PubMed Health⌕ Search

Biomedical subjects

M Nehls

Publications and source records attributed to M Nehls.

At least 19 recordsLinked to original sources
PubMed1999

Identification of a mammalian angiopoietin-related protein expressed specifically in liver.

Based on searches of EST databases for signal sequences and amphipathic helices, we have identified and cloned an angiopoietin-like gene, ANGPTL3. Multiple tissue Northern blots show that ANGPTL3 is expressed principally in the liver. ANGPTL3 is expressed early during liver development, and expression is maintained in adult liver. Human ANGPTL3 is a 460-amino-acid polypeptide with the characteristic structure of angiopoietins: a signal peptide, an extended helical domain predicted to form dimeric or trimeric coiled-coils, a short linker peptide, and a globular fibrinogen homology domain (FHD). Murine ANGPTL3 is a 455-acid polypeptide encoded by seven exons on mouse chromosome 4, spanning about 11 kb of DNA. ANGPTL3 contains the four conserved cysteines implicated in the intramolecular disulfide bonds within the FHD, but it does not contain two other cysteines that are found within the FHD of angiopoietins 1, 2, and 4. ANGPTL3 also does not contain the characteristic calcium binding motif found in the other angiopoietins. By radiation hybrid mapping and the use of surrounding genes, human ANGPTL3 maps to the 1p31 region.

Amino Acid Motifs↗
PubMed1999

Cloning, chromosomal location, and expression analysis of murine Smarce1-related, a new member of the high-mobility 365 group gene family.

Proteins containing high-mobility group (HMG) domains are segregated into two major groups. Members of one group are identified by the presence of more than one HMG domain that binds to DNA without sequence specificity, and they are usually ubiquitously expressed. In contrast, members of the other group possess a single HMG domain with high affinity to specific DNA sequences. Generally, members of the second group resemble classic tissue-specific transcriptional regulators. In contrast, Smarce1/BAF-57 is a ubiquitously expressed, novel protein with a single HMG domain that displays nonspecific DNA-binding characteristics. Additionally, as a core subunit of the mammalian SWI/SNF-like transcriptional activator complex, Smarce1/BAF-57 is also the first member of the HMG protein family that was reported to contain a kinesin-like coiled-coil (KLCC) domain. Here we report the cloning, as well as the chromosomal and phylogenetic analysis, of a novel mammalian protein that is structurally related to Smarce1, termed Smarce1-related (Smarce1r). The unique arrangement of an HMG with a KLCC domain shared with Smarce1/BAF-57 suggests a similar, albeit still unknown, function in chromatin assembly as part of a mammalian SWI/SNF-like complex. The linkage of a single nonspecific DNA-binding HMG domain with a KLCC domain makes both proteins the founding members of a third group of HMG proteins.

Amino Acid Sequence↗
PubMed1999

Structural and phylogenetic characterization of human SLURP-1, the first secreted mammalian member of the Ly-6/uPAR protein superfamily.

Members of the Ly-6/uPAR protein family share one or several repeat units of the Ly-6/uPAR domain that is defined by a distinct disulfide bonding pattern between 8 or 10 cysteine residues. The Ly-6/uPAR protein family can be divided into two subfamilies. One comprises GPI-anchored glycoprotein receptors with 10 cysteine residues. The other subfamily includes the secreted single-domain snake and frog cytotoxins, and differs significantly in that its members generally possess only eight cysteines and no GPI-anchoring signal sequence. We report the purification and structural characterization of human SLURP-1 (secreted mammalian Ly-6/uPAR related protein 1) from blood and urine peptide libraries. SLURP-1 is encoded by the ARS (component B)-81/s locus, and appears to be the first mammalian member of the Ly-6/uPAR family lacking a GPI-anchoring signal sequence. A phylogenetic analysis based on the SLURP-1 primary protein structure revealed a closer relationship to the subfamily of cytotoxins. Since the SLURP-1 gene maps to the same chromosomal region as several members of the Ly-6/uPAR subfamily of glycoprotein receptors, it is suggested that both biologically distinct subfamilies might have co-evolved from local chromosomal duplication events.

Animals↗
PubMed1999

Construction of gene targeting vectors from lambda KOS genomic libraries.

We describe a highly redundant murine genomic library in a new lambda phage, lambda knockout shuttle (lambda KOS) that facilitates the very rapid construction of replacement-type gene targeting vectors. The library consists of 94 individually amplified subpools, each containing an average of 40,000 independent genomic clones. The subpools are arrayed into a 96-well format that allows a PCR-based efficient recovery of independent genomic clones. The lambda KOS vector backbone permits the CRE-mediated conversion into high-copy number pKOS plasmids, wherein the genomic inserts are automatically flanked by negative-selection cassettes. The lambda KOS vector system exploits the yeast homologous recombination machinery to simplify the construction of replacement-type gene targeting vectors independent of restriction sites within the genomic insert. We outline procedures that allow the generation of simple and more sophisticated conditional gene targeting vectors within 3-4 weeks, beginning with the screening of the lambda KOS genomic library.

Animals↗
PubMed1998

A combined analysis of genomic and primary protein structure defines the phylogenetic relationship of new members if the T-box family.

