Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Transient Mutator”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 973 records · Page 54Linked to original sources
PubMed1999

Mutations in promoter region of thrombomodulin and venous thromboembolic disease.

The present study was designed to analyze the thrombomodulin proximal promoter region spanning nucleotides -293 to -12 to search for polymorphisms that could modify thrombomodulin gene expression in patients with venous thromboembolic disease. The study population comprised 205 patients and 394 healthy subjects of similar age and sex distribution. No polymorphisms and only 1 point mutation (G-33A) were found. The G-33A mutation was present at the heterozygous state in 2 patients and in 1 control. Being more frequent in the patients (0.97%) than in the controls (0.25%), the G-33A mutation might be a risk factor for venous thrombosis. To investigate the effect of this mutation on the thrombomodulin promoter activity, the proximal promoter region of the gene (bearing or not bearing the G-33A mutation) was inserted into a promotorless expression vector, upstream of the firefly luciferase gene, and transiently transfected into EA.hy926 endothelial cells. Under the conditions of the assay, the G-33A mutation mildly decreased the promoter activity. This study confirms that abnormalities of the thrombomodulin proximal promoter are not frequent in patients with venous thromboembolism.

Adenine↗
PubMed1996

Mutations within the gene encoding the alpha 1 (X) chain of type X collagen (COL10A1) cause metaphyseal chondrodysplasia type Schmid but not several other forms of metaphyseal chondrodysplasia.

Type X collagen is a homotrimer of alpha 1 (X) chains encoded by the COL10A1 gene. It is synthesised specifically and transiently by hypertrophic chondrocytes at sites of endochondral ossification. Point mutations and deletions in the region of the COL10A1 gene encoding the alpha 1 (X) carboxyl-terminal (NC1) domain have previously been identified in subjects with metaphyseal chondrodysplasia type Schmid (MCDS). To determine whether mutations in other regions of the gene caused MCDS or comparable phenotypes, we used PCR followed by SSCP to analyse the coding and promoter regions of the COL10A1 gene, as well as the intron/exon boundaries of five further subjects with MCDS, one subject with atypical MCDS, and nine subjects with other forms of metaphyseal chondrodysplasia. Using this approach, three of the subjects with MCDS were found to be heterozygous for the deletions 1864delACTT, 1956delT, and 2029delAC in the region of COL10A1 encoding the NC1 domain. These deletions would lead to alterations in the reading frame, premature stop codons, and the translation of truncated protein products. A fourth subject with MCDS was found to be heterozygous for a single base pair transition, T1894C, that would lead to the substitution of the amino acid residue serine at position 600 by proline within the NC1 domain. We did not, however, detect mutations in the coding and non-coding regions of COL10A1 in one subject with MCDS, the subject with atypical MCDS, and in the nine subjects with other forms of metaphyseal chondrodysplasia. We propose that the nature and distribution of mutations within the NC1 domain of COL10A1 causing MCDS argues against the hypothesis that the phenotype arises simply through haploinsufficiency but that an, as yet, unexplained mutation mechanism underlies this phenotype.

Adolescent↗
PubMed1994

Superoxide dismutase 1 with mutations linked to familial amyotrophic lateral sclerosis possesses significant activity.

Familial amyotrophic lateral sclerosis (FALS) has been linked to mutations in the homodimeric enzyme Cu/Zn superoxide dismutase 1 (SOD1). Assay by transient expression in primate cells of six FALS mutant enzymes revealed a continuum of enzymatic activity bounded by the enzyme carrying the mutation Gly-85-->Arg, which was inactive, and mutant enzyme G37R carrying the Gly-37-->Arg change, which retained full specific activity but displayed a 2-fold reduction in polypeptide stability. The G37R mutant displayed similar properties in transformed lymphocytes from an individual heterozygous for the G37R and wild-type SOD1 genes; heterodimeric enzymes composed of mutant and wild-type subunits were detected, but there was no measurable diminution in the stability and activity of the wild-type subunits. Thus, for mutants such as G37R, either surprisingly modest losses in activity (involving only the mutant subunit) can yield motor neuron death, or alternatively, mutant SOD1 may acquire properties that injure motor neurons by one or more mechanisms unrelated to the metabolism of oxygen radicals.

