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Biomedical subjects

W Doerfler

Publications and source records attributed to W Doerfler.

At least 37 records · Page 2Linked to original sources
PubMed2000

Methylation mosaicism of 5'-(CGG)(n)-3' repeats in fragile X, premutation and normal individuals.

Fragile X syndrome (FRAXA) is characterized at the molecular level by an expansion of a naturally occurring 5'-(CGG)(n)-3' repeat in the promoter and 5'-untranslated region (5'-UTR) of the fragile X mental retardation (FMR1) gene on human chromosome Xq27.3. When expanded, this region is usually hypermethylated. Inactivation of the FMR1 promoter and absence of the FMR1 protein are the likely cause of the syndrome. By using the bisulfite protocol of the genomic sequencing method, we have determined the methylation patterns in this region on single chromosomes of healthy individuals and of selected premutation carriers and FRAXA patients. In control experiments with unmethylated or M- Sss I-premethylated DNAs, this protocol has been ascertained to reliably detect all cytidines or 5-methylcytidines as unmethylated or methylated nucleotides, respectively. Analyses of the DNA from FRAXA patients reveal considerable variability in the lengths of the 5'-(CGG)(n)-3' repeats and in the levels of methylation in the repeat and the 5'-UTR. In one patient (OEl) with high repeat length hetero-geneity ( n = 15 to >200), shorter repeats (n = 20-80) were methylated or unmethylated, longer repeats ( n = 100-150) were often completely methylated, but one repeat with n = 160 proved to be completely unmethylated. This type of methylation mosaicism was observed in several FRAXA patients. In healthy females, methylated 5'-CG-3' sequences were found in some repeats and 5'-UTRs, as expected for the sequences from one of the X chromosomes. The natural FMR1 promoter is methylation sensitive, as demonstrated by the loss of activity in transfection experiments using the unmethylated or M- Sss I-premethylated FMR1 promoter fused to the luciferase gene as an activity indicator.

5' Untranslated Regions↗
PubMed2000

Neurological phenotype in Waardenburg syndrome type 4 correlates with novel SOX10 truncating mutations and expression in developing brain.

Waardenburg syndrome type 4 (WS4), also called Shah-Waardenburg syndrome, is a rare neurocristopathy that results from the absence of melanocytes and intrinsic ganglion cells of the terminal hindgut. WS4 is inherited as an autosomal recessive trait attributable to EDN3 or EDNRB mutations. It is inherited as an autosomal dominant condition when SOX10 mutations are involved. We report on three unrelated WS4 patients with growth retardation and an as-yet-unreported neurological phenotype with impairment of both the central and autonomous nervous systems and occasionally neonatal hypotonia and arthrogryposis. Each of the three patients was heterozygous for a SOX10 truncating mutation (Y313X in two patients and S251X [corrected] in one patient). The extended spectrum of the WS4 phenotype is relevant to the brain expression of SOX10 during human embryonic and fetal development. Indeed, the expression of SOX10 in human embryo was not restricted to neural-crest-derived cells but also involved fetal brain cells, most likely of glial origin. These data emphasize the important role of SOX10 in early development of both neural-crest-derived tissues, namely melanocytes, autonomic and enteric nervous systems, and glial cells of the central nervous system.

Autonomic Nervous System↗
PubMed2000

The human 20-kDa 5'-(CGG)(n)-3'-binding protein is targeted to the nucleus and affects the activity of the FMR1 promoter.

Previous reports have described the human DNA CGG repeat-binding protein (CGGBP1 or p20), which binds specifically to nonmethylated, but not to methylated, 5'-(CGG)(n)-3' repeats in the promoter of the fragile X mental retardation 1 (FMR1) gene. The results of transfection experiments into human HeLa cells using a p20-green fluorescent protein fusion construct indicate that the p20 protein is targeted to the nucleus. By deletion analyses, a nuclear localization signal has been found between amino acids 80 and 84. Deletions between amino acids 69 and 71 and between 95 and 167 interfere with 5'-(CGG)(n)-3' binding. The results of electrophoretic mobility shift assays using DNA with 5'-(CGG)(n)-3' repeats of different lengths render it likely that oligomers of the p20 protein bind to the repeat. In cotransfection experiments, the activity of the FMR1 promoter is reduced by the presence of p20. Upon transfection of the p20 cDNA construct into HeLa cells, transcription of the endogenous FMR1 gene is decreased. The green fluorescent protein-p20 fusion protein associates preferentially with the telomeres of the short arms of human chromosomes 13, 14, 15, 21, and 22. Their telomeres carry the genes for the 28 S rRNA, which contain 5'-(CGG)(n)-3' repeats. The translated region of the p20 gene from three healthy, five fragile X syndrome, and five premutation-carrying individuals has been sequenced, but mutations have not been detected.

