Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Germline alterations of the human von Hippel-Lindau (VHL) tumor suppressor gene predispose to renal cell carcinoma and a constellation of other tumor types found in VHL disease. This gene is also mutated or deleted in a high proportion of sporadic nonpapillary renal cell carcinomas. In the Eker rat model, spontaneous renal cell carcinoma develops with a high frequency. We therefore investigated the role of this tumor suppressor gene in the development of these hereditary rat tumors. By using reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis, the sequence of the rat VHL gene was determined over the portion of the gene homologous to regions where most mutations in the human VHL gene occur. The sequence homology was 90% and the amino-acid identity 99% between the rat and human genes. A developmental and tumor-specific pattern of expression for the VHL gene was found; a ubiquitous 3.2-kb transcript was expressed in all rat tissues examined (neonatal kidney, lung, liver, brain, heart, kidney, spleen, testis, and stomach), and an additional 4.5-kb transcript was expressed in neonatal kidney and cell lines derived from Eker rat renal cell carcinomas (ERC cell lines). To determine whether mutations in the VHL gene were involved in tumor development in the Eker model, RT-PCR, single-strand conformation polymorphism (SSCP) analysis, and direct sequencing were used to search for alterations in this gene in the ERC cell lines. Alterations in the VHL gene were not detected by SSCP, and these data were confirmed by direct sequencing. Transformed rat kidney epithelial cell lines derived from Fisher rats also expressed the VHL gene but like the ERC cell lines did not contain mutations in the VHL gene. These data indicate that in the rat, transformation of kidney epithelial cells and the development of solid, nonpapillary renal cell carcinoma can occur via pathways that are independent of alterations at the VHL gene locus.
Mol Carcinog 1996 Feb
PMID:Renal cell carcinoma development in the rat independent of alterations at the VHL gene locus. 859 82

The von Hippel-Lindau disease (VHL) gene is a putative tumor suppressor gene responsible for VHL, an autosomal dominantly inherited multitumor syndrome. It is also implicated in the development of sporadic tumors including clear cell renal carcinoma and central nervous system hemangioblastoma. To define the molecular basis of VHL patients in Japanese populations, we tested for germline mutations of the VHL gene in 45 unrelated Japanese VHL patients by single-strand conformation polymorphism (SSCP) analysis and Southern blot analysis. We detected 23 (51%) intragenic mutations and three (6.7%) deletions by SSCP analysis and Southern blot respectively. The intragenic mutations consisted of 14 missense mutations, seven microdeletions or insertions and two splice-site mutations. Interestingly, nine of 10 mutations in exon 1 are localized in a short region of 37 nucleotides. Five unique sites of mutation were included, which were not seen in previous studies. Unlike Western VHL patients, nonsense mutations were not found in Japanese VHL patients. If the presence of pheochromocytomas is regarded as phenotypic marker for VHl classification, the mutations found in 22 VHL patients without pheochromocytoma consisted of 11 missense mutations, six microdeletions or insertions, two splice-site alterations and three deletions. The mutations found in four VHL patients with pheochromocytomas consisted of one missense mutation at nucleotide 683 (codon 228), two missense mutations at nucleotide 712 (codon 238) and a novel 20 bp insertion at nucleotide 776 (codon 259). Although the mutations at codon 238 are the mutational hot spot found in Western VHL patients with pheochromocytomas, a 20 bp insertion of original VHL cDNA sequence, from nucleotide 777 to 796, is a unique mutation. Our results suggest that mutations in Japanese VHL patients contain some unique features compared with those in Western patients. VHL gene has a critical role for the etiology in VHL in Japanese populations as well as Western VHL.
Hum Mol Genet 1995 Dec
PMID:Germline mutations in the von Hippel-Lindau disease (VHL) gene in Japanese VHL. Clinical Research Group for VHL in Japan. 863 92

