Biomedical subjects

H Scrable

Publications and source records attributed to H Scrable.

10 recordsLinked to original sources

PubMed2001

The lac operator-repressor system is functional in the mouse.

We report the successful transfer of a fully functional lac operator-repressor gene regulatory system to the mouse. The key component is a lac repressor transgene that resembles a typical mammalian gene both in codon usage and structure and expresses functional levels of repressor protein in the animal. We used the repressor to regulate the expression of a mammalian reporter gene consisting of the tyrosinase promoter embedded with three short lac operator sequences and the tyrosinase coding sequence. Pigmentation of the mouse was controlled by the interaction of the lac repressor with the regulatable Tyrosinase transgene in a manner that was fully reversible by the lactose analog IPTG. Direct control of mammalian promoters by the lac repressor provides tight, reversible regulation, predictable levels of de-repressed expression, and the promise of reversible control of the endogenous genome.

Animals↗

PubMed1999

A genetic program for deletion of foreign DNA from the mammalian genome.

Mammalian genomes are in constant jeopardy of invasion by prokaryotic DNA sequences because of their extensive exposure to bacteria; however, mammalian genomes appear to be protected from horizontal transmission of bacterial DNA. Transgenic mice provide a convenient model system for investigating the capacity of mammalian genomes in vivo to retain, silence, and/or reject foreign DNAs. We have previously reported that bacterial genes encoding the Lac repressor (lacI) are subject to sequence-dependent methylation and silencing in the transgenic mouse. In this paper, we report that bacterially derived lacI transgenes, but not their mammalian counterparts, can also be eliminated from the somatic cell DNA of affected animals. This somatic instability is heritable, strain-dependent, and conferred in cis. Our data are consistent with a model of genome surveillance in the mouse which can lead to loss of foreign DNA and which may be analogous to restriction-modification systems that maintain the integrity of the bacterial genome.

Animals↗

PubMed1997

A genetic animal model of human neocortical heterotopia associated with seizures.

Malformations of the human neocortex are commonly associated with developmental delays, mental retardation, and epilepsy. This study describes a novel neurologically mutant rat exhibiting a forebrain anomaly resembling the human neuronal migration disorder of double cortex. This mutant displays a telencephalic internal structural heterotopia (tish) that is inherited in an autosomal recessive manner. The bilateral heterotopia is prominent below the frontal and parietal neocortices but is rarely observed in temporal neocortex. Neurons in the heterotopia exhibit neocortical-like morphologies and send typical projections to subcortical sites; however, characteristic lamination and radial orientation are disturbed in the heterotopia. The period of neurogenesis during which cells in the heterotopia are generated is the same as in the normotopic neocortex; however, the cells in the heterotopia exhibit a "rim-to-core" neurogenetic pattern rather than the characteristic "inside-out" pattern observed in normotopic neocortex. Similar to the human syndrome of double cortex, some of the animals with the tish phenotype exhibit spontaneous recurrent electrographic and behavioral seizures. The tish rat is a unique neurological mutant that shares several features with a human cortical malformation associated with epilepsy. On the basis of its regional connectivity, histological composition, and period of neurogenesis, the heterotopic region in the tish rat is neocortical in nature. This neurological mutant represents a novel model system for investigating mechanisms of aberrant neocortical development and is likely to provide insights into the cellular and molecular events contributing to seizure development in dysplastic neocortex.

Animals↗

PubMed1997

Activation of the lac repressor in the transgenic mouse.

