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PubMed2002

Genetic analysis of high-risk e6 in episomal maintenance of human papillomavirus genomes in primary human keratinocytes.

Papillomaviruses possess small DNA genomes that encode five early (E) proteins. Transient DNA replication requires activities of the E1 and E2 proteins and a DNA segment containing their binding sites. The E6 and E7 proteins of cancer-associated human papillomavirus (HPV) transform cells in culture. Recent reports have shown that E6 and E7 are necessary for episomal maintenance of HPV in primary keratinocytes. The functions of E6 necessary for viral replication have not been determined, and to address this question we used a recently developed transfection system based on HPV31. To utilize a series of HPV16 E6 mutations, HPV31 E6 was replaced by its HPV16 counterpart. This chimeric genome was competent for both transient and stable replication in keratinocytes. Four HPV16 E6 mutations that do not stimulate p53 degradation were unable to support stable viral replication, suggesting this activity may be necessary for episomal maintenance. E7 has also been shown to be essential for episomal maintenance of the HPV31 genome. A point mutation in the Rb binding motif of HPV E7 has been reported to render HPV31 unable to stably replicate. Interestingly, HPV31 genomes harboring two of the three p53 degradation-defective E6 mutations combined with this E7 mutation were maintained as replicating episomes. These findings imply that the balance between E6 and E7 functions in infected cells is critical for episomal maintenance of high-risk HPV genomes. This model will be useful to dissect the activities of E6 and E7 necessary for viral DNA replication.

Amino Acid Sequence↗
PubMed2005

Phenotypic variability in myotonia congenita.

Myotonia congenita is a hereditary chloride channel disorder characterized by delayed relaxation of skeletal muscle (myotonia). It is caused by mutations in the skeletal muscle chloride channel gene CLCN1 on chromosome 7. The phenotypic spectrum of myotonia congenita ranges from mild myotonia disclosed only by clinical examination to severe and disabling myotonia with transient weakness and myopathy. The most severe phenotypes are seen in patients with two mutated alleles. Heterozygotes are often asymptomatic but for some mutations heterozygosity is sufficient to cause pronounced myotonia, although without weakness and myopathy. Thus, the phenotype depends on the mutation type to some extent, but this does not explain the fact that severity varies greatly between heterozygous family members and may even vary with time in the individual patient. In this review, existing knowledge about phenotypic variability is summarized, and the possible contributing factors are discussed.

Chloride Channels↗
PubMed2004

Molecular pathophysiology of mucolipidosis type IV: pH dysregulation of the mucolipin-1 cation channel.

Mucolipidosis type IV (MLIV) is an autosomal recessive neurogenetic disorder characterized by developmental abnormalities of the brain and impaired neurological, ophthalmologic and gastric function. Large vacuoles accumulate in various types of cells in MLIV patients. However, the pathophysiology of the disease at the cellular level is still unknown. MLIV is caused by mutations in a recently described gene, MCOLN1, encoding mucolipin-1 (ML1), a 65 kDa protein whose function is also unknown. ML1 shows sequence homology and topological similarities with polycystin-2 and other transient receptor potential (Trp) channels. In this study, we assessed both, whether ML1 has ion channel properties, and whether disease-causing mutations in MCOLN1 have functional differences with the wild-type (WT) protein. ML1 channel function was assessed from endosomal vesicles of null (MCOLN1(-/-)) and ML1 over-expressing cells, and liposomes containing the in vitro translated protein. Evidence from both preparations indicated that WT ML1 is a multiple subconductance non-selective cation channel whose function is inhibited by a reduction of pH. The V446L and DeltaF408 MLIV causing mutations retain channel function but not the sharp inhibition by lowering pH. Atomic force imaging of ML1 channels indicated that changes in pH modified the aggregation of unitary channels. Mutant-ML1 did not change in size on reduction of pH. The data indicate that ML1 channel activity is regulated by a pH-dependent mechanism that is deficient in some MLIV causing mutations of the gene. The evidence also supports a novel role for cation channels in the acidification and normal endosomal function.

Cells, Cultured↗
PubMed1998

Mutants of the CMP-sialic acid transporter causing the Lec2 phenotype.