The thermodynamics of the binding of the Sac7d protein of Sulfolobus acidocaldarius to double-stranded DNA has been characterized using spectroscopic signals arising from both the protein and the DNA. Ligand binding density function analysis has been used to demonstrate that the fractional change in protein intrinsic tryptophan fluorescence quenching that occurs upon DNA binding is equal to the fraction of protein bound. Reverse titration data have been fit directly to the McGhee-von Hippel model [McGhee, J., & von Hippel, P. (1974) J. Mol. Biol. 86, 469-489] using nonlinear regression. Sac7d binds noncooperatively to poly(dGdC) x poly(dGdC) with an intrinsic affinity of 6.5 x 10(6) M(-1) and a site size of 4 base pairs in 1 mM KH2PO4 and 50 mM KC1 (pH 6.8). Some binding sequence preference is noted, with the binding to poly(dIdC) x poly(dIdC) over 10-fold stronger than to poly(DAdT) x poly(dAdT). The binding is largely driven by the polyelectrolyte effect and is consistent with a release of 4.4 monovalent cations from DNA upon complex formation or the formation of 5 ion pairs at the protein-DNA interface. Extrapolation of salt back-titration data to 1 M KC1 indicates a -2.2 kcal/mol nonelectrostatic contribution to the binding free energy. A van't Hoff analysis of poly(dGdC) x poly(dGdC) binding shows that the binding enthalpy is approximately zero and the process is entropically driven. The affinity decreases slightly between pH 5.4 and 8.0. There is no significant difference between the binding parameters of recombinant Sac7d and native Sac7 proteins, indicating that methylation of the native protein has no effect on the DNA binding function. The binding of Sac7d to various DNAs leads to a significant increase in the DNA long-wavelength circular dichroism (CD) band, the intensity of which shows a sigmoidal dependence on Sac7d concentration. The sigmoidal CD binding isotherm can be quantitatively modeled by a conformational transition in the DNA that is cooperatively induced when protein monomers are bound within a given number of base pairs, ranging from zero for poly(dIdC) x poly(dIdC) to 8 or less for poly(dAdG) x poly(dCdT).
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PMID:Equilibrium DNA binding of Sac7d protein from the hyperthermophile Sulfolobus acidocaldarius: fluorescence and circular dichroism studies. 867 37

We have examined the formation of the primosome subassembly of the bacteriophage T4-coded DNA replication (elongation) complex from its helicase, primase, and DNA components. Previously, we had shown that the T4 helicase (gp41) exists in solution in a stable monomer left and right arrow dimer equilibrium at physiological protein (and salt) concentrations and forms a hexamer upon activation by ATP (or GTP) binding (Dong, F., Gogol, E. P., and von Hippel, P. H.(1995) J. Biol. Chem. 270, 7462-7473). Here we report that the T4 primase (gp61) is a monomer in solution under the same conditions, and that the ATP-activated helicase binds to a single gp61 primase molecule on appropriate DNA templates to reconstitute a stable primosome. We show that: (i) the gp41 helicase alone does not form a stable complex with DNA templates, although this helicase by itself can carry out moderately processive ATP-driven translocation along single-stranded DNA (Young, M. C., Schultz, D. E., Ring, D., and von Hippel, P. H.(1994) J. Mol. Biol. 235, 1447-1458); (ii) the primase alone does form a stable complex with DNA; (iii) the helicase can bind to the primase-DNA complex in the presence of ATP or GTP to form a stable ternary complex; (iv) this complex consists of six helicase subunits and one primase subunit; and (v) the reconstituted primosome is stable for at least 10 to 20 min after NTP cleavage and dissociation of the hydrolysis products. These results strongly suggest that the functional T4 DNA replication primosome consists of an integrated 6:1 helicase-primase complex bound to DNA, and that the ATP-activated helicase hexamer remains intact throughout the processive DNA replication process.
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PMID:The ATP-activated hexameric helicase of bacteriophage T4 (gp41) forms a stable primosome with a single subunit of T4-coded primase (gp61). 870 59

The von Hippel-Lindau (VHL) disease product is thought to down-regulate transcription by antagonizing elongin-enhanced transcriptional elongation. Germline VHL gene mutations predispose to the development of retinal, cerebellar and spinal haemangioblastomas, renal cell carcinoma and phaeochromocytoma. In addition, somatic Inactivation of the VHL gene is frequent in sporadic renal cell carcinoma and haemangioblastoma. Regulation of transcript elongation is an important control mechanism for gene expression and the VHL gene might modify the expression of proto-oncogenes and growth suppressor genes during embryogenesis. We therefore investigated the expression of VHL mRNA during human embryogenesis by in situ hybridization studies at 4, 6 and 10 weeks post conception. Although VHL mRNA was expressed in all three germ layers, strong expression was noted in the central nervous system, kidneys, testis and lung. Within the kidney, VHL mRNA was differentially expressed within renal tubules suggesting that the VHL gene product may have a specific role in kidney development. Two alternatively spliced VHL mRNAs characterized by inclusion (isoform I) or exclusion (isoform II) of exon 2 are transcribed in adult tissues. To investigate if the two isoforms are differentially expressed during embryogenesis, VHL mRNA was reverse transcribed from 13 fetal tissues (8-10 weeks gestation). The quantitative distribution of VHL mRNA within fetal tissues reflected that seen by in situ hybridization and the ratio of the two VHL isoforms was similar between tissues. Although the genes regulated by the VHL gene product have not yet been identified, our findings are compatible with the hypothesis that VHL-mediated control of transcriptional elongation may have a role in normal human development.
Hum Mol Genet 1996 May
PMID:Expression of the von Hippel-Lindau disease tumour suppressor gene during human embryogenesis. 873 31

