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M Nehls

Publications and source records attributed to M Nehls.

28 records · Page 2Linked to original sources
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↗
PubMed1994

A yeast artificial chromosome contig on mouse chromosome 11 encompassing the nu locus.

Mutations at the nude locus disrupt the homing process of T cell progenitor cells to the thymic rudiment, a key aspect of T cell differentiation. Here, we map the nude locus to a set of overlapping yeast artificial chromosomes (YAC) clones covering a genetic interval of about 0.5 centi Morgan on mouse chromosome 11. These results provide a suitable starting point to molecularly clone the nude gene.

Animals↗
PubMed1994

DNA methylation represses the murine alpha 1(I) collagen promoter by an indirect mechanism.

Several lines of evidence indicate that DNA methylation plays a role in the transcriptional regulation of the murine alpha 1(I) collagen gene. To study the molecular mechanisms involved, a reporter gene construct containing the alpha 1(I) promoter and part of the first exon linked to the luciferase gene (Col3luc) was methylated in vitro and transfected into murine fibroblasts and embryonal carcinoma cells. Methylation resulted in repression of the alpha 1(I) promoter in both cell types, although it was less pronounced in embryonal carcinoma cells than in fibroblasts. The extent of repression depended on the density of methylation. DNase footprint and mobility shift assays indicated that the trans-acting factors binding to the alpha 1(I) promoter and first exon are ubiquitous factors and that their DNA binding is not inhibited by methylation. Transfection of Col3luc into Drosophila SL2 cells together with expression vectors for the transcription factors Sp1 and NF-1 showed that DNA methylation also inhibits the alpha 1(I) promoter in nonvertebrate cells, although to a much lesser extent than in murine cells. However, Sp1 and NF-1 transactivated the unmethylated and methylated reporter gene in SL2 cells equally well, confirming that these factors can bind and transactivate methylated DNA and indicating that DNA methylation represses the alpha 1(I) promoter by an indirect mechanism. This was further confirmed by cotransfection experiments with unspecific methylated competitor DNA which partially restored the activity of the methylated alpha 1(I) promoter. Our results suggest that DNA methylation can inhibit promoter activity by an indirect mechanism independent of methyl-C-binding proteins and that in vertebrate cells, chromatin structure and methyl-C-binding proteins cooperatively mediate the transcriptional inhibitory effect of DNA methylation.

Animals↗
PubMed1994

Two large insert vectors, lambda PS and lambda KO, facilitate rapid mapping and targeted disruption of mammalian genes.

The construction and the testing of two lambda phage vectors are described that greatly simplify the tasks of mapping genomic DNA and making replacement-type gene-targeting vectors for mammalian cells from a library of isogenic genomic DNA. The first vector, lambda PS, accommodates up to 20 kb and allows inserts to be automatically subcloned in plasmid form because of the presence of loxP sites flanking the insert. The second vector, lambda KO, accommodates up to 16.7 kb and allows inserts to be automatically subcloned as plasmids containing HSVtk genes that are positioned flanking the inserted genomic DNA. We have prepared highly redundant libraries from genomic DNA of 129/Sv-strain mice for the construction of targeting vectors. In our scheme, the locus of interest is characterized using a library made in lambda PS. For instance, suitable flanking probes can be derived to determine targeting events. The final targeting construct with flanking HSVtk genes is obtained using the lambda KO cloning vector. The entire procedure is exemplified by successful targeting of the X-linked mouse hprt locus.

Animals↗
PubMed1994

Exon amplification from complete libraries of genomic DNA using a novel phage vector with automatic plasmid excision facility: application to the mouse neurofibromatosis-1 locus.

The identification of transcription units in the vicinity of chromosomal lesions found in tumours is an essential step in the identification of new oncogenes. Here, we describe a lambda phage vector system for genomic exon-trapping (lambda GET), which dramatically simplifies the task of exon amplification from genomic DNA. The vector accommodates about 6.5 to 19 kb of DNA and allows inserts to be automatically subcloned as multi-copy plasmids containing splice donor and acceptor sites positioned flanking the inserted genomic DNA. RNA transcripts derived from such plasmids are processed in vivo and exons contained within the inserted genomic fragments become flanked by known sequences in the resulting mRNAs. RNA-based PCR can then be used for subsequent cloning and sequence analysis of trapped exons. We have exploited the large cloning capacity of lambda GET to construct highly redundant complete genomic libraries from Sau3AI partially digested vertebrae DNAs. Using this system, we have analysed a region of about 1 MB around the mouse neurofibromatosis-1 locus and have identified novel transcription units flanking the Nf-1 gene.