Chinese hamster ovary (CHO) mutants belonging to the Lec2 complementation group are unable to translocate CMP-sialic acid to the lumen of the Golgi apparatus. Complementation cloning in these cells has recently been used to isolate cDNAs encoding the CMP-sialic acid transporter from mouse and hamster. The present study was carried out to determine the molecular defects leading to the inactivation of CMP-sialic acid transport. To this end, CMP-sialic acid transporter cDNAs derived from five independent clones of the Lec2 complementation group, were analyzed. Deletions in the coding region were observed for three clones, and single mutants were found to contain an insertion and a point mutation. Epitope-tagged variants of the wild-type transporter protein and of the mutants were used to investigate the effect of the structural changes on the expression and subcellular targeting of the transporter proteins. Mutants derived from deletions showed reduced protein expression and in immunofluorescence showed a diffuse staining throughout the cytoplasm in transiently transfected cells, while the translation product derived from the point-mutated cDNA (G189E) was expressed at the level of the wild-type transporter and co-localized with the Golgi marker alpha-mannosidase II. This mutation therefore seems to directly affect the transport activity. Site-directed mutagenesis was used to change glycine 189 into alanine, glutamine, and isoleucine, respectively. While the G189A mutant was able to complement CMP-sialic acid transport-deficient Chinese hamster ovary mutants, the exchange of glycine 189 into glutamine or isoleucine dramatically affected the transport activity of the CMP-sialic acid transporter.

Animals↗
PubMed1999

Functional dissection of the promoter of the interphotoreceptor retinoid-binding protein gene: the cone-rod-homeobox element is essential for photoreceptor-specific expression in vivo.

The essential control elements in the interphotoreceptor retinoid-binding protein gene (IRBP) promoter are located between -156 and +19. The -156/-109 sequence contains a retina-specific DNAse I footprint and shows a positive regulatory activity in transiently transfected retinoblastoma cells. The -105/-85 sequence is G/C rich, shows a non-tissue specific DNAse I hypersensitivity, and a negative regulatory activity in retinoblastoma cells. The -76/-42 sequence shows a retinal-specific footprint and contains a "cone-rod-homeobox element" (CRXE) and a "photoreceptor conserved element" (PCE). IRBP promoter fragments with mutations in either CRXE, PCE or in both were linked to reporter genes and analyzed both by transient transfection and in transgenic mice. In retinoblastoma cells, the mutated CRXE-containing promoter shows a 60% repression of the CAT activity whereas the mutated PCE-containing promoter shows a 30% repression. In HeLa cells transfected with these promoters, co-transfection of a Crx expression vector with wild-type, but not with CRXE mutant promoter, activates CAT activity 20-fold over the background activity. Mutation of PCE alone or conversion of CRXE to PCE reduces this Crx-activated CAT activity to only 4-fold over the background activity. In the transgenic mouse experiments, none of the 12 lines with CRXE mutant promoter show significant expression of lacZ in the retina. In contrast, 9 of the 17 transgenic lines with PCE mutant promoter show photoreceptor-specific lacZ expression. Thus the Crx interaction with CRXE is essential for the photoreceptor-specific activity of the IRBP promoter in vivo. This interaction does not appear to require PCE, but is enhanced when PCE is present.

Animals↗
PubMed1995

Characterization of heterogeneous mutations causing constitutive activation of the luteinizing hormone receptor in familial male precocious puberty.

Familial male precocious puberty (FMPP) is a gonadotropin-independent disorder that is inherited in an autosomal dominant, male-limited pattern. A heterozygous mutation encoding substitution of Asp578 with Gly in transmembrane helix 6 of the G protein-coupled receptor for luteinizing hormone (LHR) has been found in affected males from nine American FMPP families. Cells expressing the mutant LHR exhibit markedly increased cyclic adenosine monophosphate (cAMP) production in the absence of agonist, suggesting that autonomous Leydig cell activity in FMPP is caused by a constitutively activated LHR. We have now analyzed genomic DNA from affected males from six additional FMPP families. PCR was used to amplify a fragment of the LHR gene encoding amino acid residues 441-594. None of the six new samples contained the Asp578-->Gly mutation, as indicated by absence of digestion with MspI. PCR products were then screened for heterozygous mutations using temperature-gradient gel electrophoresis. DNA fragments from two of the patients migrated abnormally. Direct sequencing of PCR product from one affected German male revealed a heterozygous mutation (ATG-->ATA) encoding Met571-->Ile at the cytoplasmic end of helix 6, the same mutation that has been reported in another European FMPP kindred. Affected males in the second family had a novel Thr577-->Ile mutation (ACC-->ATC). Mutations in different portions of the LHR or in a different gene may be responsible for disease in the other FMPP kindreds. Agonist binding and functional coupling of the mutant receptors to the cAMP and inositol phosphate pathways were studied by transiently expressing them in COS-7 cells. Agonist affinity was unaffected by the mutations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

