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Biomedical subjects

J A Mercer

Publications and source records attributed to J A Mercer.

34 records · Page 2Linked to original sources
PubMed1994

Molecular motors and cell motility in the brain.

The advent of video computer-enhanced microscopy has provided a new vision of cell migrations, growth cones, and fast axonal transport in the nervous system. In images obtained in studies of fast transport in isolated axoplasm from the squid giant axon, a virtual torrent of membrane traffic could be seen moving in both directions. Similarly, examination of growth cones and cell migrations in vitro and in vivo revealed properties of cell motility that were previously unsuspected. Evidence has accumulated that many of these activities are driven by a variety of microtubule and microfilament based motors.

Actins↗
PubMed1993

Molecular cloning of a mouse myosin I expressed in brain.

We have isolated two cDNAs that encode putative myosin I heavy chains by polymerase chain reaction amplification of brain cDNA with degenerate oligodeoxynucleotide primers representing myosin I-specific conserved amino acid sequences. We report the complete deduced amino acid sequence of one of these cDNAs. The sequences is most similar to those of the avian and bovine brush border myosin Is, with five putative calmodulin-binding repeats at the head-tail junction. Northern analysis demonstrates that this myosin heavy chain, unlike the brush border myosins, is expressed in many tissues.

Amino Acid Sequence↗
PubMed1992

An interspecific linkage map of mouse chromosome 15 positioned with respect to the centromere.

We have used an interspecific backcross between C57BL/6J and Mus spretus to derive a molecular genetic linkage map of chromosome 15 that includes 25 molecular markers and spans 93% of the estimated length of chromosome 15. Using a second interspecific backcross that was analyzed with a centromere-specific marker, we were also able to position our map with respect to the chromosome 15 centromere. This map provides molecular access to many discrete regions on chromosome 15, thus providing a framework for establishing relationships between cloned DNA markers and known mouse mutations and for identifying homologous genes in mice and humans that may be involved in disease.

Animals↗
PubMed1991

Novel myosin heavy chain encoded by murine dilute coat colour locus.

Hundreds of murine dilute mutations have been identified and analysed, making dilute one of the best genetically characterized of all mammalian loci. The recessive dilute (d) coat colour mutation carried by many inbred strains of mice produces a lightening of coat colour, caused by an abnormal adendritic melanocyte morphology that results in an uneven release of pigment granules into the developing hair shaft. Most dilute alleles (dilute-lethal) also produce a neurological defect, characterized by convulsions and opisthotonus, apparent at 8-10 days of age and continuing until the death of the animal at 2-3 weeks of age. The discovery that the original dilute allele (now termed dilute-viral or dV) is the result of the integration of an ecotropic murine leukaemia provirus has allowed the cloning of genomic DNA and in this study complementary DNA, from the dilute locus. The predicted dilute amino-acid sequence indicates that dilute encodes a novel type of myosin heavy chain, with a tail, or C-terminal, region that has elements of both type II (alpha-helical coiled-coil) and type I (non-coiled-coil) myosin heavy chains. Dilute transcripts are differentially expressed in both embryonic and adult tissues and are very abundant in neurons of the central nervous system, cephalic ganglia, and spinal ganglia. These results suggest an important role for the dilute gene product in the elaboration, maintenance, or function of cellular processes of melanocytes and neurons.

Amino Acid Sequence↗
PubMed1991

Selective gene expression in failing human heart. Quantification of steady-state levels of messenger RNA in endomyocardial biopsies using the polymerase chain reaction.