Amino Acid Sequence↗
PubMed2000

Characteristic features of the genotype and phenotype of hereditary spherocytosis in the Japanese population.

Hereditary spherocytosis (HS) is the most common hemolytic anemia of congenital origin in the Japanese population. Among 844 cases of 520 kindred with congenital red cell membrane disorders studied at the Kawasaki Medical School in the last 25 years (1975-1999), 407 cases (48.2%) of 215 kindred had HS. Among the recent 60 kindred with HS, autosomal dominant (AD) transmission was proven in 19. The remaining 41 non-AD HS included 1) homozygous patients with autosomal recessive inheritance, 2) HS patients with de novo gene mutations, and 3) mild HS with AD inheritance. The extent of clinical severity in the non-AD HS cases was nearly identical to that in the AD cases. The incidence of membrane protein abnormalities in our 60 Japanese HS kindred was unique: there were lower ankyrin deficiencies (7%), moderate band 3 deficiencies (20%), and much higher protein 4.2 deficiencies (45%), with 28% of unknown etiology. The incidence of membrane protein deficiencies corresponded to that determined by gene analyses; i.e., mutations mostly in band 3 and/or in protein 4.2 genes and fewer ankyrin gene mutations. In the band 3 gene, 11 mutations pathognomonic for HS were identified (3 frameshift and 8 missense mutations). There were 5 mutations of the protein 4.2 gene (3 missense mutations, 1 nonsense mutation, and 1 splicing mutation) pathognomonic for HS. On the other hand, 2 missense mutations were detected in the ankyrin gene in this study. The genetic abnormalities in our HS patients correlated well with the phenotypic ultrastructural abnormalities of red cell membranes in situ. Ankyrin mutations (ankyrin Marburg and ankyrin Stuttgart with frameshift mutations) were associated mostly with a disrupted cytoskeletal network, and band 3 mutations (band 3 Kagoshima with frameshift mutation) typically demonstrated anomalies of intramembrane particles (IMPs). Protein 4.2 mutations (homozygotes of protein 4.2 Nippon) with complete protein 4.2 deficiency showed abnormalities of both the cytoskeletal network and IMPs.

Animals↗
PubMed1999

Factors affecting de novo methylation of foreign DNA in mouse embryonic stem cells.

Integration of foreign DNA into an established host genome can lead to changes in methylation in both the inserted DNA and in host sequences and potentially alters transgene and cellular transcription patterns. This work addresses the questions of what factors influence de novo methylation, and whether the integration site or inserted DNA can affect de novo methylation. Homologous recombination was used to integrate foreign DNA into a specific gene, B lymphocyte kinase (BLK), in mouse embryonic stem (ES) cells. Two plasmids were chosen for integration; one contained the adenovirus type 2 E2AL promoter upstream of the luciferase reporter gene, and the second carried the early SV40 promoter. The methylation patterns were analyzed using HpaII and MspI restriction endonucleases for both homologously recombined and randomly integrated foreign DNA in the ES cell clones. Upon homologous reinsertion of the BLK gene into the genome of mouse ES cells, methylation patterns in this gene were reestablished. In DNA segments adjoined to the BLK gene, the de novo patterns of DNA methylation depended on the viral sequences in these clones and on the locations of the inserts, i.e. on whether the insertions resulted from homologously recombined or randomly integrated foreign DNA. In homologously recombined DNA, sequences carrying the adenovirus type 2 promoter were heavily methylated, and those with an SV40 promoter and an SV40 enhancer element remained unmethylated or hypomethylated. Upon removal of the enhancer element, these inserted constructs also became heavily methylated. In addition, all randomly integrated constructs were heavily methylated independently of the promoter and enhancer element present in the construct. These results indicate that modes and sites of integration as well as the inserted nucleotide sequence, possibly promoter strength, are factors affecting de novo methylation.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Integrated viral genomes can be lost from adenovirus type 12-induced hamster tumor cells in a clone-specific, multistep process with retention of the oncogenic phenotype.

