide sequence, polymorphism, and evolution of ovine MHC class II DQA genes

The nucleotide sequence of all exons and introns, excluding exon 1, of the ovine major histocompatibility complex (MhcOvar) genes analogous to the HLA-DQA1 and -DQA2 genes has been determined and the gene structure found to be similar to that reported for other species. The predicted amino acid sequences of the Ovar-DQA genes have been compared with the equivalent DQA genes in man, mouse, rat, rabbit, and cattle and used to determine the evolutionary relationships of the sheep class II genes to these other species. Northern blot analysis of sheep mRNA using exon specific probes for each of the two Ovar-DQA genes show that both genes are transcribed, whereas in humans there is no evidence that HLA-DQA2 is transcriptionally active. Restriction fragment length polymorphisms (RFLPs) have been used to define a polymorphic series of alleles in both Ovar-DQA genes and have indicated that the number of DQA genes is not constant in sheep as it is in humans, but varies with the haplotype.

Differential expression of growth hormone receptor messenger RNA from a second promoter

Multiple alternatively spliced 5 untranslated regions (5UTRs) have been identified in growth hormone (GH) receptor mRNA isolated from hepatic and adipocyte tissue. In the present study, the preferential utilisation of a GC-rich 5UTR, designated exon 1B, was observed following the isolation of ovine (o) GH receptor cDNA clones from a skeletal muscle cDNA library. Although exon 1B-oGH receptor mRNA was expressed in all tissues examined, marked differences in the level of expression relative to the whole GH receptor transcript pool were observed between tissues. A single genomic clone (lambda 9) was isolated that encompassed exon 1B, together with 6 kilobase pairs of 5 and 12 kilobase pairs of 3 flanking sequence. Multiple transcription initiation sites were identified using RNase protection analysis on skeletal muscle poly(A)+ RNA, a result consistent with the absence of a proximal TATA box element. A CAAT box (-37 to -33) and a putative binding site for SP1 (a GC box -68 to -63) were found in the sense orientation immediately upstream of major transcription initiation site. Transfection of a series of overlapping promoter fragments linked to the luciferase reporter gene into HuH7, CHO and HeLa cells defined a core promoter element of 134 base pairs that was sufficient for maximum promoter activity. The emerging complexity of the 5 regulatory region of the GH receptor gene was emphasised by the observation that probes derived from exon 1B and the distal 3 intron boundary do not hybridise with previously cloned genomic sequences that span the liver-specific P1 promoter and exon 2.

Functional expression of an ovine growth hormone receptor in transfected Chinese hamster ovary cells

Structural heterogeneity has been demonstrated for growth hormone (GH) receptors from a number of species, and both high and low affinity art receptors have been characterised by ligand binding studies. In the present study, we have transfected Chinese hamster ovary (CHO-K1) cells with a cDNA clone encoding a full-length transmembrane ovine (o) GH receptor, under the regulatory control of the human metallothionein IIA promoter. A stably transfected cell line was established (GHR9.5) which expresses on the cell surface a single class of receptor which binds 220,000 [125I]oGH molecules at high affinity (Kd = 0.30 nM) which is comparable to the affinity established for endogenous oGH receptors in postnatal sheep liver microsomes (Kd = 0.27 nM, Freemark et al. (1987) Endocrinology 120, 1865-1872). The expressed receptor also binds ovine placental lactogen (oPL, 205,000 binding sites per cell) with high affinity (Kd = 0.76 nM). The presence of two species of oGH receptor was detected in GHR9.5 cells using affinity cross-linking analysis (M(r) 148,000 and M(r) 73,000) and given that the oGH receptor cDNA codes for a non-glycosylated receptor of M(r) 69,914, it is likely that these cross-linked species correspond to homodimeric and monomeric forms of the oGH receptor, each binding to a single molecule of GH. Parallel cross-linking studies with sheep liver microsomes also demonstrated two oGH receptor species (M(r) 133,000 and M(r) 58,000), the difference in relative molecular weights between the transfected and endogenous receptors presumably resulting from tissue-specific post-translational modifications. In the presence of oGH, the GHR9.5 cells respond by increasing total cellular protein synthesis by 27% relative to non-GH-exposed GHR9.5 cells, indicating the functionality of the expressed receptor. We also demonstrate unequivocally that oPL, through a specific interaction with the transfected oGH receptor, is able to mediate a similar cellular response (38% protein synthesis induction). Responsiveness to oGH and oPL in the GHR9.5 cells is dependent on serum starvation prior to oGH exposure and occurs only with prolonged exposure (greater than 2 h) to oGH. This cellular stimulation occurs independently of c-fos transcription which has previously been shown to be one of the earliest events associated with GH action in tissues expressing endogenous GH receptors (Doglio et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1148-1152; Slootweg et al. (1990) J. Mol. Endocrinol. 4, 265-274).

