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P M Clissold

Publications and source records attributed to P M Clissold.

At least 19 recordsLinked to original sources
PubMed2001

JmjC: cupin metalloenzyme-like domains in jumonji, hairless and phospholipase A2beta.

On the basis of significant sequence similarity, we have identified JmjC domains in more than 100 eukaryotic and bacterial sequences. These include human hairless, mutated in individuals with alopecia universalis, retinoblastoma-binding protein 2 and several putative chromatin-associated proteins. JmjC domains are predicted to be metalloenzymes that adopt the cupin fold, and are candidates for enzymes that regulate chromatin remodelling.

Amino Acid Motifs↗
PubMed2000

Truncated adenomatous polyposis coli (APC) tumour suppressor protein can undergo tyrosine phosphorylation.

Numerous mutations in the adenomatous polyposis coli (APC) gene have been described in colorectal cancer. The vast majority introduce nonsense codons leading to the production of truncated N-terminal APC fragments. Mutations occurring before APC codon 158, have been associated with an attenuated form of familial adenomatous polyposis whereas those occurring at codon 168 or beyond lead to the characteristic form of the disease. These 10 amino acid residues of APC contain a YYAQ motif which appears to constitute a potential SH2 binding domain similar to a sequence present in tyrosine kinase receptors that activate STAT 3 when phosphorylated. We have expressed a recombinant, N-terminal APC fragment in bacterial cells, and shown that it can indeed undergo tyrosine phosphorylation in this domain. We used site-directed mutagenesis to confirm the specificity of the reaction. These observations raise the possibility that tyrosine phosphorylation may be another mechanism involved in controlling APC function.

Adenomatous Polyposis Coli Protein↗
PubMed1995

Yeast artificial chromosome cloning of the beta-catenin locus on human chromosome 3p21-22.

beta-Catenin has emerged as an important component of the adherens junctions between epithelial cells. As a result of studies of its interaction with the APC gene product, it has been implicated in the development of colorectal cancer. alpha-Catenin, beta-catenin, E-cadherin and APC appear to mediate contact inhibition in epithelia. As part of the study of the organization of the beta-catenin gene, we have isolated yeast artificial chromosomes (YACs) to characterize its intron/exon structure. YAC fluorescence in situ hybridization analysis and polymerase chain reaction analysis of somatic cell hybrid DNAs show that beta-catenin maps in the 3p21-22 region, the location of tumour-suppressor genes deleted in small-cell lung cancer (SCLC) and other disorders. beta-Catenin YACs will provide a source of microsatellite markers useful in loss of heterozygosity studies to assess the importance of beta-catenin deletions in SCLC.

Base Sequence↗
PubMed1995

Simultaneous detection of amplicon and HSV-1 helper encoded proteins reveals that neurons and astrocytoma cells do express amplicon-borne transgenes in the absence of synthesis of virus immediate early proteins.

HSV-1 amplicon vectors were used to express either a cytoplasmic (beta-galactosidase) or a membrane targeted protein (TIMP-Thy1) in primary neuronal cultures, and a human astrocytoma cell line. Whereas some cells became infected by vector particles alone others were simultaneously infected by both vector and helper particles. Our results show that IEHCMV and HSV-1 IE3 promoters are able to direct transgene expression in these cells in the absence of synthesis of helper virus transacting proteins, and stress the need of monitoring expression from both partners of an amplicon population, in order to differentiate transgene expression in cells singly infected with amplicon particles, from those infected by both amplicon and helper particles.

Astrocytes↗
PubMed1994

Recombinant glycosyl-phosphatidylinositol-anchored proteins are not associated with protein kinases in transfected thymoma cells.