In adenovirus type 12 (Ad12)-induced tumor cells, in Ad12-transformed cells and in continuously passaged cell lines from these sources, the viral DNA is integrated in multiple copies, usually at a single chromosomal location. In different tumors or cell lines, the sites of integration of Ad12 DNA are all different. Rare exceptions exist. In most instances, the integrated viral DNA resides very stably in the host cell genomes. However, upon continuous serial passage of such cell lines, the integrated viral DNA can be destabilized and lost. In two instances, i.e. in the Ad12-induced hamster tumor cell lines H1111(1) and CLAC1, we have investigated the loss of integrated viral DNA in detail. After extended serial passage, these two cell lines seemed to be devoid of Ad12 DNA sequences, as detectable by Southern blot hybridization, but continued to induce tumors after reinjection into hamsters. Cells from these two cell lines were now recloned three times, and DNAs from cultures derived from several individual clones were reinvestigated for the presence of several parts of the viral genome by the polymerase chain reaction (PCR). Some of the clones still carried parts of the Ad12 genome. However, several clones were isolated that proved free of all parts of the viral genome, except for minute segments from the right terminus of the Ad12 genome. Apparently, the loss of integrated viral DNA from these cell lines proceeded as a continuous, gradual, multistep process whose pattern could differ from cell clone to cell clone, once destabilization had been initiated. The mechanism of destabilization is not understood. Cell populations of 2 x 10(6) to 3 x 10(7), and as low as 10(2), cells from the clones, that contained only minimal remnants from the right viral DNA terminus, were reinjected into newborn or 13-20 day-old weanling Syrian hamsters (Mesocricetus auratus). Tumors developed within 5-17 days after injection. Tumor cell clones also grew in soft agar. The injection of primary hamster skin fibroblasts never elicited tumor formation. The tumor cells induced by this reinjection proved repeatedly free of Ad12 DNA both by Southern blot hybridization and by PCR, except for those cell and tumor clones that contained small segments of the right terminal E4 region of the Ad12 genome. The tumor cells, however, retained their oncogenic phenotype. The results raise questions about the cell clone-specific excision patterns of integrated foreign DNA from the recipient genome and the possibility of a hit-and-run mechanism of adenoviral oncogenesis.

Adenoviruses, Human↗
PubMed1999

Insertion of foreign DNA into an established mammalian genome can alter the methylation of cellular DNA sequences.

The insertion of adenovirus type 12 (Ad12) DNA into the hamster genome and the transformation of these cells by Ad12 can lead to marked alterations in the levels of DNA methylation in several cellular genes and DNA segments. Since such alterations in DNA methylation patterns are likely to affect the transcription patterns of cellular genes, it is conceivable that these changes have played a role in the generation or the maintenance of the Ad12-transformed phenotype. We have now isolated clonal BHK21 hamster cell lines that carry in their genomes bacteriophage lambda and plasmid pSV2neo DNAs in an integrated state. Most of these cell lines contain one or multiple copies of integrated lambda DNA, which often colocalize with the pSV2neo DNA, usually in a single chromosomal site as determined by the fluorescent in situ hybridization technique. In different cell lines, the loci of foreign DNA insertion are different. The inserted bacteriophage lambda DNA frequently becomes de novo methylated. In some of the thus-generated hamster cell lines, the levels of DNA methylation in the retrotransposon genomes of the endogenous intracisternal A particles (IAP) are increased in comparison to those in the non-lambda-DNA-transgenic BHK21 cell lines. These changes in the methylation patterns of the IAP subclone I (IAPI) segment have been documented by restriction analyses with methylation-sensitive restriction endonucleases followed by Southern transfer hybridization and phosphorimager quantitation. The results of genomic sequencing experiments using the bisulfite protocol yielded additional evidence for alterations in the patterns of DNA methylation in selected segments of the IAPI sequences. In these experiments, the nucleotide sequences in >330 PCR-generated cloned DNA molecules were determined. Upon prolonged cultivation of cell lines with altered cellular methylation patterns, these differences became less apparent, perhaps due to counterselection of the transgenic cells. The possibility existed that the hamster BHK21 cell genomes represent mosaics with respect to DNA methylation in the IAPI segment. Hence, some of the cells with the patterns observed after lambda DNA integration might have existed prior to lambda DNA integration and been selected by chance. A total of 66 individual BHK21 cell clones from the BHK21 cell stock have been recloned up to three times, and the DNAs of these cell populations have been analyzed for differences in IAPI methylation patterns. None have been found. These patterns are identical among the individual BHK21 cell clones and identical to the patterns of the originally used BHK21 cell line. Similar results have been obtained with nine clones isolated from BHK21 cells mock transfected by the Ca2+-phosphate precipitation procedure with DNA omitted from the transfection mixture. In four clonal sublines of nontransgenic control BHK21 cells, genomic sequencing of 335 PCR-generated clones by the bisulfite protocol revealed 5'-CG-3' methylation levels in the IAPI segment that were comparable to those in the uncloned BHK21 cell line. We conclude that the observed changes in the DNA methylation patterns in BHK21 cells with integrated lambda DNA are unlikely to preexist or to be caused by the transfection procedure. Our data support the interpretation that the insertion of foreign DNA into a preexisting mammalian genome can alter the cellular patterns of DNA methylation, perhaps via changes in chromatin structure. The cellular sites affected by and the extent of these changes could depend on the site and size of foreign DNA insertion.

