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Biomedical subjects

W F Dove

Publications and source records attributed to W F Dove.

At least 19 recordsLinked to original sources
PubMed1992

Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene.

Germ-line mutations of the APC gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominantly inherited disease in humans. Patients with FAP develop multiple benign colorectal tumors. Recently, a mouse lineage that exhibits an autosomal dominantly inherited predisposition to multiple intestinal neoplasia (Min) was described. Linkage analysis showed that the murine homolog of the APC gene (mApc) was tightly linked to the Min locus. Sequence comparison of mApc between normal and Min-affected mice identified a nonsense mutation, which cosegregated with the Min phenotype. This mutation is analogous to those found in FAP kindreds and in sporadic colorectal cancers.

Adenomatous Polyposis Coli↗
PubMed1992

Transient expression in Physarum of a chloramphenicol acetyltransferase gene under the control of actin gene promoters.

We cloned and sequenced two actin promoters from Physarum, and constructed plasmids carrying these promoters upstream of a bacterial chloramphenicol acetyltransferase (cat) gene. We then tested the plasmids for their ability to express cat in Physarum amoebae. We present reliable methods for introducing plasmid DNA into Physarum amoebae by electroporation, and show that expression of the cat gene in amoebae occurs in the presence, but not the absence, of one or the other Physarum actin promoter.

Actins↗
PubMed1992

Cellular and molecular analysis of plasmodium development in Physarum.

The development of an amoeba into a plasmodium involves extensive changes in cellular organisation and gene expression. The genetic basis of a number of recessive mutations that block plasmodium development has been elucidated. The stage at which development becomes abnormal has been determined for all the mutants, as has the terminal phenotype. In order to investigate the changes in gene expression that accompany plasmodium development, a cDNA library has been made using RNA isolated from cell populations in which development was occurring.

Animals↗
PubMed1992

The Min (multiple intestinal neoplasia) mutation: its effect on gut epithelial cell differentiation and interaction with a modifier system.

Min is a fully penetrant dominant mutation that leads to the development of multiple intestinal adenomas throughout the duodenal-to-colonic axis. Min/+ C57BL6/J mice have an average life-span of 120 d. Multi-label immunocytochemical studies of these lesions demonstrate patches of differentiated enterocytes, and scattered enteroendocrine, goblet and Paneth cells. Expression of endogenous marker genes within these differentiated cells can be directly correlated with the position occupied by the adenoma along the duodenal-to-colonic axis and mirrors the regional differentiation of the normal gut epithelium. The presence of multiple lineages in adenomas together with their retention of spatial information suggests that tumorigenesis in Min/+ mice may be initiated in a multipotent stem cell normally located at the base of intestinal crypts. To study the time-dependent properties of these tumors, genetic conditions were sought in which Min/+ animals could survive for up to 300 d. Min is fully penetrant in hybrids with either AKR/J or MA/MyJ. However, the hybrids demonstrate a reduction in the number of intestinal adenomas. Preliminary backcross analysis is consistent with a single major modifier locus unlinked to Min in both the AKR/J and MA/MyJ strains. The increased lifespan of the hybrid animals is also associated with the development of invasive tumors. New tumors do not arise continuously over the lifespan of these animals; instead all adenomas appear to be established by 100 d of age or sooner. These studies indicate that the Min/+ mouse is a powerful model system for analyzing the mechanisms that establish and maintain a balance between proliferation and differentiation in the continuously renewing gut epithelium and for an assessment of the multi-step hypothesis of intestinal neoplasia.

Adenoma↗
PubMed1992

Preferential expression of one beta-tubulin gene during flagellate development in Physarum.