We have introduced sequences encoding the lac repressor of Escherichia coli into the genome of the mouse. One sequence was derived from the bacterial lac operon and the other was created by re-encoding the amino acid sequence of lacI with mammalian codons. Both versions are driven by an identical promoter fragment derived from the human beta-actin locus and were microinjected into genetically identical pronuclear stage embryos. All transgenes utilizing the bacterial coding sequence were transcriptionally silent in all somatic tissues tested. The sequence re-encoded with mammalian codons was transcriptionally active at all transgene loci and expressed ubiquitously. Using methylation-sensitive enzymes, we have determined the methylation status of lac repressor transgenes encoded by either the bacterial or mammalian sequence. The highly divergent bacterial sequence was hypermethylated at all transgene loci, while the mammalian sequence was only hypermethylated at a high copy number locus. This may reflect a normal process that protects the genome from acquiring new material that has an abnormally divergent sequence or structure.

Actins↗

PubMed1993

Reassessment of breakpoints in chromosome 11p15.

Specific tumor-associated rearrangements involving the regions 11p13 and 11p15 have been extensively documented. However, cytogenetic definition of the breakpoints occurring at the boundaries of these two regions was not precise enough to correlate with the molecular data. Using probes corresponding to the genes coding for MYOD1, CTSD, LDHA, and RBTN1 and to the anonymous sequence D11S776, we have reassessed the breakpoints of three hybrids (J1.10, BID7, and NYX3.1) and confirmed the localization or more precisely mapped these four genes and the anonymous DNA marker on different subregions of 11pter-->p13, including the smallest region of 11p15.5 duplicated in a patient with Beckwith-Wiedemann syndrome.

Animals↗

PubMed1992

Control of Ha-ras-mediated mammalian cell transformation by Escherichia coli regulatory elements.

Inducible eukaryotic promoters, particularly those responsive to glucocorticoids or heavy metals, have been extensively used to study the consequences of induction of a target gene in mammalian cells. An alternative approach, intended to improve the selectivity of gene induction and to minimize perturbation of chromatin structure, is to utilize elements from prokaryotic regulatory systems that are unlikely to be shared by mammalian cells. We and others previously have shown that the lac repressor can function in mammalian cells and repress expression of a reporter gene controlled by a eukaryotic promoter containing a lac operator sequence. The reporter gene can be specifically activated by administration of the lactose analogue isopropyl beta-D-thiogalactoside. The target genes tested so far encode the biochemical and histochemical markers, chloramphenicol acetyltransferase and beta-galactosidase. As a model system to establish whether or not the lactose regulatory system can also be used to effectively modulate a cellular phenotype, NIH 3T3 cells were made transgenic for a constitutively expressed lacI gene, encoding lac repressor, and an activated human Ha-ras gene directed by a simian virus 40 promoter within which a lac operator sequence had been embedded. In the absence of inducer, cells were phenotypically untransformed. Consequent to isopropyl beta-D-thiogalactoside administration, four biological end points characteristic of a transformed phenotype were observed. Consistent with transformation, the cells assumed an altered morphology; they displayed a reduced density inhibition of growth; they acquired the capacity to grow in soft agar; and they were released from a G0 block following serum deprivation. The data demonstrate that regulation of gene expression in mammalian cells by the lactose regulatory system affords a sensitive means for modulating cellular phenotype.

3T3 Cells↗

PubMed1992

The protein product of the neurofibromatosis type 1 gene is expressed at highest abundance in neurons, Schwann cells, and oligodendrocytes.

von Recklinghausen's neurofibromatosis (NF1) is a common inherited human disease. The events leading to patient symptoms from inheritance of a defective NF1 gene are unknown. Since knowledge of the distribution of the normal NF1 gene product should improve understanding of the pathogenesis of the disease, we raised antibodies against peptides coded by portions of the recently cloned human NF1 cDNA. These antibodies specifically recognize a 220 kd protein (neurofibromin) in both human and rat spinal cord. Neurofibromin is most abundant in the nervous system. Immunostaining of tissue sections indicates that neurons, oligodendrocytes, and nonmyelinating Schwann cells contain neurofibromin while astrocytes and myelinating Schwann cells do not. These results suggest a function for neurofibromin in the normal nervous system. Some NF1 disease manifestations, such as Schwann cell tumors and learning disabilities, may result from abnormalities in the cells that express neurofibromin.