BACKGROUND: Evaluation of gene expression in failing human heart has been limited by the availability of cardiac tissue. METHODS AND RESULTS: We used the polymerase chain reaction (PCR) to assess gene expression in small quantities of failing and nonfailing human heart. PCR is a powerful new molecular biological tool that allows a small quantity of DNA to be amplified as much as 1 million-fold. Total RNA was extracted from 3-5 mg samples of human heart and reverse-transcribed to complementary DNA (cDNA). With selected oligonucleotide primers, we used PCR to amplify cDNAs encoding atrial natriuretic peptide, beta-myosin heavy chain, phospholamban, and cytoskeletal beta-actin. To quantify the relative levels of messenger RNA (mRNA) in human heart, a known amount of a control RNA was present in the reverse transcription and PCR reactions. The amount of mRNA in the sample could therefore be assessed in relation to the amount of control product. The control RNA was transcribed from a synthetic DNA template containing primers complementary to those used to amplify the cDNAs of interest. Atrial natriuretic factor mRNA could not be detected in nonfailing human heart but was abundant in ventricular myocardium from failing human heart. In contrast, steady-state levels of phospholamban mRNA decreased, whereas levels of beta-myosin heavy-chain mRNA were unchanged with heart failure. CONCLUSIONS: Alterations in gene expression in the failing human heart appear to be selective. In addition, the present study suggests that PCR provides a rapid and economical way to quantify the expression of multiple genes of interest in endomyocardial biopsy specimens and may therefore be used to advance our understanding of heart muscle disease.

Biopsy↗
PubMed1991

Patterns of Evi-1 expression in embryonic and adult tissues suggest that Evi-1 plays an important regulatory role in mouse development.

Evi-1 is a putative protooncogene first identified as a common site of retroviral integration in murine myeloid leukemias. It encodes a 145 x 10(3) Mr nuclear DNA-binding protein that contains ten zinc-finger motifs separated into two domains, as well as an acidic domain. These features suggest that Evi-1 encodes a transcriptional regulatory protein. In Drosophila, zinc-finger proteins such as Kruppel are involved in body plan patterning, and exhibit a spatially restricted pattern of expression in the embryo. To determine if Evi-1 may be involved in morphogenetic processes in the mouse embryo, we have performed in situ hybridization and Northern blot analysis on embryonic and adult mouse tissues to delineate the spatial and temporal pattern of Evi-1 expression. Our results show that Evi-1 is expressed at high levels in a few tissues in the embryo and is widely expressed, albeit at generally low levels, in the adult. Regions that exhibit high-level expression in the embryo include: the urinary system and the Mullerian ducts; the bronchial epithelium of the lung; focal areas within the nasal cavities; the endocardial cushions and truncus swellings in the heart; and the developing limbs. Expression in the limb occurs at the highest levels from 9.5 to 12.5 days, is present in both hind and forelimbs, is absent at the apical ectodermal ridge, and does not appear to establish a gradient.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

A molecular genetic linkage map of mouse chromosome 2.

Interspecific backcross mice were used to create a molecular genetic linkage map of chromosome 2. Genomic DNAs from N2 progeny were subjected to Southern blot analysis using molecular probes that identified the Abl, Acra, Ass, C5, Cas-1, Fshb, Gcg, Hox-5.1, Jgf-1, Kras-3, Ltk, Pax-1, Prn-p, and Spna-2 loci; these loci were added to the 11 loci previously mapped to the distal region of chromosome 2 in the same interspecific backcross to generate a composite multilocus linkage map. Several loci mapped near, and may be the same as, known mutations. Comparisons between the mouse and the human genomes indicate that mouse chromosome 2 contains regions homologous to at least six human chromosomes. Mouse models for human diseases are discussed.

Animals↗
PubMed1990

Lack of ecotropic virus involvement in induction of lymphomas in DBA/2J mice by 7,12-dimethylbenz(a)anthracene.

