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T Boehm

Publications and source records attributed to T Boehm.

At least 19 recordsLinked to original sources
PubMed1997

The nude gene encodes a sequence-specific DNA binding protein with homologs in organisms that lack an anticipatory immune system.

In the mouse, the product of the nude locus, Whn, is required for the keratinization of the hair shaft and the differentiation of epithelial progenitor cells in the thymus. A bacterially expressed peptide representing the presumptive DNA binding domain of the mouse whn gene in vitro specifically binds to a 11-bp consensus sequence containing the invariant tetranucleotide 5'-ACGC. In transient transfection assays, such binding sites stimulated reporter gene expression about 30- to 40-fold, when positioned upstream of a minimal promotor. Whn homologs from humans, bony fish (Danio rerio), cartilaginous fish (Scyliorhinus caniculus), agnathans (Lampetra planeri), and cephalochordates (Branchiostoma lanceolatum) share at least 80% of amino acids in the DNA binding domain. In agreement with this remarkable structural conservation, the DNA binding domains from zebrafish, which possesses a thymus but no hair, and amphioxus, which possesses neither thymus nor hair, recognize the same target sequence as the mouse DNA binding domain in vitro and in vivo. The genomes of vertebrates and cephalochordates contain only a single whn-like gene, suggesting that the primordial whn gene was not subject to gene-duplication events. Although the role of whn in cephalochordates and agnathans is unknown, its requirement in the development of the thymus gland and the differentiation of skin appendages in the mouse suggests that changes in the transcriptional control regions of whn genes accompanied their functional reassignments during evolution.

Amino Acid Sequence↗
PubMed1997

Identification of interaction partners for the basic-helix-loop-helix protein E47.

Helix-loop-helix proteins constitute a family of transcription factors with the potential to form homo- and hetero-dimers mediated by the helix-loop-helix domain. Oncogenic mutations in such genes can disrupt the equilibrium of protein-protein interactions in the affected cell. In order to assess the biological consequences of such mutations, the full complement of interacting proteins must be known. To identify proteins interacting with the basic-helix-loop-helix domain of the ubiquitously expressed E47 protein, a 'sandwich'-screening procedure was developed which distinguishes between homo- and hetero-oligomers, and specifically excludes the detection of complexes which cannot bind DNA. Nine distinct cDNAs were identified which encode proteins with apparent basic-helix-loop-helix domains, including a novel clone termed eip1 which is distantly related in the basic-helix-loop-helix domain to the Drosophila enhancer-of-split m7 protein. Using epitope-tagging, interaction of E47 basic-helix-loop-helix protein with the eip1 protein encoded by this novel cDNA was confirmed by immunoprecipitation experiments in COS7 cells. Interaction was also observed in the yeast two-hybrid system. Three cDNAs encoding proteins without basic-helix-loop-helix domains were also found to interact in the sandwich-expression screen. Interactions with human PARP and mouse replication factor 1a were confirmed using glutathione transferase-tagged cDNAs. A cDNA encoding part of the nucleolin protein sequence interacted with the E47 basic-helix-loop-helix only when fused to a beta-galactosidase tag.

Amino Acid Sequence↗
PubMed1997

Endostatin: an endogenous inhibitor of angiogenesis and tumor growth.

We previously identified the angiogenesis inhibitor angiostatin. Using a similar strategy, we have identified endostatin, an angiogenesis inhibitor produced by hemangioendothelioma. Endostatin is a 20 kDa C-terminal fragment of collagen XVIII. Endostatin specifically inhibits endothelial proliferation and potently inhibits angiogenesis and tumor growth. By a novel method of sustained release, E. coli-derived endostatin was administered as a nonrefolded suspension. Primary tumors were regressed to dormant microscopic lesions. Immunohistochemistry revealed blocked angiogenesis accompanied by high proliferation balanced by apoptosis in tumor cells. There was no toxicity. Together with angiostatin data, these findings validate a strategy for identifying endogenous angiogenesis inhibitors, suggest a theme of fragments of proteins as angiogenesis inhibitors, and demonstrate dormancy therapy.

Amino Acid Sequence↗
PubMed1997

A yeast artificial chromosome contig spanning the mouse immunoglobulin kappa light chain locus.

