PubMed Health⌕ Search

Biomedical subjects

B Royer-Pokora

Publications and source records attributed to B Royer-Pokora.

At least 37 records · Page 2Linked to original sources
PubMed1997

Determination of functional domains of the human transcription factor PAX8 responsible for its nuclear localization and transactivating potential.

The conserved structure of the transcription factors of the Pax gene family may reflect functional conservation. We have demonstrated that the human Pax8 transcription factor is organized in several functional domains and contains two regions responsible for its nuclear localization, in addition to an activating region at the carboxy terminus of the protein and an inhibitory region encoded by the exon 9 present only in a splice variant PAX8a. Regions of PAX8 determining the nuclear localization of the PAX8A/lacZ fusions contain short amino acid sequences similar to several described nuclear localization sites (NLS). These NLS were identified in the paired domain and between the octapeptide and the residual homeodomain, respectively. The activating domain is encoded by the exons 10 and 11 and its function is modulated by the adjacent domains encoded by the exons 9 and 12. The domain encoded by exon 9 significantly inhibits the function of the activating domain. Pax8 is expressed in thyroid cells and its product binds promoters of the thyroglobulin and thyroperoxidase genes through its paired domain. Thyroid cell growth and differentiation depend on thyrotropin which, by stimulating cAMP synthesis, activates the cAMP-dependent protein kinase A (PKA). We have investigated a link between thyrotropin stimulation and gene activation by Pax8. Stimulation of cAMP synthesis augments Pax8-specific transcription in thyroid cells, indicating that PKA is involved in Pax8 activation. Cotransfection of GAL4/PAX8 fusions and the catalytic subunit of PKA in A126, a PKA-deficient derivative of the PC12 pheochromocytoma cell line, synergistically activates the GAL4-specific reporter, suggesting the activating domain of PAX8 is dependent upon the catalytic subunit of the PKA. We propose that this dependence is due to a hypothetical adaptor which forms a target for PKA and interacts with the activating domain of PAX8. We show that PAX8 isolated from the thyroid cell line FTRL5 is a phosphoprotein in which phosphorylation is not dependant on cAMP pathway activation. Our results suggest that Pax8 is part of the cAMP signaling pathway and mediates thyrotropin-dependent gene activation in thyroid cells. Investigation of the PAX8 expression in a panel of Wilms' tumors shows a striking correlation between the expression of PAX8 and another transcription factor, WT1, indicating that these two genes may interact in vivo.

Animals↗
PubMed1997

Correlation of germ-line mutations and two-hit inactivation of the WT1 gene with Wilms tumors of stromal-predominant histology.

The WT1 gene, located on chromosome 11p13, is mutated in a low number of Wilms tumors (WTs). Germ-line mutations in the WT1 gene are found in patients with bilateral WT and/or associated genital tract malformations (GU). We have identified 19 hemizygous WT1 gene mutations/deletions in 64 patient samples. The histology of the tumors with mutations was stromal-predominant in 13, triphasic in 3, blastemal-predominant in 1, and unknown in 2 cases. Thirteen of 21 patients with stromal-predominant tumors had WT1 mutations and 10 of these were present in the germ line. Of the patients with germ-line alterations, six had GU and a unilateral tumor, two had a bilateral tumor and normal GU tracts, and two had a unilateral tumor and normal GU. Three mutations were tumor-specific and were found in patients with unilateral tumors without GU. These data demonstrate a correlation of WT1 mutations with stromal-predominant histology, suggesting that a germ-line mutation in WT1 predisposes to the development of tumors with this histology. Twelve mutations are nonsense mutations resulting in truncations at different positions in the WT1 protein and only two are missense mutations. Of the stromal-predominant tumors, 67% showed loss of heterozygosity, and in one tumor a different somatic mutation in addition to the germ-line mutation was identified. These data show that in a large proportion of a histopathologically distinct subset of WTs the classical two-hit inactivation model, with loss of a functional WT1 protein, is the underlying cause of tumor development.

DNA-Binding Proteins↗
PubMed1996

A LINE element is present at the site of a 300-kb deletion starting in intron 10 of the PAX6 gene in a case of familial aniridia.