Amino Acid Sequence↗
PubMed1993

Self-limiting infection by int/nef-double mutants of simian immunodeficiency virus.

Simian immunodeficiency virus (SIVmac) infectious for rhesus monkeys was altered by site-directed mutagenesis of genes influencing in vivo replication and persistence with the long-term goal to develop attenuated lentiviruses with limited replication capacity in vivo. Double mutants of SIVmac (termed delta-int 1 to 3) were generated by introducing frameshift and deletion mutations into the nef gene and into the pol gene region coding for the integrase protein. Delta-int/delta-nef viruses formed after transfection of CD(4+)-lymphocyte cell lines were unable to establish sustained replication. In contrast, both wild-type SIVmac and mutant SIVmac delta-nef (coding for a truncated NEF protein and a wild-type INT protein) replicated continuously and at a comparable rate. However, a transient and self-limiting infection of the C8166 T-cell line was observed subsequent to transfection of double mutant proviruses into HeLa-tat-III cells. Viruses attenuated by int/nef-double mutation were able to enter the T-cells, initiate synthesis of viral DNA as shown by PCR amplification of closed circular episomes, and express viral antigens in infected cells as demonstrated by immunocytochemical staining. Integration of the int mutant viruses into the chromosome was completely inhibited. Episomal viral DNA was detectable in the infected cells for up to 2 weeks, after which it disappeared. Thus, SIVmac attenuated by int and nef mutation established a transient infection of permissive cells resulting in the expression of viral antigen from episomal viral DNA over a limited period of time.

Antigens, Viral↗
PubMed1996

Sp1-mediated transcriptional activation of the human Pi class glutathione S-transferase promoter.

Previous studies in this laboratory have identified an essential AP-1 recognition sequence (C1 region; -69 to -63) in th human Pi class glutathione s-transferase (GSTP1) promoter and a negatively acting regulatory element (-105 to -86) that acts to suppress GSTP1 transcription in the human mammary carcinoma cell line, MCF7 (1). The data presented here further delineate the functional characteristics of the GSTP1 promoter by examining the significance of two potential binding sites for the transcription factor, Sp1 (-57 to -49 and -47 to -39). The introduction of mutations within these Sp1-like elements and the use of Sp1 antisera in electrophoretic mobility shift assays demonstrated that Sp1 was bound to this region of the GSTP1 promoter in three different cell lines, MCF7, VCREMS, and EJ. Moreover, these in vitro studies indicated that only one of the two putative Sp1 response elements was utilized. Transient transfection assays using GSTP1 promoter constructs that incorporated mutations of the Sp1 elements clearly demonstrated that binding of Sp1 to the GSTP1 promoter was absolutely required for optimal levels of GSTP1 transcription. In particular, disruption of the distal Sp1 recognition motif (-57 to -49) markedly reduced GSTP1 promoter activity in each cell line, thus indicating preferential binding of Sp1 to the distal site. However, insertion of the repressor binding site (-105 to -86) into these constructs suggested that Sp1 was not involved in mediating the suppressive effects of the GSTP1 transcriptional repressor in MCF7 cells, because inhibition of Sp1 binding did not alleviate repressor activity. Therefore, these studies provide strong evidence that Sp1 plays a central role in regulating basal levels of GSTP1 transcription.

Base Sequence↗
PubMed2000

c-Myb-binding sites mediate G(1)/S-associated repression of the plasma membrane Ca(2+)-ATPase-1 promoter.