Animals↗
PubMed1998

A 5'-CG-3'-rich region in the promoter of the transcriptionally frequently silenced RET protooncogene lacks methylated cytidine residues.

In a large proportion of familial and sporadic cases of Hirschsprung disease (HSCR) mutations in the RET (rearranged during transfection) protooncogene have been described. We have investigated the structure of the RET gene promoter and have analysed a region of approximately 1000 nucleotides in its promoter and 5'-upstream segments for the occurrence of 5-methyldeoxycytidine (5-mC) residues by using the bisulfite protocol of the genomic sequencing method. With an estimated sensitivity of about 93% of this technique, not a single 5-mC residue could be detected in the control region of a gene that seems to be silenced or exhibit low activity in many adult tissues. In these experiments, the DNAs of peripheral white blood cells (PWBC) from four healthy individuals, from seven patients with familial HSCR, as well as DNAs from different human tissues and from a human embryonic kidney (HEK) cell line have been included. In a DNA segment starting 790 nucleotides upstream of the transcriptional start site of the RET gene, a few 5-mC residues have been identified. This region possibly constitutes the transition site from an unmethylated promoter to a more extensively methylated region in the human genome. The data presented are remarkable in that a highly 5'-CG-3'-enriched segment of the human genome with 49 5'-CG-3' dinucleotide pairs in 400 bp within the putative promoter region is completely devoid of 5-mC residues, although this control region is not actively transcribed in most adult human tissues. By hybridization of a PCR-amplified RET protooncogene cDNA probe harboring exons 9-15 to a membrane (Clontech) containing poly-A selected RNAs from 50 different human tissues, weak RET protooncogene expression in many of the neural cell derived tissues has been detected. RNAs extracted from many other human tissues do not share sequence homologies to this 32P-labeled probe. Mechanisms other than DNA methylation obviously play the crucial role in the inactivation of the RET gene promoter in these tissues. We have also demonstrated by the in vitro premethylation of a RET promoter-chloramphenicol acetyltransferase (CAT) gene construct and transient transfection experiments into neuroblastoma cells that the transcriptional activity of the RET promoter is decreased by HpaII (5'-CCGG-3') methylation and abolished by SssI (5'-CG-3') methylation. Hence, the RET promoter region is sensitive to this regulatory signal. However in vivo, DNA methylation of the promoter region seems not to be the predominant regulatory mechanism for the RET protooncogene. Possibly, in adults the RET gene can be occasionally activated.

Adult↗
PubMed1998

New 5'-(CGG)n-3' repeats in the human genome.

We identified new, potentially unstable loci in the human genome containing 5'-(CGG)n-3' trinucleotide repeats by screening a human subgenomic library as well as a chromosome 16 library with a 5'-(CGG)17-3' oligodeoxyribonucleotide probe. Five different clones were isolated, two from the chromosome 16 library and three from the subgenomic library. Determinations of the nucleotide sequences have revealed that the E7 clone displayed, in addition to the 5'-(CGG)n-3' trinucleotide repeat, a 5'-(CAG)n-3' and a 5'-(CCT)n-3' trinucleotide repeat. Two clones, CL16-1 and P5-5, had homologies to known genes, the human casein kinase II alpha' subunit (chromosome 16) and the human calcium-activated potassium channel (chromosome 10), respectively. Clones E7 and P4 were assigned to chromosome 6, whereas CL16-8 mapped to chromosome 16. Their potential coding capacities were assessed by RNA transfer (Northern blotting) experiments. Four different transcripts were identified by using the E7 clones as hybridization probes, three of them being brain-specific. The P4 clone was expressed in placenta and skeletal muscle. Minor polymorphisms within the repeats were observed in normal and in fragile X individuals. Lung and colon carcinoma cell lines in which some microsatellites were shown to be unstable were also investigated. Expansions of the 5'-(CGG)n-3' repeats were not found.