[Functional analysis of human A gamma-globin gene promoter with-173 T-->C mutation].

OBJECTIVE: To investigate the effect of the human A gamma-globin gene-173 T-->C mutation on the binding of transacting factors to the promoter and the activity of the promoter. METHODS: Electrophoretic mobility shift assay and transient transfection assay were used in this study. RESULTS: The A gamma-globin gene-173 T-->C mutation decreased the affinity of GATA-1 to the mutant promoter fragment (-201(-)-158) 96% (P < 0.01) and the binding of Oct-1 to the same DNA fragment 55% (P < 0.05). The activity of the mutant promoter was 2-fold (P < 0.05) as strong as that of the normal one in MELGM979 cells. The mutant and the normal promoters exhibited basically same activities in K562 and Hela cells. CONCLUSIONS: The -173 T-->C mutation decreased dramatically the binding of GATA-1, implicating that GA-TA-1 may act as a negative regulator of A gamma-globin gene in adults. The -173 T-->C mutation may enhance the activity of A gamma-globin gene promoter in the adult erythroid cell environment.

Adult↗
PubMed1998

Isolation and characterization of Bacillus subtilis sigB operon mutations that suppress the loss of the negative regulator RsbX.

sigmaB, a transcription factor that controls the Bacillus subtilis general stress response regulon, is activated by either a drop in intracellular ATP or exposure to environmental stress. RsbX, one of seven sigmaB regulators (Rsb proteins) whose genes are cotranscribed with sigmaB, is a negative regulator in the stress-dependent activation pathway. To better define the interactions that take place among the Rsb proteins, we analyzed sigB operon mutations which suppress the high-level sigmaB activity that normally accompanies the loss of RsbX. Each of these mutations was in one of three genes (rsbT, -U, and -V) which encode positive regulators of sigmaB, and they all defined amino acid changes which either compromised the activities of the mutant Rsbs or affected their ability to accumulate. sigmaB activity remained inducible by ethanol in several of the RsbX- suppressor strains. This finding supports the notion that RsbX is not needed as the target for sigmaB activation by at least some stresses. sigmaB activity in several RsbX- strains with suppressor mutations in rsbT or -U was high during growth and underwent a continued, rather than a transient, increase following stress. Thus, RsbX is likely responsible for maintaining low sigmaB activity during balanced growth and for reestablishing sigmaB activity at prestress levels following induction. Although RsbX likely participates in limiting the sigmaB induction response, a second mechanism for curtailing unrestricted sigmaB activation was suggested by the sigmaB induction profile in two suppressor strains with mutations in rsbV. sigmaB activity in these mutants was stress inducible but transient, even in the absence of RsbX.

Amino Acid Sequence↗
PubMed2006

Null mutations in the filaggrin gene (FLG) determine major susceptibility to early-onset atopic dermatitis that persists into adulthood.

Atopic dermatitis (AD) is a common disease with a complex etiology in childhood and adult life. A significant proportion of childhood AD is transient, but in many cases it persists into adulthood. We have recently shown that null mutations in the filaggrin gene (FLG) are an important predisposing factor for childhood eczema and eczema-associated asthma, but persistence to adulthood has not been analyzed. Here we studied a cohort of adult patients with persistent AD, which had been present since early childhood. In this cohort, the combined allele frequency of the two common FLG null variants was 0.270 (cf. population frequency 0.046). This represents an odds ratio of 7.7 with 95% confidence interval of 5.3-10.9 and a chi2 P-value of 1.7 x 10(-53). Our data conclusively demonstrate that identification of FLG null alleles is an indicator of a poor prognosis in AD, predisposing to a form of eczema that starts in early infancy and persists into adulthood. This study helps to further define the nature of the AD phenotype associated with FLG null alleles.