We demonstrate that two Myb-binding sites of the mouse plasma membrane Ca(2+)-ATPase-1 (PMCA1) promoter are required for G(1)/S cell cycle stage-associated repression of PMCA1 promoter activity. Nuclear run-on experiments revealed G(1)/S-associated repression of PMCA1 transcription. Ribonuclease protection assays revealed two transcription initiation sites between two Myb-binding sites in the PMCA1 promoter. Gel shift assays showed that c-Myb can bind to wild-type but not point mutated Myb binding sequences of the PMCA1 promoter. Transient transfection assays using cell cycle-synchronized vascular smooth muscle cells (VSMC) and PMCA1 promoter-luciferase constructs showed a 2-fold decrease in reporter activity at G(1)/S as compared with G(0). Overexpression of wild-type c-Myb severely repressed PMCA1 promoter activity at both G(0) and G(1)/S while co-transfection of a dominant negative c-Myb, or a construct encoding an anti-c-Myb neutralizing antibody, completely abolished the repression seen at G(1)/S. Single nucleotide substitutions in the first, second, or both Myb-binding sites alleviated the G(1)/S-associated repression of PMCA1 promoter activity in transformed rat VSMC and primary mouse VSMC cultures. We conclude that c-Myb mediates G(1)/S-associated transcriptional repression of the PMCA1 Ca(2+) pump in rodent VSMC by direct binding to the PMCA1 promoter.

Animals↗
PubMed1998

Suppression of tumorigenicity of glioblastoma cells by adenovirus-mediated MMAC1/PTEN gene transfer.

Mutated in multiple advanced cancers 1/phosphatase and tensin homologue (MMAC1/PTEN) is a novel tumor suppressor gene candidate located on chromosome 10 that is commonly mutated in human glioblastoma multiforme and several other cancer types. To evaluate the function of this gene as a tumor suppressor, we constructed a replication-defective adenovirus (MMCB) for efficient, transient transduction of MMAC1 into tumor cells. Infection of MMAC1-mutated U87MG glioblastoma cells with MMCB resulted in dose-dependent exogenous MMAC1 protein expression as detected by Western blotting of cell lysates. In vitro proliferation of U87MG cells was inhibited by MMCB in comparison to several control adenoviruses at equal viral doses, implying a specific effect of MMAC1 expression. Anchorage-independent growth in soft agar was also inhibited by MMCB compared to control adenovirus. Tumorigenicity in nude mice of transiently transduced mass cell cultures was then assessed. MMCB-infected U87MG cells were almost completely nontumorigenic compared to untreated and several control adenovirus-treated cells at equal viral doses. These data support an in vivo tumor suppression activity of MMAC1/PTEN and suggest that in vivo gene transfer with this recombinant adenoviral vector has a potential use in cancer gene therapy.

Adenoviridae↗
PubMed1990

Polyadenylylation signal of the mouse thymidylate synthase gene was created by insertion of an L1 repetitive element downstream of the open reading frame.

The mouse thymidylate synthase (TS; EC 2.1.1.45) mRNA is unusual in that the poly(A) tail is added at the translation stop codon. To determine the sequence requirements for 3' processing of this mRNA, we constructed TS minigenes with deletion and point mutations in potential regulatory sequences. The minigenes were transiently transfected into cultured cells and the effect on 3' processing was determined by S1 nuclease protection assays. These analyses revealed that at least two elements are required for efficient polyadenylylation at the stop codon. The first is an upstream AUUAAA sequence. When this was changed to AUCAAA, polyadenylylation at the stop codon was blocked. However, when it was changed to the canonical AAUAAA hexanucleotide, the amount of TS mRNA increased severalfold. The second element is a stretch of 14 consecutive uridylate residues 32 nucleotides downstream of the stop codon. This U-rich region is absent from the human TS gene, which explains why the human TS mRNA is not polyadenylylated at the stop codon even though the two genes are otherwise almost identical through this region. The most surprising observation was that the U-rich region corresponds to the 3' end of a 360-nucleotide mouse L1 repetitive element that was inserted in opposite orientation to the gene more than 5 million years ago. Thus the polyadenylylation signal of the present mouse TS gene was created by the transposition of a repetitive element downstream of a cryptic polyadenylylation signal.

Animals↗
PubMed1996

A mutation in the RET proto-oncogene in Hirschsprung's disease affects the tyrosine kinase activity associated with multiple endocrine neoplasia type 2A and 2B.