Amino Acid Sequence↗
PubMed1985

Phylogenetic aspects of cardiac hormones as revealed by immunocytochemistry, electronmicroscopy, and bioassay.

A hormone family of cardiac peptides has recently been isolated and biochemically and pharmacologically characterized by the relaxation of vascular smooth muscle, diuretic and natriuretic activities. The cardiac hormones are stored in specific granules of the atrial myoendocrine cells. Since data is available only from mammals (rat, pig, man) we started a phylogenetic study by investigating representatives of the higher vertebrate classes (birds, reptiles, amphibians, bony fish) as well as an invertebrate species, the gastropod mollusc Helix pomatia. Homologous cardiac hormones of the cardiodilatin (CDD) family which exerted a dose-dependent relaxant effect on the rabbit aorta were extracted from the atria of all species studied and from the ventricles of amphibians and teleosts. The storage sites of cardiac hormones were localized by electronmicroscopy and immunocytochemistry using antisera against several sequences of pig CDD and applying the peroxidase-antiperoxidase technique. CDD-immunoreactivity (CDD-IR) was observed in myoendocrine cells in the atria of all vertebrate species studied, and in amphibians and teleosts also in the ventricles. In the snail, however, CDD-IR was present in nerve endings of the atrium and in perikarya of the subesophageal ganglion as well as in fibers of the intestinal nerve, while no CDD-IR was detected in heart muscle cells. In correlation, no "specific" granules were observed in myocardiocytes of the snail and vascular smooth muscle relaxant bioactivity was present in extracts of the subesophageal ganglion. The findings indicate that in the vertebrates studied the cardiodilatin-immunoreactive substances seem to constitute an endocrine system in the heart. In the snail, in contrast, they are present in a neuro-cardiac axis. This seems to represent a model unique in phylogeny.

Animals↗
PubMed1985

Biochemical and immunological evidence for a cardiodilatin-like substance in the snail neurocardiac axis.

Cardiac hormones, which have been isolated recently from mammalian atria, are potent regulatory peptides of blood pressure and blood volume. By using biochemical and immunological methods to determine cardiac hormones of the cardiodilatin family, this type of peptide hormone was detected in a neurosecretory system projecting from the subesophageal ganglion to the heart of the snail. The cardiodilatin-like molecule was characterized by its biological effects on mammalian vascular smooth muscle, by radioimmunoassay combined with high-performance liquid chromatography, and by immunocytochemistry. In mammals cardiodilatin-like peptides appear to serve purely endocrine functions. In contrast, in the snail they are present in a neuroendocrine system, the function of which remains to be established.

Animals↗
PubMedDate not supplied

Knock-out mouse for Canavan disease: a model for gene transfer to the central nervous system.

BACKGROUND: Canavan disease (CD) is an autosomal recessive leukodystrophy characterized by deficiency of aspartoacylase (ASPA) and increased levels of N-acetylaspartic acid (NAA) in brain and body fluids, severe mental retardation and early death. Gene therapy has been attempted in a number of children with CD. The lack of an animal model has been a limiting factor in developing vectors for the treatment of CD. This paper reports the successful creation of a knock-out mouse for Canavan disease that can be used for gene transfer. METHODS: Genomic library lambda knock-out shuttle (lambdaKOS) was screened and a specific pKOS/Aspa clone was isolated and used to create a plasmid with 10 base pair (bp) deletion of exon four of the murine aspa. Following linearization, the plasmid was electroporated to ES cells. Correctly targeted ES clones were identified following positive and negative selection and confirmed by Southern analysis. Chimeras were generated by injection of ES cells to blastocysts. Germ line transmission was achieved by the birth of heterozygous mice as confirmed by Southern analysis. RESULTS: Heterozygous mice born following these experiments have no overt phenotype. The homozygous mice display neurological impairment, macrocephaly, generalized white matter disease, deficient ASPA activity and high levels of NAA in urine. Magnetic resonance imaging (MRI) and spectroscopy (MRS) of the brain of the homozygous mice show white matter changes characteristic of Canavan disease and elevated NAA levels. CONCLUSION: The newly created ASPA deficient mouse establishes an important animal model of Canavan disease. This model should be useful for developing gene transfer vectors to treat Canavan disease. Vectors for the central nervous system (CNS) and modulation of NAA levels in the brain should further add to the understanding of the pathophysiology of Canavan disease. Data generated from this animal model will be useful for developing strategies for gene therapy in other neurodegenerative diseases.

Amidohydrolases↗