The cross-linking by antibody of some glycosyl-phosphatidyl-inositol (GPI)-anchored proteins on the plasma membrane of T cells leads to cell activation. Phosphorylation of proteins on tyrosine residues has a central role in the control of T cell activation, and non-receptor protein tyrosine kinases can be coprecipitated with immune complexes of GPI-anchored proteins in T cell lysates. In order to investigate the nature of this interaction, two recombinant GPI-anchored proteins were constructed (using the GPI signal sequence from Thy-1), and their associations with protein tyrosine kinases in stable transfectants of a mouse thymoma have been investigated. One recombinant GPI protein is the extracellular domain of the human complement receptor-1, normally an integral membrane protein, and the other is the secreted protein, human tissue inhibitor of metalloproteinases. The latter protein should be foreign to the cell surface and yet has been expressed as a GPI-anchored protein at levels equivalent to the highly expressed antigens Thy-1 and Ly6.A2 on mouse thymoma cells. Neither of the two recombinant proteins, when immunoprecipitated from NP40 lysates of transfected cells, was associated with protein tyrosine kinases in contrast with the natural endogenous GPI-anchored proteins Thy-1 and Ly6.A2 in non-transfected parental cells. Moreover, high expression of foreign recombinant GPI protein appears to interfere with the association of the natural GPI proteins with protein tyrosine kinases.

Animals↗
PubMed1993

Construction, expression and functional analysis of a glycolipid-linked form of CR1.

By genetic engineering of human CR1 cDNA and its stable transfection into cells we have produced a cell line which expresses CR1 anchored to the cell surface by a glycolipid anchor. The glycosyl-phosphatidylinositol (GPI)-CR1 protects cells intrinsically from damage mediated by complement activated through the classical pathway. Cell surface GPI-CR1 is more efficient on a molar basis than soluble CR1 in the assay, but extrinsic protection of other cells was not obtained. Soluble CR1-protected cells extrinsically in the assay but was required at nearly ten fold higher amounts than the intrinsic protection conferred by GPI-anchored CR1. Additionally, GPI-CR1 was shown to act as a co-factor to Factor I in the generation of C3c from iC3b. Since GPI-anchored proteins can incorporate spontaneously into the membranes of living cells by virtue of their lipid tails, the isolated GPI-CR1 will be used to introduce CR1 on to the surfaces of many different types of cell so that its role in immunity can be further investigated.

Animals↗
PubMed1992

A cDNA construct of tissue inhibitor of metalloproteinases (TIMP) linked to the last exon of Thy-1 confers glycophospholipid anchorage on this naturally secreted protein.

A naturally secreted protein, tissue inhibitor of metalloproteinases (TIMP), has been transiently expressed on the surface of transfected COS cells and stably on transfected murine BW 5147 thymoma cells, by linkage of the entire coding sequence of the cDNA to the last exon of Thy-1. Thy-1 is a glycophospholipid-linked protein. In COS cells the chimaeric protein can be labelled by [3H]ethanolamine, which is a component of glycophospholipid anchors. Ltk- cells cannot anchor proteins by glycan phosphatidylinositol linkage and were found to be unable to express the engineered protein extracellularly on their plasma membranes. Phosphatidylinositol-specific phospholipase C treatment released 90% of the protein from all BW 5147 cells, but very little from the COS-1 cells. It is concluded that the last exon of Thy-1 has conferred the property of glycophospholipid anchorage on the normally secreted protein TIMP.

Amino Acid Sequence↗
PubMed1989

A novel strategy for producing chimeric bispecific antibodies by gene transfection.

We have used genetic manipulation to produce chimeric bispecific antibodies. Plasmids containing variable regions of immunoglobulin from a murine hybridoma secreting anti-hepatitis B surface antigen were joined to human constant regions. These chimeric plasmids were introduced into transfectomas, secreting chimeric antibodies to iodo-hydroxy-nitrophenyl, by electroporation. Transfectomas secreting bispecific antibodies were identified. This approach has advantages over the fusion of hybridomas or chemical linking of two antibody molecules and will enable the use of bispecific antibodies in vivo.

Animals↗
PubMed1989

The difference between human C3F and C3S results from a single amino acid change from an asparagine to an aspartate residue at position 1216 on the alpha-chain of the complement component, C3.

