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Biomedical subjects

H Weiher

Publications and source records attributed to H Weiher.

33 records · Page 2Linked to original sources
PubMed1995

Functional rescue of the glomerulosclerosis phenotype in Mpv17 mice by transgenesis with the human Mpv17 homologue.

The germ line insertion of a defective retrovirus into the Mpv17 gene of mice is associated with a recessive phenotype. Mice homozygous for the integration develop glomerulosclerosis at a young age. The phenotype resembles human glomerulosclerosis in its physiological parameters as well as in histology. A human homologue of the Mpv17 gene has been identified, isolated and analyzed. We here show that this gene, which has a role in the production of reactive oxygen species, can rescue the phenotype of Mpv17 deficient mice when introduced by transgenesis. This provides formal proof for the hypothesis that the phenotype is caused by the loss of function of the Mpv17 gene. It also provides evidence for the functional conservation of the Mpv17 gene in mammals and points to a potential role of this gene in human kidney disease.

Animals↗
PubMed1995

Expression of interstitial collagenase during skeletal development of the mouse is restricted to osteoblast-like cells and hypertrophic chondrocytes.

We determined the expression pattern of the matrix metalloproteinase interstitial collagenase (MMP-1) during mouse embryo development using in situ hybridization and immunohistochemistry. Localized MMP-1 mRNA was first detected at 14.5 days postconceptus. The spatial and temporal expression was restricted to areas of endochondral and intramembranous bone formation, such as in the mandibula, maxilla, clavicle, scapula, in the vertebrae, and in the dorsal, but not the ventral part of the ribs. The highest levels of MMP-1 transcripts and MMP-1 protein were found in the metaphyses and diaphyses of the long bones. MMP-1 was expressed by hypertrophic chondrocytes and by osteoblastic cells localized along the newly formed bone trabeculae. No expression was detected in osteoclasts. Two other related members of the MMP family, stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10), were not expressed during days 7.5 and 16.5 of mouse embryogenesis. The tissue-specific expression of MMP-1 and the exclusive ability of interstitial collagenase to digest native collagen of types I, II, III, and X, the major components of bone, cartilage, and tendon, strongly suggests an important and specific function of this enzyme in bone development and remodeling.

Animals↗
PubMed1994

The glomerulosclerosis gene Mpv17 encodes a peroxisomal protein producing reactive oxygen species.

The mutant mouse strain Mpv17 carries a retroviral insert in its genome which inactivates the Mpv17 gene. At a young age these mice develop glomerulosclerosis and nephrotic syndrome which resembles human disease. We show here that the Mpv17 gene product is highly conserved and encodes a peroxisomal protein. Loss of the Mpv17 protein does not impair peroxisome biogenesis but instead leads to a reduced ability to produce reactive oxygen species (ROS). In turn, overproduction of the Mpv17 gene in transfected cells results in dramatically enhanced levels of intracellular ROS indicating a direct involvement of Mpv17 in ROS production. These data reveal a role for the Mpv17 protein in peroxisomal reactive oxygen metabolism and establish a novel link between peroxisomal ROS production and glomerulosclerosis.

Amino Acid Sequence↗
PubMed1993

The human homolog of the glomerulosclerosis gene Mpv17: structure and genomic organization.

Mice carrying a retroviral insert in both alleles of the Mpv17 gene develop glomerulosclerosis and nephrotic syndrome at young age. Thus, the Mpv17 gene is a recessive disease gene in mice and this mouse strain is a potential animal model for glomerular diseases in man. We here describe the isolation and analysis of a human homolog of this gene. By interspecies hybridisation cDNA clones representing a single RNA species were isolated from human liver. Sequence analysis revealed over 90% identify in a region coding for a protein of 176 amino acids and unknown function in both species. Cloning of the genomic locus revealed a single copy gene which we mapped to the short arm of chromosome 2 at band 2p23-p21. Determination of the intron-exon structure and the junction sequences enabled us to establish a PCR based procedure to isolate the coding region from human genomic DNA. Thus, it is now possible to analyse patients suffering from candidate diseases on the basis of a blood sample if biopsy material is not available.

Animals↗
PubMed1990

Transgenic mouse model of kidney disease: insertional inactivation of ubiquitously expressed gene leads to nephrotic syndrome.

Transgenic mouse strains carrying proviruses were generated by exposing mouse embryos to a recombinant retrovirus. Animals carrying a single provirus were intercrossed to derive mice homozygous for a given proviral insertion. Adult mice homozygous for the Mpv17 integration developed nephrotic syndrome and chronic renal failure. Histologically, affected kidneys showed progressive glomerular sclerosis. Similar lesions are seen in patients with progressive renal function deterioration. A probe to DNA sequences flanking the provirus detected a 1.7 kb RNA ubiquitously expressed during embryogenesis and in adults with high levels in kidney, brain, and heart. This RNA was not detected in tissues of homozygous animals, suggesting that the provirus interferes with RNA expression. Sequence analysis of the cDNA revealed that the gene encodes a 176 amino acid peptide containing hydrophobic regions, suggesting membrane association of the putative protein. The Mpv17 mutant is a potentially useful experimental system for studying mechanisms leading to renal disorders in man.