We have identified a 300-kb germline deletion in 11p13 in a family with aniridia but no Wilms' tumor. Cloning and sequencing of the breakpoint revealed that the deletion starts in intron 10 of the PAX6 gene and removes the C-terminal part of the proline-serine-threonine rich domain, leaving both DNA-binding domains intact. The PAX6 gene is joined head-to-head to a LINE-1 (L1) element. The L1 is truncated at the 3' end, removing part of ORF2. Sequencing of the L1 element shows that it does not encode a functional transposase and is therefore probably not an active element. These data suggest that the L1 element is normally present at the site of the distal deletion endpoint in 11p13. No extensive sequence homologies are detected at the deletion junction points; however, the PAX6 gene as well as the L1 element have runs of T nucleotides at this position, indicating that the deletion occurred by nonhomologous recombination. Several consensus recognition sequences for topoisomerase I flank the deletion site in both sequences, suggesting an involvement of this enzyme during the deletion-recombination process.

Amino Acid Sequence↗
PubMed1995

Expression of resistance-related proteins in nephroblastoma after chemotherapy.

Tumor tissues of untreated and cytostatic-agent-treated patients with nephroblastomas were investigated for expression of resistance-related proteins (P-glycoprotein, glutathione S-transferase-pi, glutathione peroxidase and topoisomerase II) to ascertain whether resistance proteins are changed after treatment. Tumor tissue was analyzed by means of mRNA. Twenty-three children were treated with actinomycin D and vincristine for 4 to 8 weeks. Eight children received no preoperative chemotherapy. In untreated patients, no expression of P-glycoprotein was seen, whereas, in the patients who were treated with actinomycin D and vincristine, 12 out of 23 tumors showed increased P-glycoprotein expression (> mean value). Although we found no difference between treated and untreated tumors for glutathione S-transferase-pi, we found significant differences in the expression of glutathione peroxidase. In the 8 untreated patients, 7 tumors showed low glutathione peroxidase (< mean value) and one high (> mean value) glutathione-peroxidase-mRNA content. With treatment, 11 tumors expressed low levels and 12 tumors high levels of mRNA. A significant positive correlation between P-glycoprotein and glutathione peroxidase was found. In addition, of the 8 untreated patients, 2 had low topoisomerase-II expression, and 6 high expression. With treatment, the expression was reduced in 18 tumors, and only 5 tumors had high levels of this protein. These results were confirmed by PCR and immunohistochemistry.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

A single ancestral gene of the human LIM domain oncogene family LMO in Drosophila: characterization of the Drosophila Dlmo gene.

Members of the human TTG/RBTN family, now renamed 'LMO' for LIM-only proteins, encode proteins with two tandem copies of a LIM motif. There are three members of this family, two have been isolated at the sites of chromosomal translocations in T-cell leukaemia. The function of the LIM motifs is at present unknown. We found that the LMO-2 gene is highly conserved between mammals, Drosophila and yeast. As a first step to obtain a model system for studying the function of the LIM motifs we have isolated the Drosophila homologue Dlmo. In contrast to mammals Drosophila appears to have only one lmo gene. A 2087 bp cDNA clone was isolated from a larval cDNA library, encoding a protein of 266 amino acids. A second transcript with an alternative 5' end was identified in RNA from embryos. The Drosophila lmo protein consists of two tandem copies of the conserved LIM domain characteristic of the human LMO family and an extended amino and carboxy terminus, which is not present in the human proteins. The amino acid sequence similarity with human LMO-1 and LMO-2 in LIM 1 is 79% and 69% and in LIM-2 90% and 60%, respectively. In addition a short stretch of 25 nucleotides with a homology of 83% between LMO-2 and Dlmo is found in the 3' UTR. Dlmo, like LMO-1, has an intron after the second LIM encoding region, which is not present in LMO-2. It is expressed maternally and at a high level in early embryogenesis as well as in adults. Interestingly we observed that the Dlmo protein is immunologically related to LMO-2 and can be detected by immunohistochemistry in early cellular blastoderm embryos. The gene was localised to a genetically well characterized region (17C on the X chromosome) opening the way for identification of mutations.

Adaptor Proteins, Signal Transducing↗
PubMed1995

The TTG-2/RBTN2 T cell oncogene encodes two alternative transcripts from two promoters: the distal promoter is removed by most 11p13 translocations in acute T cell leukaemia's (T-ALL).