Alleles↗
PubMed1998

DNA fragments with specific nucleotide sequences in their single-stranded termini exhibit unusual electrophoretic mobilities.

DNA restriction fragments, 120-650 base pairs (bp) in length, with 5'-GCGC-3', 5'-GGCC-3' or 3'-GCGC-5' single-stranded overhanging termini, give rise to diffuse bands of unusual electrophoretic mobility in non-denaturing polyacrylamide gels. This shift in electrophoretic mobility can be observed at 4-12 degreesC, not at higher temperatures, but is stabilized by 5-10 mM Mg2+, even at 37 degreesC. The nucleotide sequence in the abutting double-stranded part of the fragment does not affect this phenomenon, which is not caused by dimerization. The altered mobility may be due to the unusual terminal DNA structure, which is dependent on co-operative interactions among more than two neighboring G and C residues. The structure is stabilized by cytidine methylation. The biological role of such fragment structures in DNA repair and recombination is presently unknown.

Base Composition↗
PubMed1998

Uptake of foreign DNA from the environment: the gastrointestinal tract and the placenta as portals of entry.

Foreign DNA (deoxyribonucleic acid) is part of our environment. Considerable amounts of foreign DNA of very different origin are ingested daily with food. In a series of experiments we fed the DNA of bacteriophage M13 as test DNA to mice and showed that fragments of this DNA survive the passage through the gastrointestinal (GI) tract in small amounts (1-2%). Food-ingested M13 DNA reaches peripheral white blood cells, the spleen and liver via the intestinal epithelia and cells in the Peyer's patches of the intestinal wall. There is evidence to assume that food-ingested foreign DNA can become covalently linked to mouse-like DNA. When M13 DNA is fed to pregnant mice the test DNA can be detected in cells in various organs of the fetuses and of newborn animals, but never in all cells of the mouse fetus. It is likely that the M13 DNA is transferred by the transplacental route and not via the germ line. The consequences of foreign DNA uptake for mutagenesis and oncogenesis have not yet been investigated.

Animals↗
PubMed1998

On the fate of orally ingested foreign DNA in mice: chromosomal association and placental transmission to the fetus.

We have previously shown that, when administered orally to mice, bacteriophage M13 DNA, as a paradigm foreign DNA without homology to the mouse genome, can persist in fragmented form in the gastrointestinal tract, penetrate the intestinal wall, and reach the nuclei of leukocytes, spleen and liver cells. Similar results were obtained when a plasmid containing the gene for the green fluorescent protein (pEGFP-C1) was fed to mice. In spleen, the foreign DNA was detected in covalent linkage to DNA with a high degree of homology to mouse genes, perhaps pseudogenes, or to authentic E. coli DNA. We have now extended these studies to the offspring of mice that were fed regularly during pregnancy with a daily dose of 50 microg of M13 or pEGFP-C1 DNA. Using the polymerase chain reaction (PCR) or the fluorescent in situ hybridization (FISH) method, foreign DNA, orally ingested by pregnant mice, can be discovered in various organs of fetuses and of newborn animals. The M13 DNA fragments have a length of about 830 bp. In various organs of the mouse fetus, clusters of cells contain foreign DNA as revealed by FISH. The foreign DNA is invariably located in the nuclei. We have never found all cells of the fetus to be transgenic for the foreign DNA. This distribution pattern argues for a transplacental pathway rather than for germline transmission which might be expected only after long-time feeding regimens. In rare cells of three different fetuses, whose mothers have been fed with M 13 DNA during gestation, the foreign DNA was detected by FISH in association with both chromatids. Is maternally ingested foreign DNA a potential mutagen for the developing fetus?

Administration, Oral↗
PubMed1997

Three brothers with mental and physical retardation, hydrocephalus, microcephaly, internal malformations, speech disorder, and facial anomalies: Mutchinick syndrome.

We describe three brothers from a non-consanguineous family with microcephaly, mental and physical retardation, speech disorder, facial anomalies, and internal hydrocephalus in two of the three affected brothers. The youngest brother died at the age of 5 months. He had situs abdominalis inversus, ASD II, and had been operated for internal hydrocephalus and atresia of the biliary duct. A search in the Oxford Medical Database Dysmorphology Program suggested phenotypic similarities with two sisters described in 1972 by Osvaldo Mutchinick in Argentina. Although differences in their phenotypes exist, it is possible that the two sets of sibs represent the same, rare syndrome. This interpretation is supported by the origin of both families from the same geographic region.