Animals↗

PubMed1991

Muscle-specific gene expression in rhabdomyosarcomas and stages of human fetal skeletal muscle development.

Rhabdomyosarcomas (RMS) bear a morphological resemblance to developing striated muscle. It has been reported that two histologically distinct subtypes of RMS, embryonal and alveolar, behave differently in many clinical aspects, such as age distribution, primary site, and prognosis. We have investigated the expression of various genes, which are preferentially expressed in normal muscle tissue or cell culture (actins, myosins, and creatine kinases, and myogenic regulatory genes MyoD, myogenin, MRF4, and Myf5), in embryonal and alveolar subtypes and compared the results to the stages of developing human fetal limb muscle. The data showed that each of the RMS tumors tested, regardless of histological features, expressed MyoD1 and MRF4 transcripts. Expression of the myogenin gene was detectable in all alveolar RMS (n = 8), whereas only 5 of 8 embryonal RMS expressed myogenin transcripts. Trace levels of Myf5 transcripts were visible in all alveolar RMS and 7 of 8 embryonal RMS. The alpha-skeletal, alpha-cardiac, and beta- and gamma-cytoplasmic actin transcripts were detectable in all alveolar RMS. While the beta- and gamma-cytoplasmic actin transcripts were evident in all embryonal RMS, only 3 of 8 and 6 of 8 embryonal RMS expressed detectable levels of alpha-skeletal and alpha-cardiac actin transcripts, respectively. The embryonic form of myosin heavy chain was detectable in 1 of 8 of each type of tumor. Myosin light chain-1/3 transcripts were detectable in 4 of 8 alveolar RMS and 5 of 8 embryonal RMS. Brain creatine kinase transcripts were detectable in all alveolar RMS and 4 of 8 embryonal RMS, whereas none of the RMS samples contained detectable levels of the muscle form of creatine kinase. A comparison of the expression profiles with those of normal developing human fetal limb muscle (from 7.5 to 24 weeks' gestation) suggested that RMS resembled a relatively restricted segment of fetal muscle development. Furthermore, the data also showed a great deal of overlap in the differentiation state achieved by the embryonal and alveolar subtypes of RMS, suggesting that the clinicopathological difference between these two may not be due to malignant transformation of the cells from different positions in the normal pathway of myogenesis.

Actins↗

PubMed1989

Molecular differential pathology of rhabdomyosarcoma.

Tumors of the soft tissues are classified histogenetically according to their phenotypic resemblance to normal adult tissue. Here we describe molecular approaches that make it possible to distinguish between one class of these tumors, rhabdomyosarcoma, and other small-, round-cell tumors. We show that the ascertainment of specific genotypic changes can be used to distinguish further between the embryonal and alveolar subtypes of rhabdomyosarcoma. We tested our model in two ways: first, in a retrospective analysis of diagnostically problematic cases of undifferentiated, small-cell tumors and, second, in a blind study of pediatric tumors. Rhabdomyosarcoma was correctly identified in all cases using this strategy alone. The underlying simplicity of the strategy used to define rhabdomyosarcoma subtypes with molecular markers suggests a model by which tumors can be unequivocally identified, which may apply equally well to other human solid tumors.

Adolescent↗

PubMed1989

A model for embryonal rhabdomyosarcoma tumorigenesis that involves genome imprinting.

Embryonal rhabdomyosarcomas (malignant pediatric tumors of striated muscle origin) have been shown to arise from cells that are clonally isodisomic for loci on chromosome 11p. We determined the parental origin of alleles in this genomic region in familial and sporadic cases of this disease and found that isodisomic chromosome 11p alleles in each tumor were of paternal origin. We have developed a modification of Knudson's two-hit model from these data that is capable of explaining the preferential allele retention and of resolving the apparent contradiction between such specific and early events in several embryonal tumors and discrepancies in the inheritance of predisposition in some of these diseases.

Alleles↗