The microbial eukaryote Physarum polycephalum displays several distinct cell types in its life cycle, including amoebae, flagellates and plasmodia. Despite its relative simplicity, Physarum has a tubulin gene family of complexity comparable to that of Drosophila. We have identified beta-tubulin cDNAs from Physarum that are derived from the betA beta-tubulin locus and encode beta 1A tubulin. We have also identified a partial cDNA for the unlinked betB beta-tubulin gene, which encodes beta 1B tubulin. The polypeptide sequences encoded by betA and betB show 99% identity, but the nucleotide sequences show only 85% identity, consistent with an ancient duplication of these genes. The betB gene is expressed in amoebae, flagellates and plasmodia, whereas betA is expressed only in amoebae and flagellates. During the amoeba-flagellate transition the level of betA transcript increases over 100-fold, while the level of betB transcript changes very little. Thus Physarum has a mechanism for regulating the level of discrete beta-tubulin transcripts differentially during flagellate development. A need for this differential regulation could account for the maintenance of the virtually isocoding betA and betB beta-tubulin genes.

Amino Acid Sequence↗
PubMed1991

Meiotic mapping of murine chromosome 17: the string of loci around l(17)-2Pas.

We describe a genetic analysis of l(17)-2Pas, an embryonic lethal mutation on murine chromosome 17. Males transmitted the l(17)-2 allele to only 38% of their offspring, whereas females transmitted this allele at 50%. Two-point crosses revealed tight linkage between l(17)-2 and brachyury (T), and deletion mapping placed l(17)-2 outside of the hairpin-tail deletion (Thp). To map this mutation more precisely, we intercrossed hybrid mice that carry distinct alleles at many classical and DNA loci on chromosome 17 and obtained 172 animals recombinant in the T to H-2 region. Strong positive interference was observed over the 14 cM interval from T to H-2K. Thus, a single recombinant can be informative; one such recombinant places l(17)-2 distal of the molecular marker D17Leh66D. Robust genetic maps can be constructed with multilocus crosses that share anchor loci. DNA markers can be interpolated onto these maps retrospectively.

Animals↗
PubMed1991

Pleiotropic action of the murine quaking locus: structure of the qkv allele.

The spontaneous allele quakingviable (qkv) exerts effects on myelination and spermiogenesis. The defects generated by qkv were not separated in a multilocus mapping cross that provided a mapping resolution of 0.1 centiMorgans (cM). Furthermore, no distortions suggestive of a large chromosomal anomaly associated with qkv were apparent. One plausible interpretation is that the quaking locus contains more than one functional domain, either organized into overlapping genes or expressed by alternative splicing mechanisms. The cloning needed to analyze this locus will be enhanced by the very high resolution of the meiotic mapping cross reported here. The recombinational distances on this qkv map were compressed compared with those previously reported in a high-resolution map for qkl-1, an embryonic lethal allele of quaking induced by ethylnitrosourea. Additional crosses confirmed prior reports that the sex and the genetic background of the heterozygous parent can affect recombinational distances. These joint effects on recombination are strong enough to account for the discrepancy between the two maps. This variability of two-factor map values leads to the preferred multilocus map-building protocol discussed in the accompanying paper.

Animals↗
PubMed1991

Fission yeast promoter-probe vectors based on hygromycin resistance.

We have constructed fission yeast vectors that carry either complete or 5'-truncated alleles of the hph gene, encoding hygromycin B phosphotransferase. We show that plasmid-borne hph can be expressed in fission yeast to confer hygromycin resistance. The vectors permit selection or screening in fission yeast for promoter activity of DNA fragments from other species. We used the vectors to identify several genomic sequences from Physarum that provide promoter function in fission yeast.

Base Sequence↗
PubMed1991

Variable pathways for developmental changes of mitosis and cytokinesis in Physarum polycephalum.

