Nicholas Hunt

Expression of the human relaxin gene in the corpus luteum of the menstrual cycle and in the prostate

DNA-RNA hybridization has been used to assess the presence of relaxin gene transcripts in human luteal tissues of pregnancy and the menstrual cycle, as well as in the human testis and prostate. The results imply a substantial capacity for hormone biosynthesis in the mid to late luteal phase of the ovary in non-pregnant women. In men the prostate has been shown also to express relaxin gene transcripts, though levels are low. The testis appears negative. The results suggest that functions for relaxin must be sought also outside pregnancy.

The chicken vasotocin gene.

cDNA clones corresponding to the vasotocin precursor polypeptide were isolated from a chicken hypothalamic library and sequenced. The derived amino‐acid sequence indicates a precursor of comparable structural organization to that described for members of the vasotocin/vasopressin gene family from other species. Unlike in mammals the C‐terminal glycopeptide moiety appears not be cleaved off from the neurophysin. Subsequent screening of a chicken genomic library permitted an analysis also of the vasotocin gene structure and exonic composition. The 5’region upstream of the first exon was sequenced and revealed an unusual pattern of 49 repetitive ‐YYCYCYAAAYY‐ motifs, together with a polyadenyl region supporting a bend in the DNA, and a long pyrimidine‐rich sequence. Three AP2‐like elements, identified in the mammalian vasopressin gene, were also observed in the immediate upstream region. There was no obvious homology to the promoter regions of the known oxytocin genes, nor to any other sequence deposited in available databases, nor to other known cis‐elements.

A Novel Endozepine-Like Peptide (ELP) is Exclusively Expressed in Male Germ Cells

A cDNA clone encoding a novel endozepine-like peptide (ELP) was isolated from mouse testes, sequenced, and its mRNA expression characterized by northern and in situ hybridization. ELP mRNA was found exclusively in the late spermatid stages of spermatogenesis in the testes of sexually mature mice and in no other tissue or cell type examined. It was also expressed in rat, bovine, porcine and sheep testes. Mouse ELP-encoding cDNA was used to construct expression vectors for the production of ELP in bacteria, and the purified bacterial protein used to raise polyclonal antibodies in rats. These antibodies identified the predicted endogenous ELP in extracts of mouse testis and epididymis and in no other tissue. Immunohistochemistry confirmed that the ELP antigen was present only in late spermatids and spermatozoa, particularly within the cytoplasmic droplet which is retained by the mature spermatozoa during their transit into the epididymis. We conclude that ELP is an intracytoplasmic peptide exclusively expressed in post-meiotic spermatozoa and which may be involved in the energy metabolism of the mature sperm.

Vasopressin biosynthesis in rodent Leydig cells

Local biosynthesis of the peptide hormone vasopressin is demonstrated in vitro in Leydig cells derived from rat and mouse testis. Cycloheximide-sensitive production of the nonapeptide was shown for rat Leydig cells in primary culture. A polymerase chain reaction technique demonstrated the presence of functionally constituted vasopressin mRNA in rat and mouse testis, primary mouse Leydig cells and in rat and mouse Leydig tumour cell lines (MA10, R2C). Stimulation of cells with gonadotropins, however, had no effect either on peptide production or on levels of specific mRNA. Similarly, treatment of the MA10 cell line with a phorbol ester, or with rat atriopeptin, which activate other second messenger pathways, had no influence on vasopressin mRNA levels. The results are discussed in terms of an autocrine regulatory system which would provide the cell with information about its microenvironment.

EXPRESSION OF RECOMBINANT HUMAN THYROID PEROXIDASE BY THE BACULOVIRUS SYSTEM AND ITS USE IN ELISA SCREENING FOR DIAGNOSIS OF AUTOIMMUNE THYROID DISEASE

The cDNAs coding for human full-length and soluble thyroid peroxidase (TPO) were constructed, cloned into a baculovirus transfer vector and used for infection of Spodoptera frugiperda (Sf9) cells. The soluble TPO lacking 87 amino acids of the C-terminal transmembrane and intracisternal domains was designed as a fusion protein with a histidine-hexapeptide as an affinity ligand at its C-terminus. Whereas the recombinant full-length TPO was expressed mainly in an insoluble form in Sf9 cells, the recombinant soluble TPO was almost completely secreted into the culture medium. Both the full-length and the soluble TPO were purified by conventional methods and by a specific affinity chromatography using metal chelating matrix respectively, and tested for their autoantigenicity towards anti-TPO autoantibodies. The ELISA established with the purified recombinant soluble TPO as antigen demonstrated its specificity, practicability and reproducibility in screening of anti-TPO autoantibodies in sera of autoimmune thyroid patients. High correlation (r = 0.89, n = 175) was obtained between the soluble TPO and natural TPO prepared from human thyroid glands. Pathological sera (n = 200) were positively assayed with a significance of 91%.