Abnormalities, Multiple↗
PubMed1997

Clonal origin of adenovirus type 12-induced hamster tumors: nonspecific chromosomal integration sites of viral DNA.

The mechanism of tumor induction by human adenovirus type 12 (Ad12) in newborn Syrian hamsters (Mesocricetus auratus) has been investigated further. Tumors were produced in newborn hamsters by the s.c. injection of CsCl-purified Ad12. In 60-70% of the surviving animals, tumors have been observed between 33 and 47 days after injection. In 60 independently elicited tumors, the patterns of Ad12 DNA integration have been studied by restriction enzyme analyses and Southern blot hybridization using Ad12 DNA or its terminal DNA fragments as hybridization probes. Moreover, the integrated viral genomes have been localized on different hamster chromosomes by the fluorescence in situ hybridization technique using either nonradioactively labeled digoxigenin probes and fluorescent antibodies or biotinylated probes and fluorescent avidin. In all of the tumors, 20 and more copies of viral DNA have been found covalently linked to cellular DNA, as apparent from the off-size restriction fragments that do not comigrate during electrophoreses with any of the known virion DNA fragments. In Ad12-induced tumors or Ad12-transformed hamster cell lines, there is no evidence for the persistence of nonintegrated, free viral DNA copies. In general, the multiple copies of Ad12 DNA are inserted into a single chromosomal cellular site, which is different for each tumor. Only in one tumor cell line have the integrated Ad12 DNA copies been localized on two different chromosomes. The off-size fragment patterns generated by restriction and Southern blotting experiments are also unique and different for each tumor. These off-size fragments represent the sites of linkage between viral and cellular DNA but may also contain rearranged viral DNA sequences. We conclude that, upon Ad12 tumor induction in hamsters, Ad12 DNA does not integrate at specific insertion sites or nucleotide sequences into the cellular genome. It is still possible that selective elements exist at the sites of viral DNA insertion, e.g., specific chromatin structures due to transcriptional activity. The data presented also demonstrate (a) that each tumor cell carries Ad12 DNA; (b) that the insertion site appears to be the same in each tumor cell in a given tumor; (c) that, upon continued passage of the tumor cells in culture, the chromosomal site of Ad12 DNA insertion does not change, at least up to 25-32 passages, which correspond to about 75-96 cell generations beyond the tumor stage; and (d) that the integrated Ad12 DNA is localized on different chromosomes in individual tumors. Hence, the Ad12-induced tumors are of clonal origin. Another peculiarity of this viral tumor system is the frequent occurrence of more than one tumor in one animal. By the criteria established above, each individual tumor is characterized by its specific chromosomal integration site and restriction pattern. Thus, multiple induced tumors in one animal exhibit separate and individual clonality. During the time of maximally 7 weeks of observation of Ad12-induced tumors in the animals, metastases into different organ systems have not been observed.

Adenoviruses, Human↗
PubMed1997

Rapid protein sequencing by tandem mass spectrometry and cDNA cloning of p20-CGGBP. A novel protein that binds to the unstable triplet repeat 5'-d(CGG)n-3' in the human FMR1 gene.

The autonomous expansion of the unstable 5'-d(CGG)n-3' repeat in the 5'-untranslated region of the human FMR1 gene leads to the fragile X syndrome, one of the most frequent causes of mental retardation in human males. We have recently described the isolation of a protein p20-CGGBP that binds sequence-specifically to the double-stranded trinucleotide repeat 5'-d(CGG)-3' (Deissler, H., Behn-Krappa, A., and Doerfler, W. (1996) J. Biol. Chem. 271, 4327-4334). We demonstrate now that the p20-CGGBP can also bind to an interrupted repeat sequence. Peptide sequence tags of p20-CGGBP obtained by nanoelectrospray mass spectrometry were screened against an expressed sequence tag data base, retrieving a clone that contained the full-length coding sequence for p20-CGGBP. A bacterially expressed fusion protein p20-CGGBP-6xHis exhibits a binding pattern to the double-stranded 5'-d(CGG)n-3' repeat similar to that of the authentic p20-CGGBP. This novel protein lacks any overall homology to other known proteins but carries a putative nuclear localization signal. The p20-CGGBP gene is conserved among mammals but shows no homology to non-vertebrate species. The gene encoding the sequence for the new protein has been mapped to human chromosome 3.

Amino Acid Sequence↗