The TTG-2 gene has been identified at the site of chromosomal translocations in acute T-cell leukemia's (T-ALL). These breakpoints map to a region between 2 and 30 kb upstream of TTG-2 in chromosome 11p13. To establish the role of these translocation breakpoints in the deregulation of TTG-2 in T-ALL we have determined the complete structure of this gene. Isolation of new TTG-2 cDNA clones from fetal liver identified an alternative transcript (TTG-2a) containing two new noncoding 5' exons. Analysis of exon/intron boundaries, identified 6 exons spread over 35 kb in 11p13. The gene encodes two alternative transcripts initiating from two promoters. TTG-2a, from promoter 1 (P1) and TTG-2b, from promoter 2 (P2) differ in the length of the 5' untranslated region, but encode the same protein. A high level of TTG-2a was present in fetal liver and spleen, whereas in adult kidney a low level of TTG-2a and a high level of TTG-2b was found. The transcription start site for TTG-2a was identified by RNase protection experiments and it displayed sequence homology to an initiator element (inr). P1 lacks a TATA box, but binding sites for SP1 and GATA-1 are present. This new genomic organisation revealed that all known chromosomal translocations map upstream of P2, removing P1 and putative upstream regulatory sequences leaving P2 intact. These results show that chromosomal translocations disrupt the TTG-2 gene itself, further confirming its role in the development of T-ALL.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Mutations of p53 in Wilms' tumors.

Mutations of p53 frequently occur in a wide variety of cancers including lung, breast, gastrointestinal, brain, and hematologic malignancies. These alterations apparently contributed to development of the malignant phenotype. Wilms' tumor is one of the most common solid tumors in childhood. The frequency of p53 alterations in this tumor is unknown. We analyzed 66 Wilms' tumor samples for p53 mutations by single-stand conformational polymorphism (SSCP) following polymerase chain reaction (PCR). Samples with an abnormal SSCP pattern were reamplified and analyzed by direct sequencing method. Mutations of p53 were found in three (5%) of 66 Wilms' tumors within the coding region (exons 2-11), showing that the frequency of p53 mutations was low. Two mutations substituted amino acids residues and one encoded a stop codon. Two of the mutations were located in the mutational hotspots (exons 5 and 6); the other was in exon 10. These data suggest that p53 mutations are infrequent in the development of Wilms' tumors.

Base Sequence↗
PubMed1994

Keratin 9 gene mutations in epidermolytic palmoplantar keratoderma (EPPK).

We have isolated the gene for human type I keratin 9 (KRT9) and localised it to chromosome 17q21. Patients with epidermolytic palmoplantar keratoderma (EPPK), an autosomal dominant skin disease, were investigated. Three KRT9 mutations, N160K, R162Q, and R162W, were identified. All the mutations are in the highly conserved coil 1A of the rod domain, thought to be important for heterodimerisation. R162W was detected in five unrelated families and affects the corresponding residue in the keratin 14 and keratin 10 genes that is also altered in cases of epidermolysis bullosa simplex and generalised epidermolytic hyperkeratosis, respectively. These findings provide further evidence that mutations in keratin genes may cause epidermolysis and hyperkeratosis and that hyperkeratosis of palms and soles may be caused by different mutations in the KRT9 gene.

Amino Acid Sequence↗
PubMed1993

Exon skipping due to a mutation in a donor splice site in the WT-1 gene is associated with Wilms' tumor and severe genital malformations.

A germline WT-1 point mutation is described in a patient with unilateral Wilms' tumor, nephritis and ambiguous external genitalia. The patient was diagnosed as a possible case of Denys Drash syndrome (DDS). Analysis of the WT-1 exons and intron borders revealed a G to C transversion in the +1 position of the splice donor consensus sequence in intron 6. Two transcripts of abnormal size were identified in tumor RNA. Sequencing of the altered WT-1 mRNA revealed that this point mutation leads to exon-skipping, resulting in transcripts either missing exon 6 or exons 5 and 6. The normally occurring alternative splicing of exon 5 in the WT-1 gene is not affected by this mutation. The reading frame is changed when either both exons 5 and 6 or exon 6 alone are missing and a stop codon follows immediately downstream in exon 7. Most mutations identified in DDS are missense mutations located in the zinc finger (ZF) region (exons 7, 8 and 9) but recently a patient with a germline mutation in exon 6 leading to premature chain termination was described. Therefore the site of the mutation in the WT-1 gene in this patient cannot exclude the possibility that he has DDS.