Adolescent↗
PubMed2005

Aquagenic wrinkling of the palms in patients with cystic fibrosis homozygous for the delta F508 CFTR mutation.

BACKGROUND: Aquagenic wrinkling of the palms (AWP) is a rare condition characterized by the rapid and transient formation of edematous whitish plaques on the palms on exposure to water (the so-called hand-in-the-bucket sign). The changes may be asymptomatic or accompanied by pruritic or burning sensations. First described in 1974 in patients with cystic fibrosis--and still primarily reported in these patients--this condition has been previously described only in females. Specific mutations in CFTR, the gene responsible for cystic fibrosis, have not been reported previously in patients with AWP. OBSERVATIONS: We describe 2 patients with AWP, both of whom are homozygous for the delta F508 mutation in CFTR: a 17-year-old boy--the first male reported to have this condition--who has had AWP for 3 years and a 13-year-old girl who has had AWP for 6 months. CONCLUSIONS: Cystic fibrosis should be considered in patients with AWP, and patients with cystic fibrosis should be asked about symptoms of this condition. Although the etiology of AWP is unknown, the association with cystic fibrosis, and with marasmus and cyclooxygenase-2 inhibitors, suggests that exposure of the skin to abnormally high concentrations of salt may play a role in its pathogenesis.

Adolescent↗
PubMed1997

Pathophysiology of Kostmann syndrome: the G-CSF receptor issue.

Kostmann Syndrome is defined as a chronic neutropenia, dating from early childhood, characterized typically by a granulopoeisis impairment at the promyelocyte stage. The origin is not yet understood. G-CSF receptor anomaly -the intra-cellular carboxy terminal region- was noted in a few patients (6 out of 54), initially in two patients who later developed secondary leukemia. More follow-up, with other patients, led us to consider the mutation of the G-CSF receptor sometimes as a transient event, not systematically resulting in malignancy. This finding directs research toward intra-cellular signaling pathway in a pathology that raises questions both of granulopoeisis and leukemogenesis.

Chromosomes, Human, Pair 7↗
PubMed2005

Subcellular localization and trafficking of polycystins.

Polycystin-2 is a member of the transient receptor potential (TRP) family of ion channels that is mutated in autosomal dominant polycystic kidney disease. Although its function as a non-selective cation channel has been demonstrated in several model systems, the precise subcellular localization of polycystin-2 (TRPP2) in tubular epithelial cells has remained controversial. Recent evidence suggests that the subcellular localization of TRPP2 is regulated by multiple protein interactions. This review will summarize our current knowledge about polycystin trafficking and highlight the experimental data that supports a compartment-specific function of 'cystogenic' proteins.

Animals↗
PubMed1992

Identification of amino acid residues of rat angiotensin II receptor for ligand binding by site directed mutagenesis.

To determine the specific mechanism of ligand binding to angiotensin (Ang II) receptor AT1, mutagenized rat receptor cDNAs were expressed transiently in COS-7 cells and the effect of the mutations on the binding to peptidic and non-peptidic ligands was analyzed by Scatchard plots. Mutation of Lys199 to Gln in the intramembrane domain strongly reduced the affinity to both [125I] Ang II and [125I]-1Sar, 8Ile-Ang II whereas mutation of two other Lys had little effect, indicating involvement of Lys199 in binding ligands. Replacement of each of four Cys in the extracellular domain markedly reduced binding affinity, indicating the importance of two putative disulfide bridges in the formation of active receptor conformation. Substitution of Asp for Asn in N-glycosylation had no effect on ligand binding or expression of the receptor. These studies indicate mutated receptors are expressed in the plasma membrane and are amenable for further detailed studies.

Angiotensin II↗
PubMed1995

Phosphoinositide-mediated phototransduction in Drosophila photoreceptors: the role of Ca2+ and trp.