The development of a uninucleate ameba into a multinucleate, syncytial plasmodium in myxomycetes involves a change from the open, astral mitosis of the ameba to the intranuclear, anastral mitosis of the plasmodium, and the omission of cytokinesis from the cell cycle. We describe immunofluorescence microscopic studies of the amebal-plasmodial transition (APT) in Physarum polycephalum. We demonstrate that the reorganization of mitotic spindles commences in uninucleate cells after commitment to plasmodium formation, is completed by the binucleate stage, and occurs via different routes in individual developing cells. Most uninucleate developing cells formed mitotic spindles characteristic either of amebae or of plasmodia. However, chimeric mitotic figures exhibiting features of both amebal and plasmodial mitoses, and a novel star microtubular array were also observed. The loss of the ameba-specific alpha 3-tubulin and the accumulation of the plasmodium-specific beta 2-tubulin isotypes during development were not sufficient to explain the changes in the organization of mitotic spindles. The majority of uninucleate developing cells undergoing astral mitoses (amebal and chimeric) exhibited cytokinetic furrows, whereas cells with the anastral plasmodial mitosis exhibited no furrows. Thus, the transition from astral to anastral mitosis during the APT could be sufficient for the omission of cytokinesis from the cell cycle. However, astral mitosis may not ensure cytokinesis: some cells undergoing amebal or chimeric mitosis contained unilateral cytokinetic furrows or no furrow at all. These cells would, most probably, fail to divide. We suggest that a uninucleate committed cell undergoing amebal or chimeric mitosis can either divide or else form a binucleate cell. In contrast, a uninucleate cell with a mitotic spindle of the plasmodial type gives rise only to a binucleate cells. Further, the decision to enter mitosis after commitment to the APT is independent of the developmental changes in the organization of the mitotic spindle and cytokinesis.

Cell Division↗
PubMed1990

A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse.

In a pedigree derived from a mouse treated with the mutagen ethylnitrosourea, a mutation has been identified that predisposes to spontaneous intestinal cancer. The mutant gene was found to be dominantly expressed and fully penetrant. Affected mice developed multiple adenomas throughout the entire intestinal tract at an early age.

Adenoma↗
PubMed1990

Pahhph-5: a mouse mutant deficient in phenylalanine hydroxylase.

Mutant mice exhibiting heritable hyperphenylalaninemia have been isolated after ethylnitrosourea mutagenesis of the germ line. We describe one mutant pedigree in which phenylalanine hydroxylase activity is severely deficient in homozygotes and reduced in heterozygotes while other biochemical components of phenylalanine catabolism are normal. In homozygotes, injection of phenylalanine causes severe hyperphenylalaninemia and urinary excretion of phenylketones but not hypertyrosinemia. Severe chronic hyperphenylalaninemia can be produced when mutant homozygotes are given phenylalanine in their drinking water. Genetic mapping has localized the mutation to murine chromosome 10 at or near the Pah locus, the structural gene for phenylalanine hydroxylase. This mutant provides a useful genetic animal model affected in the same enzyme as in human phenylketonuria.

Animals↗
PubMed1990

Variable pathways for developmental changes in composition and organization of microtubules in Physarum polycephalum.

The development of uninucleate amoebae into multinucleate plasmodia in myxomycetes is called the amoebal-plasmodial transition (APT). During the APT in Physarum polycephalum the ability to form flagellar axonemes is lost; the astral, open mitosis is replaced by the anastral, closed mitosis; and cytoskeletal microtubules disappear. These changes are accompanied by alterations in the repertoire of expressed tubulins. Using immunofluorescence microscopy we have studied the timing of loss and accumulation of developmentally regulated tubulin isotypes in relation to other cellular events during the APT. We specifically asked whether changes in the composition of microtubules are correlated with changes in their organization. The plasmodium-specific beta 2-tubulin can first be detected in microtubules of uninucleate cells after they become committed to plasmodium formation. However, rare cells are observed that exhibit beta 2-tubulin at earlier or only at later stages of development. Amoeba-specific acetylated alpha 3-tubulin disappears gradually during development. Individual cells differ in the timing of loss of this isotype: alpha 3-tubulin is present in the majority of uninucleate cells, in a fraction of binucleate and quadrinucleate cells, and is absent from larger multinucleate cells. Cytoplasmic microtubules in uninucleate cells are organized by a single microtubule-organizing center (MTOC) juxtaposed to the nucleus. Binucleate cells and quadrinucleate cells exhibit variable numbers of MTOCs. Cytoplasmic microtubules persist during the APT until the stage of plasmodia containing at least 100 nuclei. The lack of a strict correlation between the changes in tubulin composition and changes in organization of microtubular structures indicates that accumulation of beta 2-tubulin and disappearance of alpha 3-tubulin isotypes are not sufficient to bring about reorganization of microtubules during development. Individual cells in a developing population differ not only in the succession of accumulation and loss of developmentally regulated tubulins, but also in the sequences of other cellular changes occurring during the APT.