Amino Acid Sequence↗
PubMed1987

Retrovirus integration and chromatin structure: Moloney murine leukemia proviral integration sites map near DNase I-hypersensitive sites.

The chromatin conformation of mouse genome regions containing Moloney murine leukemia proviral intergration sites in two Mov mouse strains and randomly selected integration sites in virus-infected mouse 3T3 fibroblasts was analyzed. All integrations have occurred into chromosomal regions containing several DNase-hypersensitive sites, and invariably the proviral integration sites map within a few hundred base pairs of a DNase-hypersensitive site. The probability that this close association between proviral integration sites and DNase-hypersensitive sites was due to chance was calculated to be extremely low (2 X 10(-4]. Because the proviral integrations analyzed were not selected for an altered phenotype, our results suggest that DNase-hypersensitive regions are preferred targets for retrovirus integration.

Animals↗
PubMed1987

Two distinct sequence elements mediate retroviral gene expression in embryonal carcinoma cells.

Moloney murine leukemia virus (M-MuLV) and M-MuLV-derived retroviral vectors are not expressed in early mouse embryos or in embryonal carcinoma cells. M-MuLV-derived mutants or M-MuLV-related variants which transduce the neomycin phosphotransferase gene can, however, induce drug resistance in embryonal carcinoma cells with high efficiency. In this study we investigated the sequences critical for retroviral gene expression in two different embryonal carcinoma cell lines, F9 and PCC4. We show that two synergistically acting sequence elements mediate expression in embryonal carcinoma cells. One of these is located within the U3 region of the viral long terminal repeat, and the second one is in the 5' untranslated region of the retrovirus. The latter element, characterized by a single point mutation, affects the level of stable RNA in infected cells, suggesting a regulatory mechanism similar to that of human immunodeficiency virus in human T cells.

Animals↗
PubMed1985

Replication-competent Moloney murine leukemia virus carrying a bacterial suppressor tRNA gene: selective cloning of proviral and flanking host sequences.

A bacterial suppressor tRNA gene was introduced into the long terminal repeat of the Moloney murine leukemia virus (Mo-MuLV) proviral genome to construct a retrovirus that allows easy cloning of the provirus with flanking host sequences. A replication competent virus, Mo-MuLV sup containing a tRNA amber suppressor gene, was derived that replicates to high titers in tissue culture cells and stably transduces the bacterial gene. The recombinant virus can efficiently replicate in vivo when microinjected into midgestation embryos or when injected into newborn mice and displays the same tissue tropism as wild-type Mo-MuLV. The suppressor gene in Mo-MuLV sup is functional in bacteria and allows efficient recovery of proviral genomes. This was shown by ligation of DNA from infected cells to phage lambda Charon 4A arms and selective growth of recombinant phages on su- host cells. All recovered phages contained Mo-MuLV proviral sequences and, because of the high cloning capacity of phage lambda, 1-11 kilobases of flanking host DNA. This virus should facilitate studying virus-host interactions in tissue culture cells and in animals.

Animals↗
PubMed1984

A transcription enhancer acts in vitro over distances of hundreds of base-pairs on both circular and linear templates but not on chromatin-reconstituted DNA.

We have analyzed the effect of nucleosome formation and of the simian virus (SV40) enhancer on the efficiency of in vitro transcription. In a whole cell extract made from HeLa cells, nucleosome assembly on DNA results in the formation of chromatin-like complexes. However, transcription was detectable only when the DNA templates were partially or totally depleted of nucleosomes. On nucleosome-free templates, when the SV40 enhancer was present upstream from the complete SV40 early or rabbit beta-globin promoters, there was a five- to tenfold stimulation of specific transcription. When present upstream from its homologous promoter, the SV40 enhancer activated SV40 early transcription independently of its orientation with respect to the coding sequence. Point mutations known to impair the SV40 enhancer function in vivo had a similar effect in vitro. The extent of the enhancing effect was the same with linear or circular templates. When the SV40 enhancer was inserted upstream from the rabbit beta-globin gene, the activation of transcription was reduced with increasing distance between the enhancer and beta-globin upstream promoter elements, but was still significant over a distance of more than 400 base-pairs.

Chromatin↗
PubMed1984

An enhancer sequence from bovine papilloma virus DNA consists of two essential regions.

A comprehensive structural analysis of an enhancer sequence from bovine papilloma virus DNA is presented based on the construction and functional analysis of 20 mutant derivatives. The results, obtained in CV-1 tissue culture cells, show that this enhancer is a small genetic element--only 40 bp in length--that contains two essential regions. The two regions exhibit homology to each other and to DNA fragments from other viral genomes that also act as enhancers in the assay used. However, there is a certain latitude in the sequences that have enhancer activity in CV-1 cells, even within the critical regions. The results are discussed with respect to the model that enhancers are binding sites for tissue specific transcription factors. The formation of Z-DNA might be involved in the enhancement process. However, single base pair transitions in an 8 base pair stretch of alternating purines and pyrimidines within the BPV enhancer which conserve this pattern destroy enhancer function.