The importance of computer-assisted analysis of a non-linear binding phenomenon through Scatchard equation has been widely acknowledged. While several user-friendly softwares [LIGAND, SCTFIT, ALLFIT] are available for determining the binding parameters of nonlinear Scatchard phenomenon, there is no easily available software covering the class of phenomena described by the McGhee and von Hippel formalism [J. Mol. Biol. 86, 469-489 (1974)]. We report here user-friendly software, SCATPLOT, developed in Turbo BASIC, for the numerical estimation of binding parameters of a non-cooperative ligand-substrate interaction doing best fit to the experimental data on the basis of McGhee and von Hippel equation for a nonlinear Scatchard plot. A new parameter has also been incorporated to guide the process of least square analysis and subsequent determination of binding parameters.
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PMID:SCATPLOT: a computer program for determination of binding parameters of non-linear non-cooperative ligand-substrate interactions. 899 43

Inherited predisposition to phaeochromocytoma (MIM No 171300) occurs in multiple endocrine neoplasia type 2 (MEN 2) (MIM No 171400), von Hippel-Lindau (VHL) disease (MIM No 199300), and neurofibromatosis type 1 (NF1) (MIM No 162200). In addition, familial phaeochromocytoma alone has also been reported and we and others have identified germline VHL mutations in five of six kindreds analysed previously. Germline mutations in the RET proto-oncogene, which encodes a receptor tyrosine kinase, and in the VHL tumour suppressor gene cause MEN 2 and VHL disease, respectively. To further investigate the genetics of phaeochromocytoma predisposition, we analysed three groups of patients with no evidence of VHL disease, MEN 2 or NF1: Group A, eight kindreds with familial phaeochromocytoma; Group B, two patients with isolated bilateral phaeochromocytoma; and Group C, six cases of multiple extra-adrenal phaeochromocytoma or adrenal phaeochromocytoma with a family history of neuroectodermal tumours. Germline missense VHL mutations were identified in three of eight kindreds with familial phaeochromocytoma. A germline VHL mutation was also characterised in one of the two patients with bilateral phaeochromocytoma. No VHL or RET mutations were detected in the final group of patients with multiple extra-adrenal phaeochromocytoma or adrenal phaeochromocytoma with a family history of neuroectodermal tumours. The absence of germline VHL and RET gene mutations in many of these families suggested that other phaeochromoeytoma susceptibility loci may exist. Glial cell line-derived neurotrophic factor (GDNF) has been recently identified as a natural ligand for RET. Thus, it seems plausible that GDNF is a good candidate gene to play a role in phaeochromocytoma susceptibility. We searched for germline mutations in GDNF in 16 cases of familial phaeochromocytoma (groups A, B and C) and looked for evidence of somatic change in GDNF in 28 sporadic phaeochromocytomas, 12 MEN 2 phaeochromocytomas and five VHL phaeochromocytomas. No GDNF mutations were identified in patients with familial phaeochromocytoma disease, but a c277C-->T (R93W) sequence variant was identified in one of 28 sporadic tumours. This candidate mutation was identified in the germline and tumour tissue but was not present in 104 control GDNF alleles. GDNF sequence variants including R93W have been suggested previously to represent low penetrance susceptibility mutations for Hirschsprung disease and the R93W was not identified in 376 control alleles studied by others. These findings suggest that although GDNF mutations do not appear to have a major role in the pathogenesis of familial or sporadic phaeochromocytomas, allelic variation at the GDNF locus may modify phaeochromocytoma susceptibility.
Hum Mol Genet 1997 Jul
PMID:Genetic predisposition to phaeochromocytoma: analysis of candidate genes GDNF, RET and VHL. 921 74