A single contig spanning the entire mouse immunoglobulin kappa light chain (Igk) locus on chromosome 6 has been established using yeast and bacterial artificial chromosome clones. Detailed mapping of the Igk locus indicates that a member of the Igk-V2 gene family, located about 3.5 megabases upstream of the Igk-J-C complex, is the most distal functional Igk-V gene. Sequence analyses of Igk-V genes and anonymous DNA segments provide indications for internal duplications at the 5' end of the Igk-V locus and identify the likely origin of Igk-V orphon gene clusters located elsewhere in the mouse genome.

Animals↗
PubMed1997

Characterization of mouse and human nude genes.

The differentiation of primitive epithelial precursor cells in the thymic primordium into subcapsular, cortical, and medullary epithelial cells of the mature thymus requires the activity of the nude gene product Whn. Whn is also required for proper keratinization of the hair shaft. We determined the nucleotide sequence of a 58 kilobase region on mouse chromosome 11 that encompasses the mouse nude gene and part of the two neighboring genes, encoding a sodium/dicarboxylate co-transporter and the retinal protein 4. Using cross-hybridization, the human orthologue of the mouse nude gene was isolated. The human WHN protein also consists of 648 amino acids, 85% of which are identical to the mouse protein. Like the mouse gene, the human gene consists of eight coding exons and utilizes two alternative first exons in a tissue-specific fashion. Sequences upstream of the two alternative first exons display promoter activity in heterologous reporter assays. Whereas both promoters appear to be active in skin (albeit at different levels), only the most upstream element is active in the thymus, indicating that transcriptional activity of the whn gene is subject to complex regulation. Nucleotide sequence database comparisons reveal that among other winged-helix genes, the HTLF and HTLFL1 genes are most closely related to whn, although the exon/intron structure of the human HTLF gene in the DNA binding domain differs from that of whn.

Alternative Splicing↗
PubMed1996

Expression of peptides encoded by exons in cloned mammalian DNA.

New synthetic approaches, such as combinatorial chemistry, provide a rich source of potential drug candidates. At the same time, the human genome initiative and other large-scale sequencing projects provide a large number of novel drug targets. However, the functional analysis of thousands of new genes remains a major challenge for the future. A systematic strategy for genome-wide functional analysis of genes could employ the fact that at least some modules in multi-domain proteins are encoded in individual exons. Exon amplification provides information about coding regions of most genes that is independent of their transcriptional status; exon amplification from entire mammalian genomes has been demonstrated. Here, we describe the development of an exon-trap system, lambdaGEE (for genomic exon expression), that couples exon amplification with the expression of exon-encoded peptides.

Animals↗
PubMed1996

The whn transcription factor encoded by the nude locus contains an evolutionarily conserved and functionally indispensable activation domain.

Mutations in the whn gene are associated with the phenotype of congenital athymia and hairlessness in mouse and rat. The whn gene encodes a presumptive transcription factor with a DNA binding domain of the forkhead/ winged-helix class. Two previously described null alleles encode truncated whn proteins lacking the characteristic DNA binding domain. In the rat rnu allele described here, a nonsense mutation in exon 8 of the whn gene was identified. The truncated whnrnu protein contains the DNA binding domain but lacks the 175 C-terminal amino acids of the wild-type protein. To facilitate the identification of functionally important regions in this region, a whn homolog from the pufferfish Fugu rubripes was isolated. Comparison of derived protein sequences with the mouse whn gene revealed the presence of a conserved acidic protein domain in the C terminus, in addition to the highly conserved DNA binding domain. Using fusions with a heterologous DNA binding domain, a strong transcriptional activation domain was localized to the C-terminal cluster of acidic amino acids. As the whnrnu mutant protein lacks this domain, our results indicate that a transactivation function is essential for the activity of the whn transcription factor.

Amino Acid Sequence↗
PubMed1996

Two genetically separable steps in the differentiation of thymic epithelium.

The development of the thymus depends initially on epithelial-mesenchymal and subsequently on reciprocal lympho-stromal interactions. The genetic steps governing development and differentiation of the thymic microenvironment are unknown. With the use of a targeted disruption of the whn gene, which recapitulates the phenotype of the athymic nude mouse, the WHN transcription factor was shown to be the product of the nude locus. Formation of the thymic epithelial primordium before the entry of lymphocyte progenitors did not require the activity of WHN. However, subsequent differentiation of primitive precursor cells into subcapsular, cortical, and medullary epithelial cells of the postnatal thymus did depend on activity of the whn gene. These results define the first genetically separable steps during thymic epithelial differentiation.