Cytoskeleton↗
PubMed1989

Mapping to molecular resolution in the T to H-2 region of the mouse genome with a nested set of meiotic recombinants.

We describe a meiotic fine-structure mapping strategy for achieving molecular access to developmental mutations in the mouse. The induction of lethal point mutations with the potent germ-line mutagen N-ethyl-N-nitrosourea has been reported. One lethal mutation of prime interest is an allele at the quaking locus on chromosome 17. To map this mutation, quaking(lethal-1), we have intercrossed hybrid mice that carry distinct alleles at many classical and DNA marker loci on proximal chromosome 17. From this cross we have obtained 337 animals recombinant in the T to H-2 region. This number of crossovers provides a mapping resolution in the size range of single mammalian genes if recombinational hot spots are absent. DNA samples obtained from these recombinant animals can be used retrospectively to map any restriction fragment length polymorphism in the region. This set of DNA samples has been used to map the molecular marker D17RP17 just distal of quaking(lethal-1). With the nested set of crossover DNA samples and appropriate cloning techniques, this tightly linked marker can be used to clone the quaking locus.

Animals↗
PubMed1989

The localization of the divergent beta 2-tubulin isotype in the microtubular arrays of Physarum polycephalum.

The beta 2-tubulin isotype of Physarum polycephalum is only 83% identical in amino acid sequence with the constitutively expressed beta 1B-tubulin and the myxamoeba-specific beta 1A-tubulin isotypes. A polyclonal antibody specific for beta 2-tubulin was used to monitor the subcellular distribution of the beta 2-tubulin antigen in the mitotic spindle of the mature plasmodium - the sole microtubular array in that stage of Physarum. By immunofluorescence, the beta 2-tubulin antigen was detected throughout this anastral mitotic spindle, at all stages of mitosis. Physarum myxamoebae contain astral mitotic spindles and cytoskeletal microtubules. No beta 2-tubulin antigen was detected in the myxamoebal stage. However, as cultures of myxamoebae developed into plasmodia, the beta 2-tubulin antigen was found in the astral mitotic spindles and cytoskeletons in developing cells. Thus, the presence of the plasmodial beta 2-tubulin isotype in a mitotic spindle does not determine a closed, anastral mitosis.

Fluorescent Antibody Technique↗
PubMed1988

Activation of a beta-tubulin gene during early development of the plasmodium in Physarum polycephalum.

Uninucleate amoebae of Physarum polycephalum strain CL undergo apogamic development to form multinucleate plasmodia via an intermediate stage of large, uninucleate cells irreversibly committed to plasmodial development. This amoebal-plasmodial transition involves major changes in tubulin gene expression and the organization of microtubular structures. We analysed the expression of the betC locus, which encodes the plasmodial-specific beta 2-tubulin, during plasmodial development. A key question addressed was the timing of expression of betC in relation to the last open mitosis of the amoeba and the first closed mitosis of the plasmodium during the transition. Culture conditions were improved to yield partly synchronous differentiating cultures containing 50-60% committed cells, in order to facilitate biochemical analysis of development. Northern blotting indicated that betC RNA was virtually absent from amoebae and from early differentiating cultures. However, betC transcripts could already be detected in differentiating cultures containing only 0.1% of committed cells; the relative amount of betC transcripts increased as the percentage of committed cells in differentiating cultures increased. In fully developed plasmodia, there was at least a 330-fold increase in the betC transcript level compared to that in amoebae. We conclude that betC is activated during the amoebal-plasmodial transition immediately before or during the commitment event. Small amounts of beta 2-tubulin polypeptide could first be detected by Western blotting around the stage of the first closed mitosis. Thus beta 2-tubulin may participate in the first closed mitosis that committed cells undergo during their development into plasmodia.

Cell Differentiation↗