DBA/2 mice carry a single endogenous ecotropic murine leukemia provirus, Emv-3, that is replication defective because of a single nucleotide substitution in codon 3 of p15gag. However, when weanling DBA/2 mice are treated percutaneously with 7,12-dimethylbenz(a)anthracene (DMBA), ecotropic virus replication is induced in almost all of the treated mice. Previous studies have shown that this induction results from DMBA-induced reverse mutations in codon 3 that allow efficient virus replication. In addition to ecotropic virus replication, DMBA also induces lymphomas in 100% of the treated mice. These results have raised the possibility that ecotropic virus replication is causally associated with the development of lymphomas in DBA/2 mice, perhaps via the insertional activation or mutation of cellular proto-oncogenes. To test this possibility, we compared lymphoma incidence after percutaneous DMBA treatment in DBA/2J-dv/dv mice, which carry two copies of Emv-3, with lymphoma incidence in DBA/2J-d+18J/d+18J mice, which lost both copies of Emv-3 by homologous recombination involving the long terminal repeat sequences. The results of this study conclusively demonstrated that Emv-3 is not causally associated with the development of DMBA-induced lymphomas in DBA/2J mice. Interestingly, histopathological and molecular analyses of the lymphomas indicated that the majority of the lymphomas in both strains of mice were of the B-cell lineage. This was unanticipated, since the majority of chemically induced lymphomas in other inbred strains are thymic lymphomas, presumably of the T-cell lineage. Thus, DBA/2 mice appear to present a unique model system for the investigation of chemically induced B-cell lymphomas in mice.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1990

Mechanism of chemical activation of expression of the endogenous ecotropic murine leukemia provirus Emv-3.

DBA/2 mice carry a single endogenous ecotropic murine leukemia provirus, Emv-3. This provirus is defective; it is very poorly expressed in young DBA/2 mice. The defect in Emv-3 is caused by a single base substitution in codon 3 of p15gag. The resulting amino acid substitution inhibits myristylation of the gag precursor and subsequent virus assembly. Despite this defect, percutaneous treatment of DBA/2 mice with the carcinogen and mutagen 7,12-dimethylbenz[a]anthracene (DMBA) induces ecotropic murine leukemia virus replication in virtually all treated mice. We hypothesized that this induction is the result of a DMBA-induced reverse mutation in codon 3 of p15gag which allows for efficient myristylation. We tested this hypothesis by isolating ecotropic viruses from DMBA-treated mice and determining the DNA sequences of selected regions of p15gag, including codon 3. In support of the above-described model, all of the viruses examined contained single nucleotide substitutions in codon 3. In addition, most of the replication-competent viruses that were sequenced appeared to result from simple mutation of Emv-3 rather than recombination with other endogenous murine leukemia viruses. These studies may provide a basis for development of a sensitive assay for the mutagenic activity of a variety of chemical carcinogens in vivo.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

Pathogenesis of murine cytomegalovirus infection: identification of infected cells in the spleen during acute and latent infections.

Spleen cells which replicate murine cytomegalovirus (MCMV) during acute infection in vivo were identified by electron microscopy and combined immunocytochemical staining and in situ cytohybridization. Most infected cells, as defined by in situ hybridization for viral RNA with MCMV-specific probes, were shown to be positive for factor VIII-related antigen and negative for Ia, Thy-1, and F4/80 antigens. Electron microscopic ultrastructural observations indicated that the infected cells in the spleen are predominantly sinusoidal-lining cells. We also studied reactivation of MCMV from latently infected mice by cocultivation of spleen cells with mouse embryo fibroblasts. Virus was only recovered from cells in preparations of stromal (or reticular) fragments, and not from spleen cell suspensions. Neither removal of immunoglobulin-bearing cells from the stromal fragments by panning nor depletion of Thy-1- and Ia-bearing stromal cells by treatment with monoclonal antibodies and complement reduced the frequency of reactivation of MCMV. These data suggest that T lymphocytes, mature B lymphocytes, and other Ia-bearing cells are not predominant reservoirs of latent MCMV.

Acute Disease↗
PubMed1988

Developmental regulation of ovarian-specific Mos expression.

To gain better insight into the physiologic role of the Mos proto-oncogene in mice we have been studying the cell type and developmental specificity of its expression. It was previously shown that in adult mice, Mos is transcribed predominantly in ovaries and in haploid spermatids of the testes. Using in situ hybridization techniques we now show that in the ovary, Mos is expressed in oocytes, but not in somatic cells. In these analyses Mos transcripts are not detected in primary resting oocytes, but accumulate soon after the oocyte enters the growth phase. High levels of Mos RNA are present throughout oocyte growth and maturation. Mos RNA is also abundant in ovulated eggs prior to fertilization. Following fertilization, however, there is a dramatic loss of Mos RNA, as evidenced by the failure to detect hybridization in late one-cell embryos. The narrow developmental window for Mos transcription defined by this study suggests a role for ovarian Mos in one or more of the processes of oocyte growth, meiotic maturation, ovulation, or fertilization.