T-box genes form an ancient family of putative transcriptional regulators characterized by a region of homology to the DNA-binding domain of the murine Brachyury (T) gene product. This T-box domain is conserved from Caenorhabditis elegans to human, and mutations in T-box genes have been associated with developmental defects in Drosophila, zebrafish, mice, and humans. Here we report the identification of three novel murine T-box genes and an investigation of their evolutionary relationship to previously known family members by studying the genomic structure of the T-box. All T-box genes from nematodes to humans possess a characteristic central intron that presumably was inherited from a common ancestral precursor. Two additional intron positions are also conserved with the exception of two nematode T-box genes. Subsequent intron insertions, potential deletions, and/or intron sliding formed a structural basis for the divergence into distinct subfamilies and a substrate for length variations of the T-box domain. In mice, the 11 T-box genes known to date can be grouped into seven subfamilies. Genes assigned to the same subfamily by genomic structure show related expression patterns. We propose a model for the phylogenetic relationships within the gene family that provides a rationale for classifying new T-box genes and facilitates interspecific comparisons.

Amino Acid Sequence↗
PubMed1997

The structural integrity of ROR alpha isoforms is mutated in staggerer mice: cerebellar coexpression of ROR alpha1 and ROR alpha4.

The recessive mouse mutation staggerer (sg) disturbs the normal development of cerebellar Purkinje cells and affects certain functions of the immune system. To identify the causative gene, we constructed high-resolution genetic and physical maps of the staggerer locus on mouse chromosome 9. The transcription unit of the orphan nuclear receptor ROR alpha was identified in the critical interval. Our mutational analysis confirms a recent report that the sg phenotype may be caused by a genomic deletion in the common coding region of the ROR alpha isoforms. Of the four different isoforms of the ROR alpha gene that are generated by a combination of alternative promoter usage and exon splicing that differ in their DNA-binding properties, isoforms ROR alpha1 and ROR alpha4 are specifically coexpressed in the murine cerebellum and human cerebellum. Thus, at least two isoforms of the murine ROR alpha gene are affected by the genomic deletion associated with the staggerer phenotype. Our finding of cerebellum-specific coregulation suggests that distinct sets of target genes regulated by the ROR alpha1 and ROR alpha4 isoforms are required for Purkinje cell development.

Amino Acid Sequence↗
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

Suppression of apoptosis by nitric oxide via inhibition of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases.

Physiological levels of shear stress alter the genetic program of cultured endothelial cells and are associated with reduced cellular turnover rates and formation of atherosclerotic lesions in vivo. To test the hypothesis that shear stress (15 dynes/cm2) interferes with programmed cell death, apoptosis was induced in human umbilical venous cells (HUVEC) by tumor necrosis factor-alpha (TNF-alpha). Apoptosis was quantified by ELISA specific for histone-associated DNA-fragments and confirmed by demonstrating the specific pattern of internucleosomal DNA-fragmentation. TNF-alpha (300 U/ml) mediated increase of DNA-fragmentation was completely abrogated by shear stress (446 +/- 121% versus 57 +/- 11%, P <0.05). This anti-apoptotic activity of shear stress decreased after pharmacological inhibition of endogenous nitric oxide (NO)-synthase by NG-monomethyl-L-arginine and was completely reproduced by exogenous NO-donors. The activation of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like cysteine proteases was required to mediate TNF-alpha-induced apoptosis of HUVEC. Endothelial-derived nitric oxide (NO) as well as exogenous NO donors inhibited TNF-alpha-induced cysteine protease activation. Inhibition of CPP-32 enzyme activity was due to specific S-nitrosylation of Cys 163, a functionally essential amino acid conserved among ICE/CPP-32-like proteases. Thus, we propose that shear stress-mediated NO formation interferes with cell death signal transduction and may contribute to endothelial cell integrity by inhibition of apoptosis.

Apoptosis↗
PubMed1996

Shear stress inhibits apoptosis of human endothelial cells.

Physiological levels of shear stress alter the genetic program of cultured endothelial cells and reduce endothelial cell turnover in vivo. To test the hypothesis that shear stress interferes with programmed cell death, apoptosis was induced in human umbilical venous endothelial cells by growth factor withdrawal or incubation with tumor necrosis factor alpha (TNFalpha) for 18 h. Apoptosis was quantified by ELISA specific for histone-associated DNA fragments and confirmed by demonstrating the specific pattern of internucleosomal DNA fragmentation detected by electrophoresis and immunohistochemical staining. The TNFalpha (300 U/ml)-mediated increase in DNA fragmentation was completely abrogated by shear stress. Furthermore, shear stress dose-dependently reduced DNA fragmentation induced by growth factor withdrawal with maximal effect at 45 dyn/cm2. Inhibition of the CPP32-like proteases with Ac-DEVD-CHO (100 microM) revealed similar anti-apoptotic effects. In contrast, CPP32-independent induction of endothelial cell apoptosis by C2-ceramide (50 microM) was not prevented by shear stress. Thus, we propose that shear stress interferes with common cell death signal transduction involving the CPP32-like protease family and may contribute to endothelial cell integrity by inhibition of apoptosis.

Apoptosis↗
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↗
PubMed1995

Dynamic changes in gene expression during in vitro differentiation of mouse embryonic stem cells.

The expression pattern of protein tyrosine kinases (PTK) and phosphatases (PTP) was determined during the first eight days of in vitro differentiation of mouse embryonic stem (ES) cells. DNA fingerprinting of catalytic domains amplified from cDNA revealed dynamic changes in expression of previously described genes. A novel PTP is expressed in undifferentiated ES cells, and is down-regulated during in vitro differentiation.

Animals↗
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↗