We demonstrate that a Hirschsprung (HSCR) mutation in the tyrosine kinase domain of the RET proto-oncogene abolishes in cis the tyrosine-phosphorylation associated with the activating mutation in multiple endocrine neoplasia type 2A (MEN2A) in transiently transfected Cos cells. Yet the double mutant RET2AHS retains the ability to form stable dimers, thus dissociating the dimerization from the phosphorylation potential. Co-transfection experiments with single and double mutants carrying plasmids RET2A and RET2AHS in different ratios drastically reduced the phosphorylation levels of the RET2A protein, suggesting a dominant-negative effect of the HSCR mutation. Also, the phosphorylation associated with the multiple endocrine neoplasia type 2B (MEN2B) allele was affected in experiments with single and double mutants carrying plasmids co-transfected under the same conditions. Finally, analysis of the enzymic activity of MEN2A and MEN2B tumours confirmed the relative levels of tyrosine phosphorylation observed in Cos cells, indicating that this condition, in vivo, may account for the RET transforming potential.

Animals↗
PubMed2003

Electron transfer in cyanobacterial photosystem I: I. Physiological and spectroscopic characterization of site-directed mutants in a putative electron transfer pathway from A0 through A1 to FX.

The Photosystem I (PS I) reaction center contains two branches of nearly symmetric cofactors bound to the PsaA and PsaB heterodimer. From the x-ray crystal structure it is known that Trp697PsaA and Trp677PsaB are pi-stacked with the head group of the phylloquinones and are H-bonded to Ser692PsaA and Ser672PsaB, whereas Arg694PsaA and Arg674PsaB are involved in a H-bonded network of side groups that connects the binding environments of the phylloquinones and FX. The mutants W697FPsaA, W677FPsaB, S692CPsaA, S672CPsaB, R694APsaA, and R674APsaB were constructed and characterized. All mutants grew photoautotrophically, yet all showed diminished growth rates compared with the wild-type, especially at higher light intensities. EPR and electron nuclear double resonance (ENDOR) studies at both room temperature and in frozen solution showed that the PsaB mutants were virtually identical to the wild-type, whereas significant effects were observed in the PsaA mutants. Spin polarized transient EPR spectra of the P700+A1- radical pair show that none of the mutations causes a significant change in the orientation of the measured phylloquinone. Pulsed ENDOR spectra reveal that the W697FPsaA mutation leads to about a 5% increase in the hyperfine coupling of the methyl group on the phylloquinone ring, whereas the S692CPsaA mutation causes a similar decrease in this coupling. The changes in the methyl hyperfine coupling are also reflected in the transient EPR spectra of P700+A1- and the CW EPR spectra of photoaccumulated A1-. We conclude that: (i) the transient EPR spectra at room temperature are predominantly from radical pairs in the PsaA branch of cofactors; (ii) at low temperature the electron cycle involving P700 and A1 similarly occurs along the PsaA branch of cofactors; and (iii) mutation of amino acids in close contact with the PsaA side quinone leads to changes in the spin density distribution of the reduced quinone observed by EPR.

Binding Sites↗
PubMed2004

Identification of one exon deletion of intestinal alkaline sphingomyelinase in colon cancer HT-29 cells and a differentiation-related expression of the wild-type enzyme in Caco-2 cells.

Sphingomyelin (SM) metabolism in the gut has been implicated in colonic tumorigenesis. Intestinal alkaline sphingomyelinase (alk-SMase) hydrolyses SM in the intestinal content and at the brush border. The enzyme activity is decreased in the tissues of human colorectal tumours. This study examines whether site or chain-mutation of alk-SMase occurs in colon cancer HT-29 cells and Caco-2 cells. Total RNA was isolated and the cDNA of alk-SMase was amplified by RT-PCR. The size of the cDNA from HT-29 cells was smaller than that of the wild-type cDNA. DNA sequencing identified a deletion of exon 4 in alk-SMase cDNA in HT-29 cells. No mutation in genomic alk-SMase DNA from exon 3 to 5 was identified. The exon 4 deletion was caused by a shift of RNA splice site in chromosome 17q25. In Caco-2 cells, no mutation of alk-SMase cDNA was identified. Transient expression in COS-7 cells showed that the enzyme from the cDNA in HT-29 cells had little alk-SMase activity whereas that in Caco-2 cells was as active as the wild-type alk-SMase. The deleted region included residue His353, which is predicted to form a substrate-binding site of alk-SMase. H353A substitution resulted in a protein with no alk-SMase activity. In monolayer cultured Caco-2 cells and HT-29 cells the alk-SMase activities were low. However, to culture the cells under polarizing conditions increased alk-SMase activity and reduced SM level in Caco-2 cells. The alk-SMase activity varied in parallel with alkaline phosphatase activity. In conclusion, we identified an inactive deletion in alk-SMase in HT-29 cells, and a differentiation-related expression of the enzyme in Caco-2 cells. The results provide a molecular mechanism related to previous findings of reduced alk-SMase activity in human colon cancers.

