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Biomedical subjects

M J Page

Publications and source records attributed to M J Page.

53 records · Page 3Linked to original sources
PubMed1990

Molecular cloning of a human thyrotropin receptor cDNA fragment. Use of highly degenerate, inosine containing primers derived from aligned amino acid sequences of a homologous family of glycoprotein hormone receptors.

Autoantibodies to the thyrotropin (TSH) hormone receptor (TSH-R) are present in the sera of patients with thyroid autoimmune disease which are pathogenetic leading to hyperthyroidism of Graves' disease. Considerable interest has been focused on the cloning of the human TSH-R, which has until very recently, proven exceedingly difficult due to the very low receptor level expression on thyroid cells. We have used polymerase chain reaction and highly degenerate, inosine containing oligonucleotides derived from sequence alignments of the transmembrane regions 2 and 7 of a number of G-binding protein receptors including the lutropin/choriogonadotropin (LH/CG) receptors to amplify various cDNAs from human thyroid cDNA. Sequencing analysis of 27 different clones revealed that they fall into eight different groups. The very recent publication of the complete nucleotide sequence of the human TSH-R revealed that one of the groups (GT1) containing seven clones which had been sequenced belong to the human TSH-receptor. The sequence of all 7 GT1 clones was identical and in complete concordance with transmembrane regions 2 and 7 of the published TSH-R sequence. Our results show that by designing oligonucleotides to common transmembrane regions of G-binding proteins where the primers are biased in their sequence to the LH/CG receptors it is possible to amplify the TSH-R receptor sequence.

Amino Acid Sequence↗
PubMed1990

Expression of hybrid malaria antigens in insect cells and their engineering for correct folding and secretion.

Hybrid proteins containing selected regions of the major surface antigens of the sporozoite and merozoite stages of Plasmodium falciparum were expressed in insect cells using baculovirus vectors. A recombinant protein containing the signal peptide from the precursor to the major merozoite surface antigens (PMMSA) fused to a fragment from the carboxy (C) terminus of the same gene was recognized by monoclonal antibodies specific for reduction-sensitive conformational epitopes within the C-terminal fragment, suggesting that correct disulphide cross-linking of cysteine residues within this region had occurred. Addition of 26 copies of the tetrapeptide repeat from the circumsporozoite protein (CSP) resulted in a protein recognized by anti-CSP antiserum as well as the conformation specific monoclonal antibodies. Deletion of the C-terminal putative anchor sequence from both proteins resulted in secretion of protein in a fully soluble form antigenically indistinguishable from the anchor containing products. Correct conformation was not observed when the proteins were expressed as polyhedrin fusions without the signal peptide. These data indicate that the PMMSA signal peptide is recognized in insect cells and that correct assembly of disulphide cross-links is dependent upon targeting the protein to the endoplasmic reticulum.

Animals↗
PubMed1990

Mapping of autoantigenic epitopes on recombinant thyroid peroxidase fragments using the polymerase chain reaction.

Cloned cDNA templates of thyroid peroxidase (TPO) have been used in conjunction with the polymerase chain reaction (PCR) to express selected segments of the thyroid microsomal/peroxidase antigen (TMA/TPO) as recombinant protein in E. coli. Six small, different recombinant fragments averaging 120 amino acid residues and one large fragment (269 amino acids) of TPO which together encompass 80% of the extracellular region of the molecule have been produced and autoantibody (aAb) binding sites analysed by immunoblotting. A minimum of six independent, sequential antigenic determinants have been localized on the recombinant proteins and these map to the amino terminal, the central core region and the carboxyl terminal of the TPO molecule. More accurately, the six antigenic sites reside on overlapping recombinant TPO preparations termed R1a + R1b (residues 1 to 160) R1c (residues 145 to 250), R2b (residues 457 to 589), R3a (residues 577-677), R3b (residues 657-767) and R3c (residues 737-845). The large fragment of TPO termed R3 (residues 577-845) encompassing R3a, R3b and R3c also reacts with the aAbs. Different sera from patients with autoimmune thyroid disease contain antibodies to TMA/TPO which differ in their fine specificity. The use of recombinant molecular biological techniques together with PCR to prepare small segments of a large autoantigen as recombinant protein will now allow studies to progress on autoepitope mapping of the precise amino acid sequences of the TPO molecule with the use of synthetic peptides.

Autoantibodies↗
PubMed1990

The Ha-ras protein, p21, is modified by a derivative of mevalonate and methyl-esterified when expressed in the insect/baculovirus system.

Using the insect/baculovirus expression system, we demonstrate the incorporation of [3H]mevalonate and [3H]methyl groups into recombinant c-Ha-ras protein (p21). Unlike the post-translational palmitoylation of p21 expressed in this system, the modification by mevalonate is not removed by hydroxylamine suggesting the absence of a thioester linkage. It is highly likely that the insect expression system recognizes the C-terminal CAAX Motif in p21, incorporates the mevalonate into the recently described polyisoprenylation modification and carboxyl-methylates the protein.

Amino Acid Sequence↗
PubMed1989

Expression and characterization of the Ha-ras p21 protein produced at high levels in the insect/baculovirus system.