Base Sequence↗
PubMed1993

Localization of the human CD59 gene by fluorescence in situ hybridization and pulsed-field gel electrophoresis.

We have localized the human CD59 gene encoding a membrane protein that inhibits cell lysis by the membrane-attack complex of the homologous complement system. Using chromosomal in situ suppression hybridization and pulsed-field gel electrophoresis, we mapped this gene to chromosome 11p13, distal to the breakpoint of acute T-cell leukemia and proximal to the locus of the Wilms' tumor gene on a 30-kb SacII fragment.

Antigens, CD↗
PubMed1993

Characterization of the transcriptional regulatory region of the human WT1 gene.

Specific control of the expression of the Wilms tumor gene WT1 is important for normal development of the kidney. In order to characterise the transcriptional control region of the WT1 gene we have isolated genomic clones spanning the upstream region, the first WT1 exon and the 5' end of the Wit1 gene. DNA sequencing revealed that the WT1 promoter lacks a TATA box or CCAAT motif and has a GC content of 71%. Four transcriptional start sites are clustered within a 32 bp region. GC-boxes are present at nucleotide positions -413, -160, +84 and +158. DNAase I protection assays with purified Sp1 protein revealed the existence of 11 different binding sites in the WT1 promoter. WT1 and Wit1 promoter activities were tested in COS-7 cells with luciferase reporter gene constructs either containing or lacking an SV40 enhancer. WT1 promoter activity was found in a fragment extending from 449 bp upstream to 201 bp downstream of the WT1 start site. It was 26 fold lower in the absence of the SV40 enhancer than in the presence. Cotransfection with a Sp1 expression vector stimulated both constructs 3-4 fold. Wit1 promoter activity was identified in a DNA fragment extending from 200 bp upstream of the putative Wit1 TATA box to 130 bp downstream. Several potential recognition sites for WT1/EGR, Pax-8, and GAGA-like transcription factors are present in the WT1 promoter.

Base Sequence↗
PubMed1992

Tissue restricted expression and chromosomal localization of the YB-1 gene encoding a 42 kD nuclear CCAAT binding protein.

YB-1 cDNA clones were isolated by binding site screening of a Hela expression library using a human papillomavirus type 18 enhancer oligonucleotide. YB-1 belongs to a family of transcription factors which bind to recognition sequences containing a core CCAAT element. YB-1 bound to its single stranded recognition sequence on the sense strand but not to the anti-sense strand. A synthetic peptide antiserum derived from the predicted YB-1 amino acid sequence identified a 42 kD nuclear protein in immunoblots. A protein with the same size was detected by binding site blotting experiments using the HPV18 enhancer oligonucleotide which bound YB-1. YB-1 gene expression was restricted in tissues from a human 24 week old fetus. High levels of YB-1 mRNA were present in heart, muscle, liver, lung, adrenal gland and the brain, in contrast, low amounts of YB-1 mRNA were found in thymus, kidney, bone marrow and spleen. In pancreas, bladder, stomach and testis YB-1 mRNA could not be detected by Northern hybridization. Finally, we have identified four YB-1 related loci in the mouse genome and have mapped these loci to four different mouse chromosomes by interspecific backcross analysis.

Amino Acid Sequence↗
PubMed1992

Wilms' tumor-specific methylation pattern in 11p13 detected by PFGE.

The analysis of 2,550 kb from 11p13 in Wilms' tumor (WT) material revealed two regions that differed significantly in their methylation between tumor and normal tissue. In WT a hypomethylated area defined by an NruI and MluI recognition site 100-150 kb centromeric of the WT gene WT1 (site A) and hypermethylation defined by an NruI and SacII site 100 kb telomeric of the WT1 gene were found (site B). The degree of methylation in region B varied between 20 and 100% in the different samples; the highest level was observed in tumors without preoperative chemotherapy. The CpG island 5' of the WT1 gene was partially methylated in 2/29 tumors analyzed. The methylation state of regions A and B could reflect the normal pattern present in a subset of embryonal kidney cells, from which WT develops.

Blotting, Northern↗
PubMed1992

RNA polymerase chain reaction detects different levels of four alternatively spliced WT1 transcripts in Wilms' tumors.