Alkaline Phosphatase↗
PubMed2004

A beta-catenin-dependent pathway regulates expression of cytochrome P450 isoforms in mouse liver tumors.

Phenobarbital (PB) is a model tumor promoter in the rodent liver. In the mouse, the promotional effect of PB results from a selective stimulation of clonal outgrowth of hepatocytes harboring activating mutations in the beta-catenin (catnb) gene. Glutamine synthetase (GS), a downstream target in the Wnt/beta-catenin/T-cell factor (TCF) signaling pathway, is strongly up-regulated in catnb-mutated mouse liver tumors and may serve as a marker for their identification. Here we show that the levels of several cytochrome P450 (CYP) isoenzymes are also altered in GS-positive liver tumors. Immunohistochemical and western blotting analyses demonstrated that GS-positive, catnb-mutated tumors showed levels of CYP1A, CYP2B, CYP2C and CYP2E1, which were similar or slightly enhanced in comparison with non-tumoral liver tissue. This contrasts with tumors without catnb mutations, which exhibited decreased levels of these CYP isoforms. Real-time RT-PCR revealed that the differences in CYP levels in the tumors corresponded to changes in the respective mRNAs. Mouse hepatoma cells were transiently transfected with an expression vector encoding an S33Y-mutated beta-catenin protein, which was functional with regard to transactivation of a beta-catenin/TCF-responsive (topflash) reporter construct. Co-transfected with luciferase reporter vectors containing either the regulatory upstream sequence of the CYP2B1 gene or three dioxin-responsive core elements were activated by S33Y-beta-catenin. These results indicate that mutation of catnb leads to transcriptional activation of CYP isoenzymes in mouse liver tumors. As CYPs are involved in both the activation and the inactivation of several clinically important anticancer drugs, our findings may be relevant for chemotherapy of human cancers, where activation of beta-catenin-dependent signaling by mutation of the gene or alternative mechanisms is frequently observed.

Animals↗
PubMed2005

A key role for transmembrane prolines in calcitonin receptor-like receptor agonist binding and signalling: implications for family B G-protein-coupled receptors.

Calcitonin receptor like-receptor is a family B G-protein coupled receptor (GPCR). It requires receptor activity modifying protein (RAMP) 1 to give a calcitonin gene-related peptide (CGRP) receptor. Little is known of how members of this receptor family function. Proline residues often form important kinks in alpha-helices. Therefore, all proline residues within the transmembrane helices of the receptor (Pro241, Pro244 in helix 4, Pro275 in helix 5, Pro321 and Pro331 in helix 6) were mutated to alanine. Pro241, Pro275, and Pro321 are highly conserved throughout all family B GPCRs. The binding of CGRP and its ability to stimulate cAMP production were investigated in mutant and wild-type receptors after transient transfection into COS-7 cells with RAMP1. The P321A mutation significantly decreased the pEC(50) for CGRP and reduced its affinity but did not change cell-surface expression. Antagonist binding [CGRP(8-37) and 1-piperidinecarboxamide, N-[2-[[5amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl) (BIBN4096BS)] was little altered by the mutation. Adrenomedullin-mediated signaling was disrupted when P321A was coexpressed with RAMP1, RAMP2, or RAMP3. The P331A mutant produced a moderate reduction in CGRP binding and receptor activation. Mutation of the other residues had no effect on receptor function. Thus, Pro321 and Pro331 are required for agonist binding and receptor activation. Modeling suggested that Pro321 induces a bend in helix 6, bringing its C terminus near that of helix 3, as seen in many family A GPCRs. This is abolished in P321A. P321A-I325P, predicted to restore this conformation, showed wild-type activation. Modeling can also rationalize the effects of transmembrane proline mutants previously reported for another family B GPCR, the VPAC(1) receptor.