Developmental and tissue-specific regulation of ovine insulin-like growth factor II (IGF-II) mRNA expression

Insulin-like growth factor II (IGF-II) is believed to be involved in the development of the fetus. Northern and dot-blot analysis of RNA isolated from different sheep tissues at various stages of development were undertaken, revealing that the ovine IGF-II gene is expressed as a multitranscript family (6.0, 5.1, 5.0, 4.7, 3.8, 2.9, 2.3, 1.9, 1.6, 1.3 kb). Evidence that the ovine IGF-II gene may be regulated in a developmental, tissue-specific, co-ordinate or independent manner is presented. The developmental profile of IGF-II gene expression correlates with plasma levels (Mesiano et al. (1989) Endocrinology 124, 1485-1491), and suggests that the rapid fall in plasma concentration at term can be attributed to regulation at the transcriptional level. With the exception of the kidney, IGF-II expression was down-regulated at birth in all tissues examined. As in man but not rat, an adult liver-specific transcript was detected and attributed to different 5 untranslated regions in the fetal and adult IGF-II mRNAs. The finding of IGF-II transcripts in all tissues examined supports evidence from other species of autocrine/paracrine roles for IGF-II in the development of the fetus.

COMPARISON OF INTRAHEPATIC LYMPHOCYTES FROM NORMAL AND GROWTH HORMONE TRANSGENIC MICE WITH CHRONIC HEPATITIS AND LIVER CANCER

Mice expressing an ovine growth hormone–mouse metallothionein promoter fusion gene (METoGH mice) develop chronic hepatitis which becomes progressively more severe over time, hepatocellular adenomas, and eventually carcinoma in the oldest animals. T‐lymphocyte expression of activation/memory‐associated markers was compared between liver and blood lymphocytes isolated from METoGH and non‐transgenic mice at 7, 10 and 12 months of age. The percentage of intrahepatic lymphocytes (IHL) which were CD4+ was markedly diminished in METoGH mice at all times. CD4+ and CD8+ IHL in METoGH mice expressed Ly‐6A/6D at increased density, and were CD45RBlo at later time‐points. Ly‐6C+ and NK1.1+ CD4+ cells, which are common in normal mouse liver, were found at decreased frequency in METoGH livers. Further analysis demonstrated that, as a proportion of total T‐cell receptor (TCR)αβ cells, NK1.1+ TCRαβint CD4+ cell numbers (NKT cells) were diminished in the livers of METoGH mice. Observations made in METoGH mice support the hypothesis that sustained liver inflammation and hepatocellular injury may be linked to liver cancer. Additionally, it is possible that the relative lack of NKT cells may create an environment permissive for the growth of liver tumours.

Methylation and expression of a metallothionein promoter ovine growth hormone fusion gene (MToGH1) in transgenic mice.

We have examined transgene methylation in the DNA from the livers of a pedigree of mice carrying three copies of an integrated MToGH1 transgene. Utilizing the methylation-sensitive isoschizomersMsp I andHpa II, Southern blot analysis revealed that all second generation animals derived from a transgenic female had hypermethylated DNA, whereas first generation animals sired by a transgenic male displayed a range of methylation phenotypes ranging from no methylation to hypermethylation of the transgene sequences. Of the mice that exhibited hypermethylation of the transgene in CpG dinucleotides (CmCGG), a minority of these animals also exhibited apparent CpC methylation (i.e. inhibition ofMsp I cutting, presumably blocked by methylation of the outer C of CCGG). Methylation was also examined in the inner C of CC(A/T)GG sequences in the MToGH1 transgene using the isoschizomer pairBstN I andEcoR II. A minority of MToGH1 animals in the F1 generation showed clear evidence of methylation in these sites as well as in the inner and outer Cs of CCGG sites. An examination of MToGH1 expression in terms of oGH levels in serum revealed that there was a high degree of variation in the levels of circulating oGH between animals of this pedigree. There was a weak inverse relationship between the serum level of oGH and the extent of methylation of the transgene. In particular, mice exhibiting CpC together with CpG methylation were found to have very low levels of circulating oGH. Our results highlight the nature and complexity of epigenetic factors associated with transgene sequences which may ultimately influence expression of introduced genes in the mammalian genome.

Production of methionyl-minus ovine growth hormone in Escherichia coli and one-step purification

The overproduction of ovine growth hormone (oGH) in Escherichia coli is described, achieved in part by alteration of the codon usage for nine of the first 15 amino acids (aa) of the mature hormone. Recombinant oGH (re-oGH), representing 12% of the total cellular protein, was isolated from inclusion bodies by solubilisation using the cationic surfactant cetyltrimethylammonium chloride (CTAC). The hormone was refolded and subsequently purified to greater than 95% homogeneity in a single step using preparative reverse phase high performance liquid chromatography. The aa sequence analysis revealed that the N-terminus of the E. coli-derived polypeptide was identical to that of pituitary-derived oGH, and re-oGH displayed potent somatotropic activity in vivo.

Removal of 3’Untranslated Sequences Dramatically Enhances Transient Expression of Ovine Follicle‐Stimulating Hormone Beta Gene Messenger Ribonucleic Acid

The influence of the 3’untranslated (3′‐UT) region of the ovine follicle‐stimulating hormone (FSH) β mRNA on the level of transcript expression was studied. Only very low levels of FSH β mRNA were detected following transient transfection of COS cells with a eukaryotic expression vector containing a full‐length ovine FSH β cDNA that includes 1.1 kilobases (kb) of 3′‐UT. In contrast, deletion of all but 135 basepairs (bp) of the 3′‐UT resulted in a striking increase in FSH β mRNA expression following transfection of the truncated cDNA construct. These observations suggest that sequences within the 3′‐UT of the ovine FSH β mRNA may play a significant role in the post‐transcriptional regulation of FSH β expression.