The von Hippel-Lindau tumor suppressor gene (VHL) has a critical role in the pathogenesis of clear-cell renal cell carcinoma (RCC), as VHL mutations have been found in both von Hippel-Lindau disease-associated and sporadic RCCs. Recent studies suggest that vascular endothelial growth factor (VEGF) mRNA is upregulated in RCC- and von Hippel-Lindau disease-associated tumors. We have therefore assessed the effect of the VHL gene product on VEGF expression. VEGF promoter-luciferase constructs were transiently cotransfected with a wild-type VHL (wt-VHL) vector in several cell lines, including 293 embryonic kidney and RCC cell lines. wt-VHL protein inhibited VEGF promoter activity in a dose-dependent manner up to 5- to 10-fold. Deletion analysis defined a 144-bp region of the VEGF promoter necessary for VHL repression. This VHL-responsive element is GC rich and specifically binds the transcription factor Sp1 in crude nuclear extracts. In Drosophila cells, cotransfected VHL represses Sp1-mediated activation but not basal activity of the VEGF promoter. We next demonstrated in coimmunoprecipitates that VHL and Sp1 were part of the same complex and, by using a glutathione-S-transferase-VHL fusion protein and purified Sp1, that VHL and Sp1 directly interact. Furthermore, endogenous VEGF mRNA levels were suppressed in permanent RCC cell lines expressing wt-VHL, and nuclear run-on studies indicated that VHL regulation of VEGF occurs at least partly at the transcriptional level. These observations support a new mechanism for VHL-mediated transcriptional repression via a direct inhibitory action on Sp1 and suggest that loss of Sp1 inhibition may be important in the pathogenesis of von Hippel-Lindau disease and RCC.
Mol Cell Biol 1997 Sep
PMID:The von Hippel-Lindau tumor suppressor gene product interacts with Sp1 to repress vascular endothelial growth factor promoter activity. 927 38

von Hippel-Lindau (VHL) gene mutations occur throughout three exons including the exon-intron boundaries in human VHL disease-associated and sporadic renal cell carcinomas. To explore the possible role of the VHL gene in chemically induced rat kidney tumors originating from various cell types, more than 150 bp of Fischer 344 and Noble rat VHL intron sequences flanking the three exons was determined by dideoxy sequencing. Five primer sets were selected for polymerase chain reaction amplification of the coding regions of rat VHL exons 1-3 and the exon-intron boundaries. Tissues from 10 renal eosinophilic epithelial tumors induced by N-nitrosoethyl(2-hydroxyethyl)amine, 10 nephroblastomas induced by N-nitroso-N-ethylurea, and seven renal mesenchymal tumors induced by N-nitrosomethyl(methoxymethyl)amine were examined for VHL mutations by polymerase chain reaction-single-strand conformation polymorphism analysis. No mutation was detected in any tumor type, indicating that VHL mutations are not involved in the pathogenesis of rat kidney tumors arising from the distal region of the renal tubules, the metanephric blastema, or stromal tissues of the cortex.
Mol Carcinog 1997 Aug
PMID:Polymerase chain reaction-single-strand conformation polymorphism analysis for the VHL gene in chemically induced kidney tumors of rats using intron-derived primers. 929 Jun 99

We used isothermal titration calorimetry and fluorescence spectroscopy to investigate the thermodynamics of non-sequence-specific DNA-binding by the Sso7d protein from the archaeon Sulfolobus solfataricus. We report the Sso7d-poly(dGdC) binding thermodynamics as a function of buffer composition (Tris-HCl or phosphate), temperature (15 to 45 degrees C), pH (7.1 to 8.0), osmotic stress and solvent (H2O/2H2O), and compare it to poly (dAdT) binding; and we have previously also reported the salt concentration dependence. Binding isotherms can be represented by the McGhee-von Hippel model for non-cooperative binding, with a binding site size of four to five DNA base-pairs and binding free energies in the range DeltaG degrees approximately -7 to DeltaG degrees approximately -10 kcal mol-1, depending on experimental conditions. The non-specific nature of the binding is reflected in similar thermodynamics for binding to poly(dAdT) and poly(dGdC). The native lysine methylation of Sso7d has only minor effects on the binding thermodynamics. Sso7d binding to poly(dGdC) is endothermic at 25 degrees C with a binding enthalpy DeltaH degrees approximately 10 kcal mol-1 in both phosphate and Tris-HCl buffers at pH 7.6, indicating that DeltaH degrees does not include large contributions from coupled buffer ionization equilibria at this pH. The binding enthalpy is temperature dependent with a measured heat capacity change DeltaCp degrees=-0.25(+/-0.01) kcal mol-1 K-1 and extrapolations of thermodynamic data indicate that the complex is heat stable with exothermic binding close to the growth temperature (75 to 80 degreesC) of S. solfataricus. Addition of neutral solutes (osmotic stress) has minor effects on DeltaG degrees and the exchange of H2O for 2H2O has only a small effect on DeltaH degrees, consistent with the inference that complex formation is not accompanied by net changes in surface hydration. Thus, other mechanisms for the heat capacity change must be found. The observed thermodynamics is discussed in relation to the nature of non-sequence-specific DNA-binding by proteins.
J Mol Biol 1998 Mar 06
PMID:Thermodynamic characterization of non-sequence-specific DNA-binding by the Sso7d protein from Sulfolobus solfataricus. 950 Sep 18


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