Novel splicing variants of the human thyrotropin receptor encode truncated polypeptides without a membrane-spanning domain.

The thyrotropin receptor is of fundamental importance to normal thyroid function and is considered to be the predominant antigen affected by the autoantibodies of Graves’ autoimmune hyperthyroidism. The identification of the epitopes on the receptor to which the autoantibodies bind or the mechanism by which the autoantibodies arise remain to be established. In this report we have analysed in detail thein vivo transcription of the human TSH receptor gene (hTSH-R), demonstrating the presence of numerous novel TSH receptor transcripts. Northern blot analysis of mRNA from human thyroid tissue using a radiolabelled cDNA probe specific for the extracellular domain of the hTSH-R revealed the presence of small polyadenylated mRNAs, in addition to the full-length hTSH-R mRNA. A PCR strategy devised to clone transcripts with 3′ polyadenylation and 5′ hTSH-R specific sequences was used to clone five different hTSH-R transcripts (hTSH-R. ST1 to ST5; 250bp-1.7 kb) from human thyroid tissue. Sequence analysis demonstrated that the small transcripts arose by alternative splicing of the hTSH-R mRNA. The transcripts were associated with polysomes and were demonstrated in human thyroid tissue from patients suffering from Graves’ disease, sporadic goiter as well as in healthy lobes of thyroid tissue.In situ hybridization demonstrated that two of the alternative transcripts adopted a tissue distribution pattern identical to that of the full-length hTSH-R transcript. The two major truncated transcripts ST4 and ST5 contained unique sequences at the 3′ end of the mRNAs and thus potentially represent the molecular origin of soluble TSH receptor variants which have been postulated on numerous occasions.

Cis-Active Elements of Friend Spleen Focus-Forming Virus: From Disease Induction to Disease Prevention

The polycythemic strain of the Friend spleen focus-forming virus (SFFVp) is a replication-defective, acutely transforming retrovirus inducing a bistage erythroleukemia in susceptible mice. The first stage of the disease is an acute polyclonal erythroblastosis induced by the proliferation-promoting effect of gp55. gp55 is expressed from a spliced subgenomic message of SFFVp and stimulates the cellular receptor for erythropoietin. Using a selectable SFFVp that otherwise mimics the specificity of the disease, we demonstrate that the kinetics of the polyclonal expansion depends on the transcriptional strength of the retroviral cis-active elements. By exchanging gp55 for apathogenic genes, we show that SFFVp enhancer and splice signals can be successfully utilized for the development of retroviral vectors mediating very efficient transgene expression in hematopoietic cells. Apathogenic selectable SFFVp-based vectors carrying distinct enhancer alterations are a valuable tool to analyze transcriptional control of leukemia viruses in the absence of oncogenic proteins. Moreover, they might have therapeutic potential.

Erythropoietin and the ENV gp55 of the Spleen Focus Forming Virus (SFFV) Interact Differently with Erythroid Cells in the Mouse and in Tissue Culture

Erythropoietin (Epo) is a glycoprotein hormone, which is the major regulator of mammalian erythropoiesis. Epo promotes the survival, proliferation and differentiation of erythroid progenitor cells by binding to and activation of the specific cell surface receptor (Epo-R) (D’ Andrea et al. , 1989; reviewed by Youssoufian et al. , 1993). The recently cloned cDNA of the murine Epo-R encodes a protein of 507 amino acids that contains a single hydrophobic membrane spanning domain. The Epo-R is a member of the cytokine receptor family, which include the receptors for granulocyte colonystimulating factor (G-CSF), interleukin-3 (IL-3), IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF) and several other growth factors (D’Andrea et al. , 1990; Bazan, 1990). Receptor polypeptides in this family share a set of four conserved Cys residues that stabilize the structure of the membrane-distal sub-domain and a five-residue motif located close to the transmembrane domain, Trp-Ser-X-Trp-Ser (WSXWS), that was predicted to be an essential component of the ligand-binding site of cytokine receptors (D’Andrea et al. , 1990; Bazan, 1990). For several members of the cytokine receptor family, generation of high-affinity receptors requires the formation of hetero-oligomers (reviewed by Sato and Miyajima, 1994).

Retroviral Vectors for Gene Transfer and Expression in Haematopoietic Cells

Our aim is to design vectors which are 1) generally useful to transfer and express genes in any given murine or human cell and 2) show limited expression in specialised cells. Retro-viruses are at present the best studied vector system for gene transfer into eukaryotic cells. We have utilised a series of naturally occuring acutely oncogenic murine retroviruses with well defined specificities of transformation to design such vectors.