The third component of the human C system, C3, exhibits two common genetic variants. These variants have been characterized by high voltage agarose electrophoresis and are designated C3 fast (C3F) and C3 slow (C3S). C3F occurs at appreciable frequencies only in Caucasian populations and has been shown to be associated with an increased incidence of certain diseases, such as partial lipodystrophy, IgA nephropathy, and Indian childhood cirrhosis. It has been shown that C3F differs from C3S with regard to isoelectric point as well as its ability to bind macrophages. The availability of a full-length cDNA probe for human C3 has made it possible to study the polymorphism at the genomic level. We have used RNA/RNA hybridization to demonstrate that the difference between C3F and C3S occurs in the C3d region. We subsequently used oligonucleotide-primed DNA amplification to show that C3F arises from a point mutation at codon 1216 converting a deoxyadenosine for a deoxyguanosine. The result of this point mutation at the translational level is the substitution of an asparagine residue in C3S for an aspartic acid residue in C3F. It is known that C3d contains the binding site for CR2 as well as the internal thioester site and multiple protease cleavage sites. The identification of the structural basis of the differences between C3F and C3S will assist our continuing studies of the mechanism of the functional differences between the two alleles and the disease associations of C3F. It also allows us to use DNA based techniques to allotype C3 in subjects with little or no C3 in their serum.

Amino Acid Sequence↗
PubMed1984

Structure of mouse major urinary protein genes: different splicing configurations in the 3'-non-coding region.

The multigene family which codes for the mouse major urinary proteins (MUPs) consists of approximately 35 genes. Most of these are members of two different groups, Group 1 and Group 2, which can be distinguished by nucleic acid hybridisation. Here we describe the structure of a Group 1 gene and show that two size classes of MUP mRNA which are found in mouse liver result from different splicing events in the 3'-non-coding region and contain different polyadenylation sites. Short mRNA is approximately 750 nucleotides long, contains six exons, and is the main product of the Group 2 genes. Long mRNA is approximately 880 nucleotides long, contains seven exons and is the main product of the Group 1 genes. Five exons and part of the sixth are common to long and short mRNA and contain the coding region. This codes for an acidic protein of 180 amino acids containing an 18 residue signal peptide. A comparison of the mouse sequence with a homologous rat alpha 2u-globulin sequence shows that the rate of evolutionary divergence of the two proteins has been high. Silent sites have diverged four times more rapidly than replacement sites, showing that there has been selection against change in the protein sequence.

Amino Acid Sequence↗
PubMed1984

Messenger RNAs coding for mouse major urinary proteins are differentially induced by testosterone.

We have investigated the sexual dimorphism of the mouse major urinary proteins (MUPs) by isoelectric focusing (IEF). In each of two inbred strains which have different male patterns (C57BL and BALB/c), the females show a simpler pattern with fewer prominent components. The main female component is different in each strain, and these may be the products of allelic structural genes. Treatment with testosterone induces the excretion of MUPs with the male pattern. The in vitro translation of hybrid-selected MUP mRNA was studied in the same way. Again, the female patterns were simpler than the male, and the patterns obtained with female mRNA from the two inbred strains were different. When the females were treated with testosterone, a male or male-like pattern was obtained. We argue that some MUP structural genes are quite actively transcribed in females and that the transcription of others is dependent to different degrees upon induction by testosterone.

Animals↗
PubMed1982

Two main groups of mouse major urinary protein genes, both largely located on chromosome 4.

Fourteen different major urinary protein (MUP) genomic clones from BALB/c mice were isolated. By restriction site mapping, six of these form two sets of three overlapping clones. By the criterion of cross-hybridization, the 10 different genes fall into two groups of four (Group 1) and three (Group 2) genes, while three genes fall into neither group. Southern blot analysis of genomic DNA with Group 1 and Group 2 plasmid subclones shows that the haploid mouse (BALB/c) genome contains approximately 15 Group 1 genes, 12 Group 2 genes and at least seven MUP genes that belong to neither group. An analysis of mouse-Chinese hamster hybrid cell lines shows that most, if not all, Group 1 and Group 2 genes are located on mouse chromosome 4.

Animals↗
PubMed1982

Variation in mouse major urinary protein (MUP) genes and the MUP gene products within and between inbred lines.