Amino Acid Sequence↗
PubMed2004

Activation of the Drosophila TRP and TRPL channels requires both Ca2+ and protein dephosphorylation.

The Transient Receptor Potential (TRP) proteins constitute a large and diverse family of channel proteins, which is conserved through evolution. TRP channel proteins have critical functions in many tissues and cell types, but their gating mechanism is an enigma. In the present study patch-clamp whole-cell recordings was applied to measure the TRP- and TRP-like (TRPL)-dependent currents in isolated Drosophila ommatidia. Also, voltage responses to light and to metabolic stress were recorded from the eye in vivo. We report new insight into the gating of the Drosophila light-sensitive TRP and TRPL channels, by which both Ca2+ and protein dephosphorylation are required for channel activation. ATP depletion or inhibition of protein kinase C activated the TRP channels, while photo-release of caged ATP or application of phorbol ester antagonized channels openings in the dark. Furthermore, Mg(2+)-dependent stable phosphorylation event by ATPgammaS or protein phosphatase inhibition by calyculin A abolished activation of the TRP and TRPL channels. While a high reduction of cellular Ca2+ abolished channel activation, subsequent application of Ca2+ combined with ATP depletion induced a robust dark current that was reminiscent of light responses. The results suggest that the combined action of Ca2+ and protein dephosphorylation activate the TRP and TRPL channels, while protein phosphorylation by PKC antagonized channels openings.

Animals↗
PubMed1994

Role of the extracellular regions of the parathyroid hormone (PTH)/PTH-related peptide receptor in hormone binding.

The PTH/PTH-related peptide receptor is a member of a newly discovered family of G-protein-coupled receptors. Strikingly conserved features among these receptors include the positioning of eight extracellular cysteines and several other residues that are located predominantly within the membrane-embedded region. Deletion mutants or receptors with point mutations of the highly conserved cysteine residues were transiently expressed in COS-7 cells to evaluate PTH binding and PTH-stimulated cAMP production. Deletion of residues 61-105, which are encoded by exon E2 in the PTH/PTH-related peptide receptor gene, did not affect receptor function. An epitope derived from Haemophilus influenza hemagglutinin was, therefore, introduced into this portion of most receptors to allow the independent assessment of cell surface expression. PTH binding capacity was not reduced by the deletion of residues 258-278 in the first extracellular loop. Receptors with deletion of either residues 31-47 in the amino-terminal extension or residues 431-440 in the third extracellular loop failed to bind PTH, although expression of the receptor on the cell surface was only marginally reduced. Most other receptor mutants, including those in which each of the six cysteines in the amino-terminus was replaced by serines, failed to be processed and/or expressed appropriately, whereas the substitution of cysteine-281 or -351 had a less severe effect. The combined replacement of both cysteines concomitantly increased PTH binding and cell surface expression, suggesting the formation of a disulfide bond between these two residues. Our data indicate that residues near the amino-terminus and within the third extracellular loop are necessary for ligand binding, whereas more than 25% of the receptor's extracellular region appears not to be involved.

Amino Acid Sequence↗
PubMed2005

Mutation of Cys105 inhibits dimerization of p12CDK2-AP1 and its growth suppressor effect.