A Wilms' tumor susceptibility gene (WT1) localized to 11p13 was recently isolated and shown to be altered in some sporadic Wilms' tumors. This gene encodes a DNA-binding protein with four zinc fingers (ZFs) in the carboxy-terminal region and a glutamine/proline (Gln/Pro)-rich domain near the 5' end. Two alternative splice sites were described, splice I in the Gln/Pro-rich domain (51 bp) and splice II between ZFs 3 and 4 (9 bp). Using RNA polymerase chain reaction (PCR) we show that Wilms' tumors contain all four possible transcripts, which are also identified in normal adult and embryonic kidney cells. The transcripts containing the 9-bp ZF insert were always predominant in tumors and normal cells. The presence of all four WT1 transcripts in tumors and expressing tissues suggests that each encoded protein isoform has an important role for the function of the WT1 gene.

Adult↗
PubMed1991

Direct pulsed field gel electrophoresis of Wilms' tumors shows that DNA deletions in 11p13 are rare.

In order to search for small tumor-specific deletions in 11p13 we analysed DNA isolated from 30 fresh Wilms' tumor (WT) samples with pulsed field gel electrophoresis. For these studies we have isolated new probes from the ends of several Notl fragments. Using these and previously described probes from 11p13 we first completed and extended the existing map of the 11p13 region. The analysis of the tumor material showed that (I) tumor-specific deletions were very rare: one homozygous deletion out of 30 tumors analysed, (2) hemizygous deletions were not observed in any of the tumors. The homozygous deletion in one patient spans 220 kb and is composed of a tumor-specific translocation associated with a deletion on one chromosome and a deletion of about 220 kb on the other chromosome at the same site. The WT-33 Wilms' tumor candidate gene maps to this deleted segment. A small constitutional deletion of 1,300 kb was identified in a patient with WT and genital tract malformations. These results suggest that in the majority of sporadic WT loss of gene function is due to subtle alterations in the gene, e.g., point mutations or very small deletions.

Alleles↗
PubMed1991

TTG-2, a new gene encoding a cysteine-rich protein with the LIM motif, is overexpressed in acute T-cell leukaemia with the t(11;14)(p13;q11).

We have cloned 70 kb of DNA from chromosome 11p13 at the site of a recurrent translocation in T-cell leukaemia (T-ALL): t(11;14)(p13;q11). The translocation involves the TCR-delta gene on 14q11 and a new site on 11p13. Two new and 10 previously identified translocations all mapped within 25 kb on 11p13, the 11p13 T-cell translocation cluster (11p13 ttc). A search for expressed sequences surrounding the breakpoint cluster region on 11p13 identified a gene telomeric of all breakpoints which is overexpressed in three T-ALL samples with a t(11;14). The gene T-cell translocation gene (TTG-2) encodes a small cysteine-rich protein. Forty-eight per cent of the amino acids are identical with another translocation-deregulated gene, TTG-1 (T-cell translocation gene 1 or rhombotin) in 11p15. There are two copies of a cysteine-rich motif in both proteins. Two tandem copies of the same cysteine-rich motif are also present in the recently described lin-11, isl-1 and mec-3 gene products, and one motif is found in the CRIP protein. Therefore the proteins encoded by these two translocation-deregulated genes belong to this new class of cysteine-rich proteins with the 'LIM' motif, which are important in normal development.

Amino Acid Sequence↗
PubMed1989

Molecular cloning of the translocation breakpoint in T-ALL 11;14 (p13;q11): genomic map of TCR alpha and delta region on chromosome 14q11 and long-range map of region 11p13.

Using chromosome walking techniques, overlapping lambda and cosmid clones from the T cell receptor alpha (TCR alpha) region have been isolated; these span the entire J alpha region and parts of the TCR delta gene. Molecular analysis of the acute childhood leukemia cells (T-ALL) 8511 revealed a rearrangement on one chromosome 14 in J alpha 58 kb 5' of C alpha; this does not result in production of alpha message. The translocation was identified 90 kb 5' of C alpha at the previously identified J delta 2 element. A probe derived from the 5' region of the translocation breakpoint hybridized to DNA from a mouse-human cell hybrid containing chromosome 11 as the only human chromosome. This probe was used to isolate cosmid clones from chromosome 11. Several rare cutting restriction enzyme sites were found in close vicinity to the translocation breakpoint, and a long-range map spanning 1000 kb of chromosome region 11p13 was established. Analysis of the DNA from 15 cases of sporadic and familial Wilms' tumor did not reveal any changes, indicating that the translocation breakpoint does not reside in this gene.

Blotting, Southern↗