Normal and mutated cDNAs of Ha-ras have each been cloned into a standard (pAc373) and a novel (p36C) baculovirus transfer vector and introduced via homologous recombination into the genome of Autographa californica nuclear polyhedrosis virus immediately downstream of the polyhedrin promoter. Spodoptera frugiperda cells infected with recombinant virus containing the normal Ha-ras gene express very high levels of ras p21 protein (approximately 20% of total cell protein), whereas the mutant protein was expressed at considerably lower levels. Molecular analysis showed that this was most likely due to a post-transcriptional event. The expression vector p36C produced considerably higher levels of recombinant p21 compared to the more commonly used pAc373. The majority of the normal ras p21 protein is soluble, cytoplasmic, and appears to be nonacylated. However, about 10% of the p21 associates with the membrane fraction of infected cells and migrates as a slightly faster band on gels. Furthermore, this band is sensitive to hydroxylamine treatment and shows specific incorporation of [3H]palmitate, strongly suggesting that it is the palmitoylated form of p21, which is the biologically active form of the protein. Both the soluble and membrane-associated p21 have been purified to homogeneity under nondenaturing conditions, the latter in the presence of detergents. The isolation of native palmitoylated p21 has not been reported previously. The difference in hydrophobicity between these two proteins has been demonstrated by Triton X-114 partitioning. The use of the insect/baculovirus expression system to express relatively high levels (20 mg/liter) of palmitoylated p21 should aid experiments to resolve the structural and functional properties of this molecule.

Amino Acid Sequence↗
PubMed1986

Steroid regulation of transfected genes in mouse mammary tumour cells.

The regulation of mouse mammary tumour virus (MMTV) RNA by glucocorticoid hormones is well-established and has provided much information on how steroid hormones work. However, we have shown that androgens can also control MMTV RNA accumulation in S115 mouse mammary tumour cells. This novel androgen action could be explained on the basis that the MMTV long terminal repeat (LTR) can respond to several classes of steroid if appropriate receptors are present in the cells. We have used transfection experiments to demonstrate that androgens can act directly on the LTR in S115 cells. Hormonal regulation of transfected chimaeric genes into these cells was effected by androgen and glucocorticoid but not by oestrogen or progesterone, corresponding to the receptor status of the cells. Furthermore, hormonal control was also conferred by the LTR on expression of an independent cotransfected adjacent gene under its own separate promoter, suggesting that effects of an LTR can stretch to neighbouring genes in a type of hormone-enhancer insertion mechanism.

Androgens↗
PubMed1985

Expression of amplified human beta interferon genes using heavy metal induction in Chinese hamster ovary cells.

An expression cassette consisting of the human beta-interferon (beta-IFN) cDNA fused to the human metallothionein (MeT)IIA promoter has been linked to a selectable mouse dihydrofolate reductase gene (dhfr) and used to transform dhfr-deficient Chinese hamster ovary (CHO) cells. Transformants resistant to increasing concentrations of methotrexate (Mtx) were isolated and found to secrete beta-IFN either constitutively or upon induction with cadmium (up to 325 000 units beta-IFN/10(6) cells/24 h). Molecular analysis demonstrates a large increase in beta-IFN-specific DNA sequences and beta-IFN mRNA levels in amplified cell lines, with initiation of transcription occurring at the authentic start point for the MeT promoter.

Animals↗
PubMed1984

Use of gene transfer to study expression of steroid-responsive genes.

The ability to introduce DNA into eucaryotic cells has provided a means to analyse the expression of cloned genes. By manipulating the genes in vitro using recombinant DNA techniques it is possible to identify regulatory DNA sequences which are important for individual steps in gene expression. This review will summarize how these techniques have been applied to study steroid-responsive genes and what we have learned about steroid hormone action.

Bovine papillomavirus 1↗
PubMed1983

Androgen-regulated expression of a cloned rat prostatic c3 gene transfected into mouse mammary tumor cells.

The expression of the rat prostatic steroid binding protein gene C3 is androgen-responsive in vivo in the rat prostate. Recombinant transfection vectors were constructed containing a cloned C3 rat gene ligated directly into the dominant acting selection vector pSV2-gpt, and were used to transfect androgen-responsive mouse mammary tumor cells (S115 cells). Transformants containing intact copies of the C3 gene were isolated and shown by primer extension analysis to produce C3 transcripts that had authentic 5' termini. The steady state level of the transcripts was under androgen control in three cell lines tested which had low copy numbers of the intact C3 gene, but was not regulated in a cell line which had a very high C3 gene copy number. Despite this difference in the androgenic regulation of integrated C3 rat genes, both type of cell lines still demonstrated androgen-regulated growth characteristics.

Androgen-Binding Protein↗
PubMed1983

Serum regulation of the estrogen responsiveness of the human breast cancer cell line MCF-7.

Initially after receiving MCF-7 cells, we were able to confirm their estrogen responsiveness. We observed significant increases in thymidine incorporation, in thymidine kinase activity, and in cell numbers in response to 10(-8) M estradiol. Subsequently, however, the cells failed to show a response to estradiol. A growth response to estradiol could be restored by increasing the serum concentration in the medium. Cells grown in 15% serum (calf or human) responded to estradiol with increased rates of growth and thymidine incorporation and increased activities of thymidine kinase and DNA polymerase. We suggest that there is present in serum a "factor" which can influence the expression of a growth response to estradiol.

Blood↗
PubMed1982

Effect of androgen on the transcription of rat prostatic binding protein genes.

The mechanisms whereby androgens modulate mRNA levels for the C1, C2 and C3 prostatic binding protein genes in the rat ventral prostate have been investigated. Transcription rates for the 3 genes were lowered 2-3-fold upon castration but found to increase again upon re-administration of testosterone. Although this demonstrated a direct effect of androgens at the transcriptional level for these genes, the rates of mRNA synthesis were insufficient to account for previously observed steady-state measurements. This was confirmed by analysis of the nuclear steady-state levels for each mRNA following hormonal manipulation of the rats in vivo. It is concluded that besides direct transcriptional regulation, androgens must also exert a major effect of the half-life of these mRNAs in the rat ventral prostate.

Androgen-Binding Protein↗