The mouse major urinary proteins (MUPs) and the unprocessed in vitro translation products of MUP mRNA were each resolved by isoelectric focusing (IEF). The urinary MUPs showed about 15 distinct components, and the unprocessed MUPs about 20. In each case wide variation was observed in the relative intensities of individual bands. A comparison of three inbred lines (C57BL, BALB/c and JU) showed inter-line variation in the patterns both of the urinary MUPs and of the unprocessed MUPs. A series of experiments was carried out with a cloned MUP cDNA probe. All three inbred lines contain the same number (about 20) of MUP genes per haploid genome. In Southern blot analysis of genomic DNA the MUP genes displayed complex patterns which we interpret as showing variation on a common basic MUP gene sequence. For each combination of restriction enzymes tested, one size of fragment carried more than half of the total label, and this fragment was always the same in the three inbred lines. Inter-line differences were observed in the patterns of some of the less reactive fragments. MUP mRNA consists of at least two distinct species with sizes of 1 and 1.2 kb, which reacted with the probe in a label ratio of about 0.5 to 1. In the three inbred lines this ratio was essentially the same.

Animals↗
PubMed1982

Variation between mouse major urinary protein genes isolated from a single inbred line.

We describe ten Charon 4A genomic DNA clones from BALB/c mice which include at least seven different major urinary protein (MUP) genes. We have established the orientation of all seven sequences, and have placed six of them in precise register by means of restriction site maps and Southern blot hybridization with cloned cDNA sequences. Four of the seven genomic sequences (family I sequences) form hybrids with six independent cDNA clones that have a high thermal stability and hybridize more strongly with mRNA from three inbred mouse lines. Hybrids between the remaining three genomic sequences and the cDNA clones have a lower thermal stability and hybridize less strongly with mRNA from the three inbred lines. Homologies between different cloned sequences extend over as much as 15 kb. No clone contains parts of two MUP genes, and no homology has been detected between the 3' flanking region of one MUP gene and the 5' flanking region of another.

Animals↗
PubMed1981

Molecular cloning of cDNA sequences transcribed from mouse liver endoplasmic reticulum poly(A)mRNA.

Poly(A)mRNA was isolated from the endoplasmic reticulum (ER) of male BALB/c mice and used as the template for cDNA synthesis. Double-stranded (ds) cDNA was cloned in a bacterial plasmid and 21 clones were selected for further study. Six clones have been identified by hybrid selection of mRNA, translation of the mRNA in a template-dependent system, polyacrylamide gel electrophoresis and specific immunoprecipitation. Four code for the major urinary protein of the mouse (MUP), one for serum albumin and one for alpha 1-antitrypsin. A seventh codes for an unidentified polypeptide with Mr = 27000. Initial-rate hybridisation kinetics of immobilised cloned sequences with end-labelled mRNA showed that serum albumin mRNA is most abundant in the population of mRNA species present in the endoplasmic reticulum fraction of female BALB/c liver. MUP mRNA is also very abundant. The alpha 1-antitrypsin mRNA and the mRNA specifying the 27000-dalton protein are about 5 times less abundant than serum albumin mRNA on a molar basis.

Animals↗
PubMed1981

Comparison of poly(A)-mRNA prepared from membranes and free polyribosomes of mouse liver.

We have compared poly(A)-mRNA isolated from mouse liver endoplasmic reticulum (ER) membranes (ER mRNA) with that from free polyribosomes (free mRNA) by translation in a cell-free system and by mRNA--cDNA reassociation analysis. The partitioning of certain translation products between the two fractions suggests that the physical contamination of the free mRNA by the ER is 10%, and contamination of the ER by the free is 1%. Reassociation crossreactions suggest that no sequences are totally absent from either fraction. In particular, the rare sequences are shared between the two fractions. Among the more abundant sequences, we detect three groups: (i) a group of about 10 sequences that are relatively abundant in both fractions; (ii) a group that occurs mostly in the free fraction; (iii) a group of about 12 sequences, 6 or 7 of them encoding secretory polypeptides. The members of this group are largely or entirely confined to the ER fraction. Secretory proteins are almost entirely absent from the products of th free mRNA fraction. Quantitation of individual mRNA sequences by hybridization with cDNA clones shows that a range of sequence concentrations exists within the abundant class of ER mRNA.

Animals↗