Animals↗
PubMed1984

Avian erythroblastosis virus E26: nucleotide sequence of the tripartite onc gene and of the LTR, and analysis of the cellular prototype of the viral ets sequence.

An intact 5.7-kb provirus of the avian erythroblastosis virus E26 has been molecularly cloned for comparisons with avian myeloblastosis virus (AMV) and other avian tumor viruses. E26 and AMV transform hemopoietic cells exclusively. Both cause myeloblastosis, but E26 also causes erythroblastosis. Sequence analysis of the proviral DNA showed that: The tripartite transforming gene of E26 forms a contiguous reading frame of 1046 codons, including 272 gag, 283 mybE, and 491 ets codons. No subgenomic ets-specific mRNA was detected in E26-infected cells. By contrast, the onc gene of AMV consists almost entirely of a mybA sequence expressed via subgenomic mRNA that extends over the 5' and 3' ends of mybE. mybE is only slightly diverged from the mybA homolog of AMV and even less from the cellular proto-myb sequence with no characteristic mutation that sets apart the two viruses from proto-myb. The U5 region of the long terminal repeat (LTR) of E26 and AMV are colinear and differ only in scattered point mutations. The U3 region of the E26 LTR is different from that of AMV but is colinear and closely related with that of avian carcinoma virus MH2 and also with that of Prague Rous sarcoma virus (RSV), except for an unexpected 16-nucleotide substitution of 22 RSV nucleotides. Upstream of the 3' LTR, the c region of E26 appears to be the same as that of RSV for 70 nucleotides and very similar to those of AMV and MH2 for about 20 to 30 nucleotides. Since the U3s of E26, MH2 and RSV are very closely related and neither MH2 nor RSV show a particular erythroblast tropism, it is possible that the U3 does not play a critical role in the erythroblast tropism of E26. Electrophoretic size analyses of chicken DNA digested with restriction enzymes indicate that DNA fragments totaling over 50 kb hybridize with viral ets DNA.

Alpharetrovirus↗
PubMed1983

Multiple point mutations affecting the simian virus 40 enhancer.

Enhancers, or activators, dramatically increase the transcriptional activity of certain eukaryotic genes. A series of multiple point mutations affecting the simian virus 40 (SV40) enhancer-activator region were generated in order to define the nucleotide sequence required for this function. Three independent assays provided information leading to the identification of nucleotides essential for enhancer function. One class leads to a decrease in gene expression, while the second completely abolishes functional activity. One critical replacement appears to be the first G (guanine) in a sequence TGGAAAG (T, thymine, A, adenine) located in the 5' region of the 72 base-pair repeat of SV40. Comparison of this sequence with nucleotide sequences in other known enhancers leads to the identification of potential related core elements.

Base Sequence↗
PubMed1983

Bovine papilloma virus contains an activator of gene expression at the distal end of the early transcription unit.

Bovine papilloma virus (BPV) contains a cis-acting DNA element which can enhance transcription of distal promoters. Utilizing both direct and indirect transient transfection assays, we showed that a 59-base-pair DNA sequence from the BPV genome could activate the simian virus 40 promoter from distances exceeding 2.5 kilobases and in an orientation-independent manner. In contrast to the promoter 5'-proximal localization of other known viral activators, this element was located immediately 3' to the early polyadenylation signal in the BPV genome. Deletion of these sequences from the BPV genome inactivated the transforming ability of BPV recombinant plasmids. Orientation-independent reinsertion of this 59-base-pair sequence, or alternatively of activator DNA sequences from simian virus 40 or polyoma virus, restored the transforming activity of the BPV recombinant plasmids. Furthermore, the stable transformation frequency of the herpes simplex virus type 1 thymidine kinase gene was enhanced when linked to restriction fragments of BPV DNA which included the defined activator element. This enhancement was orientation independent with respect to the thymidine kinase promoter. The enhancement also appeared to be unrelated to the establishment of the recombinant plasmids as episomes, since in transformed cells these sequences are found linked to high-molecular-weight DNA. We propose that the enhancement of stable transformation frequencies and the activation of transcription units are in this case alternate manifestations of the same biochemical events.

Base Sequence↗
PubMed1982

Segment-specific mutagenesis: extensive mutagenesis of a lac promoter/operator element.

A method for highly efficient segment-specific mutagenesis is described. The method uses as target for sodium bisulfite mutagenesis the DNA single strands of a DNA restriction fragment that had been separated by cloning into base-complementary regions of a pair of phage fd vectors. After repair synthesis in vitro, the mutagenized DNA fragment is recovered by cloning into a nonmutated plasmid vector and analyzed for sequence and by functional tests. By using this method, the nucleotide sequence of a 109-base pair restriction fragment containing the lac promoter/operator from Escherichia coli was extensively modified. More than 90% of the 235 isolates obtained showed a change in phenotype; all of 22 analyzed for their nucleotide sequence were found to carry multiple C leads to T point mutations in up to 60% of the possible target positions. Nevertheless, few isolates showed major changes in promoter activity relative to the nonmutated promoter element, which indicates a high degree of flexibility in the promoter sequence.

Base Sequence↗