p12(CDK2-AP1) (p12) is a CDK2-associated protein that negatively regulates its kinase activity. Growth arrest of normal diploid cells by contact inhibition resulted in an induction of p27(kip1) and reduction of CDK2 levels. Interestingly, we observed concomitantly in growth-arrested cells, there was a reduction of nuclear p12 and the appearance of a nuclear 25-kDa molecule (p25) recognized by anti-p12 polyclonal antibody. Biochemical analysis showed that bacterial His-tagged p12 could be converted into a dimeric p25 in a reducing agent-dependent manner, and mutating the only cysteine residue of p12 (Cys(105) --> Ala(105)) abolished the dimerization. Transient transfection of wild type p12 into U2OS cells showed a reducing agent-sensitive dimerization that was also abolished by the C105A mutation. Furthermore, reduction of p12 expression by a short interfering RNA resulted in a parallel reduction of p25. These data supports the possibility that p25 is a homodimeric form of p12 through the cysteine residue. More interestingly, transient transfection of p12 (C105A) into the normal diploid lung fibroblast CCD18LU cells resulted in a reduction of the growth-inhibitory effect of p12 and abolished the inhibitory effect of p12 on CDK2 kinase activity. In addition, we found that the C105A mutation did not alter nuclear localization of p12, but it prevented association with CDK2. Taken together, our data suggest that p12 forms a nuclear homodimers in contact inhibited normal diploid cells and dimerization of p12 is a necessary process for the growth inhibition effect by p12.

Alanine↗
PubMed2005

TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage.

Mutations in the gene MCOLN1 coding for the TRP (transient receptor potential) family ion channel TRP-ML1 lead to the lipid storage disorder mucolipidosis type IV (MLIV). The function and role of TRP-ML1 are not well understood. We report here that TRP-ML1 is a lysosomal monovalent cation channel. Both native and recombinant TRP-ML1 are cleaved resulting in two products. Recombinant TRP-ML1 is detected as the full-length form and as short N- and C-terminal forms, whereas in native cells mainly the cleaved N and C termini are detected. The N- and C-terminal fragments of TRP-ML1 were co-immunoprecipitated from cell lysates and co-eluted from a Ni2+ column. TRP-ML1 undergoes proteolytic cleavage that is inhibited by inhibitors of cathepsin B (CatB) and is altered when TRP-ML1 is expressed in CatB-/- cells. N-terminal sequencing of purified C-terminal fragment of TRP-ML1 expressed in Sf9 cells indicates a cleavage site at Arg200 downward arrow Pro201. Consequently, the conserved R200H mutation changed the cleavage pattern of TRP-ML1. The cleavage inhibited TRP-ML1 channel activity. This work provides the first example of inactivation by cleavage of a TRP channel. The significance of the cleavage to the function of TRP-ML1 is under investigation.

Animals↗
PubMed1997

Differential effects of the splice acceptor at nucleotide 3295 of human papillomavirus type 31 on stable and transient viral replication.

In human papillomavirus type 31 (HPV-31), the E1--E4 and E5 open reading frames are expressed from polycistronic mRNAs. The major polycistronic mRNAs which encode E1--E4 and E5 are spliced messages which utilize a splice acceptor at nucleotide (nt) 3295 (SPA3295). Our laboratory recently developed a recombinant system for the synthesis of HPVs following immortalization of primary keratinocytes with cloned HPV-31 genomes (M. G. Frattini et al., Proc. Natl. Acad. Sci. USA 93:3062-3067, 1996). These immortalized cell lines are capable of maintaining HPV-31 DNA as episomes and induce the synthesis of virions in organotypic raft culture. In this study, we used these methods to begin an analysis of the roles of E1--E4 and E5 in HPV pathogenesis by mutating the major splice at nt 3295. Mutation of SPA3295 did not significantly alter the ability of HPV-31 genomes to replicate transiently in keratinocytes, nor did the mutation affect the immortalization potential of HPV-31. However, genomes carrying the SPA3295 mutation were not stably maintained as viral episomes, and the resulting immortalized keratinocyte cell line contained multiple, integrated copies of the mutated HPV-31 DNA. Northern analysis indicated that cell lines immortalized with the mutant HPV-31 expressed transcripts which were similar in size and abundance to wild-type messages, including those transcripts which rely on utilization of SPA3295. RNase protection and reverse transcription-PCR revealed that mutation of SPA3295 resulted in the utilization of a cryptic splice acceptor at nt 3298. These data suggest that the requirements for stable maintenance of HPV genomes are more stringent than those for transient replication and that factors which define these requirement rely on the major splice acceptor at nt 3295.

Alternative Splicing↗