Alleles↗
PubMed1996

Genomic Organization of the ATM gene.

The ATM gene was recently identified and found to be responsible for the genetic disorder ataxiatelgiectasia. The major ATM transcript is 13 kb. Using long-distance PCR, we determined the genomic structure of this gene and identified all of its exon-intron boundaries. The ATM gene spans approximately 150 kb of genomic DNA and consists of 66 exons. The initiation codon falls within exon 4. The last exon is 3.8 kb and contains the stop codon and a 3'-untranslated region of about 3600 nucleotides.

Ataxia Telangiectasia↗
PubMed1995

An intragenic deletion in the human PTPN6 gene affects transcriptional activity.

An intragenic deletion in the human PTPN6 gene is described. The PTPN6 gene maps to chromosome 12p12-13 and is shown to possess two alternative first exons. A 1.7-kb deletion occurring in the intron between the two alternatively used first exons is the result of an illegitimate recombination between two Alu-type repeats. The deletion increases the transcriptional activity of the distal promotor.

Base Sequence↗
PubMed1995

YAC/P1 contigs defining the location of 56 microsatellite markers and several genes across a 3.4-cM interval on mouse chromosome 11.

The characterization of three YAC/P1 contigs from adjacent segments of the central region of mouse Chromosome (Chr) 11 is described. These contigs are based upon 63 YACs and 40 P1 recombinants. From these clones, 185 end sequences were obtained, of which 147 sequences could be converted into sequence-tagged sites and mapped within the three contigs. Deletions were detected in 16 out of 63 YACs; 19 of 63 YACs were found to be chimeric. No such aberrations were found in P1 recombinants. A total of 22 public and 34 newly developed microsatellite markers were unambiguously localized to and ordered in the contigs. In the cryb1/Nf1 interval of the central contig, several new genes have been identified by exon trapping and precisely localized with respect to known STS markers.

Amino Acid Sequence↗
PubMed1994

New member of the winged-helix protein family disrupted in mouse and rat nude mutations.

Mutations at the nude locus of mice and rats disrupt normal hair growth and thymus development, causing nude mice and rats to be immune-deficient. The mouse nude locus has been localized on chromosome 11 (refs 3, 4) within a region of < 1 megabase. Here we show that one of the genes from this critical region, designated whn, encodes a new member of the winged-helix domain family of transcription factors, and that it is disrupted on mouse nu and rat rnuN alleles. Mutant transcripts do not encode the characteristic DNA-binding domain, strongly suggesting that the whn gene is the nude gene. Mutations in winged-helix domain genes cause homeotic transformations in Drosophila and distort cell-fate decisions during vulval development in Caenorhabditis elegans. The whn gene is thus the first member of this class of genes to be implicated in a specific developmental defect in vertebrates.

Alleles↗
PubMed1994

The sequence complexity of exons trapped from the mouse genome.

BACKGROUND: A central issue in genome analysis is the identification and characterization of coding regions. Estimating the coding complexity of vertebrate genomes by measuring the kinetic complexity of mRNA populations and by sequence analysis of cDNAs is limited by the fact that any given source of mRNA represents a very biased sample of all genes. Exon trapping is a method that enables the identification of genes irrespective of their transcriptional status. RESULTS: Exons were trapped from the entire mouse genome, and the resulting fragments cloned. About 7% of a random sample of exons taken from this library have significant structural homology or sequence similarity to previously sequenced genes. Using cDNAs derived from several stages of mouse development, evidence for expression of about 62% of this sample of exons was found. These data suggest that the great majority of 'exons' in the library are derived from genes. We estimate that the fraction of the genome contained in trapped exons is 2.4%; this corresponds to a sequence complexity of about 72 megabases. CONCLUSIONS: The library of exons trapped from the entire mouse genome probably represents one of the least biased and most comprehensive libraries of mouse coding regions, and should therefore prove very useful for finding genes during genome mapping and sequencing.

Amino Acid Sequence↗