Age Factors↗
PubMed1986

Pathogenesis of acute murine cytomegalovirus infection in resistant and susceptible strains of mice.

We have characterized the progress of acute murine cytomegalovirus (MCMV) infection in the spleen, liver, and salivary gland of susceptible (BALB/c) and resistant (C3H) strains of mice after intraperitoneal inoculation. Viral replication was analyzed by virus titration, infectious-center assays, and in situ cytohybridization with cloned subgenomic fragments of the MCMV genome. The most striking differences between strains were observed in the spleen. At 24 h postinfection (p.i.), both strains had a similar number of infected spleen cells. At 48 h p.i., BALB/c mice showed marked dissemination of the splenic infection which continued until 96 h p.i. In contrast, the number of infected C3H spleen cells did not increase from the 24-h level but declined later on. This early block in dissemination of MCMV infection in C3H mouse spleens was not a result of the H-2k haplotype, as BALB.K (H-2k) mice, which show an intermediate level of resistance to MCMV infection, exhibited dissemination of the infection between 24 and 48 h p.i., albeit at a reduced level. However, between 72 and 96 h p.i., we observed a decline in the number of infected spleen cells in BALB.K mice similar to that observed in C3H mice. We also demonstrated by Southern blot analysis of DNA from the infected spleen cells that the termini of the MCMV genome fuse after in vivo infection.

Animals↗
PubMed1983

Molecular cloning and restriction endonuclease mapping of the murine cytomegalovirus genome (Smith Strain).

We have cloned EcoRI and HindIII fragments of the Smith strain of murine cytomegalovirus (MCMV) in the plasmid vector pACYC184. These cloned fragments were used to establish a restriction endonuclease map of the genome with respect to the EcoRI and HindIII sites. The map was constructed on the basis of data derived from cross-hybridizations of EcoRI and HindIII cloned fragments, double-digestions of the cloned fragments with EcoRI and HindIII, and hybridization of cloned HindIII fragments to Southern blots of MCMV DNA cleaved with EcoRI. From our mapping data, we have determined that the length of the MCMV genome is approximately 240 kbp. The genome does not appear to undergo inversions and lacks detectable repeated sequences. One HindIII cloned fragment was obtained which contained both HindIII termini. The existence of this fragment may be related to the mode of replication of the MCMV genome.

Animals↗
PubMed1983

Transcription in mouse embryo cells permissively infected by murine cytomegalovirus.

The sites of transcription and abundance of steady-state cytoplasmic viral RNA in murine cytomegalovirus (Smith strain) infected mouse embryo cells were analyzed. Cloned subgenomic DNA fragments were labeled with 32P and hybridized to filters containing polyadenylated RNA extracted from cells at immediate-early, early, and late times in the infection. The pattern of transcription was distinctive at each time point. Immediate-early transcription occurred primarily at 0.770-0.816 map units. Minor sites of immediate-early transcription were clustered at 0.671-0.861 map units and at the termini of the genome (0.944-0.002 map units). Early transcripts were detected from the entire genome with the exception of a fragment at 0.278-0.305 map units. The site of major immediate-early transcription at 0.770-0.816 map units was represented less abundantly while the concentration of RNA from most other sites of immediate-early transcription was increased at the early time point. The most abundant site of early transcription was at 0.840-0.861 map units. RNA from late in the infection hybridized to all subgenomic fragments. The highest concentration of late RNA transcripts was detected with fragments located at 0.444-0.770 map units. In contrast, late RNA transcripts from both ends of the genome were present at a concentration equal to or lower than that seen at the early time point, and the concentration of late RNA from the major early site (0.840-0.861 map units) was significantly decreased. We also detected uninfected mouse cell RNA with three separate subgenomic EcoRI fragments.

Animals↗