Drosphoinate photoreceptors, represent a paradigm for the genetic dissection of phototransduction and, more generally for Ca2+ signaling. As in most invertebrates, phototransduction in Drosophila is mediated by the phosphoinositide (PI) cascade and is completely blocked by null mutations of the norpA gene which encodes a phospholipase C-beta isoform. The light-activated conductance in Drosophila is normally highly permeable to Ca2+, but in null mutants of the trp gene Ca2+ permeability is greatly reduced. Furthermore, the trp gene sequence shows homologies with voltage gated Ca2+ channels, suggesting that trp encodes a light-sensitive channel subunit. Ca2+ influx via these channels is instrumental in light adaptation, and profoundly influences phototransduction via positive and negative feedback at multiple molecular targets including protein kinase C. The mechanism of activation of the light-sensitive channels remains unresolved. A requirement for Ca2+ release from internal stores is suggested by the finding that Drosophila photoreceptors cannot sustain a maintained response under various conditions which might be expected to result in depletion of Ca2+ stores. However, Ca2+ release cannot be detected by Ca2+ indicator dyes and raising Ca2+ by photorelease of caged Ca2+ fails to mimic excitation. Recent studies, both in situ and with heterologously expressed trp protein, suggest that the trp-dependent channels may be activated by a process analogous to 'capacitative Ca2+ entry', a widespread, but poorly understood mode of PI-regulated Ca2+ influx in vertebrate cells.

Animals↗
PubMed1995

Role of the pyrimidine-rich tract on the translation of 'chimeric' polio-hepatitis A mRNAs with engineered 5'-terminal untranslated regions.

The 5'-terminal untranslated region (5'-UTR) of picornavirus RNA contains a series of cis-acting elements required for the internal initiation of translation, including a pyrimidine-rich tract (PRT), which in entero- and rhinoviruses is located about 20 nts upstream from a silent AUG triplet in the vicinity of the translation initiation site. In hepatitis A virus (HAV) RNA, the PRT is only 12 nts upstream from the legitimate AUG initiation codon, and a second, longer PRT in a region far removed from the translation initiation site. This 5'-distal PRT includes a 'core' sequence 80% homologous to the PRT of poliovirus RNA. A 'chimeric' polio-hepatitis A mRNA was constructed in which the sequences extending between nucleotides 45 and 156 of HAV RNA replaced the corresponding ones in poliovirus 5'-UTR. The construction was extended with poliovirus sequences up to position 1809. The recombinant mRNA so generated carried two copies of the PRT. In vitro translation in lysates of truncated poliovirus mRNAs generated a single peptide of Mr = 39 kDa, while the chimeric mRNA generated a series of short peptides as a result of fortuitous (or aberrant) initiation events. A more extensive substitution in the chimeric 5'-UTR which removed the 3'-most PRT brought by the poliovirus sequences, restored the translation at the authentic initiation site. Point mutations were engineered in the 5'-most PRT of the chimeras, and bi-cistronic plasmids were constructed in which either the parental poliovirus 5'-UTR or the chimeric ones were introduced in the intergenic region. Upon transfection of COS-1 cells, the chimeric polio-hepatitis A 5'-UTR containing two PRTs did not express the reporter gene. Removal of the 3'-distal PRT or point mutations engineered into the 5'-most PRT partially restored the transient expression of the reporter gene, consistent with the notion that a single (and only a single) functionally active PRT in a proper context is required to secure the internal initiation of translation of bi-cistronic mRNAs in vivo.

Base Sequence↗
PubMed1998

Allelic polymorphisms in the transcriptional regulatory region of apolipoprotein E gene.

In this work, we explored the existence of genetic variants within the apolipoprotein E gene transcriptional regulatory region, using a denaturing gradient gel electrophoresis screening of a region comprising nucleotides -1017 to +406. Upon a population study, three new polymorphic sites (-491, -427 and -219) and two mutations were found. Functional effects of the polymorphisms, assayed by transient transfection and electrophoretic mobility shift assays in a human hepatoma cell line, showed that polymorphisms at sites -491 and -219 of the APOE promoter produce variations in the transcriptional activity of the gene, most probably through differential binding of nuclear proteins.

Adolescent↗