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A Villasante

Publications and source records attributed to A Villasante.

31 records · Page 2Linked to original sources
PubMed1986

Oncogene activation and surface markers in mouse lymphomas induced by radiation and nitrosomethylurea.

Thymic lymphomas have been induced by gamma-radiation and treatment with the chemical nitrosomethylurea in different mice strains. As indicated by the NIH 3T3 focus forming assay, a significant percentage of the tumors contain activated oncogenes of the ras family (K or N). Cloning and sequencing has enabled us to identify single base mutations as the only significant alteration present in the activated oncogenes. These alterations result in the substitution of amino-acid 12 or 61 of the p21 product of the ras genes. With the use of synthetic oligonucleotides it has been found that the tumors do not all contain the same mutation and in one case so far the normal allele is absent.

Animals↗
PubMed1986

Brain-specific expression of MAP2 detected using a cloned cDNA probe.

We describe the isolation of a set of overlapping cDNAs encoding mouse microtubule associated protein 2 (MAP2), using an anti-MAP antiserum to screen a mouse brain cDNA expression library cloned in bacteriophage lambda gt11. The authenticity of these clones was established by the following criteria: (a) three non-identical clones each expressing a MAP2 immunoreactive fusion protein were independently isolated from the expression library; each of these clones cross-hybridized at the nucleic acid level; (b) anti-MAP antiserum was affinity purified using nitrocellulose-bound fusion protein; these antibodies detected only MAP2 in an immunoblot experiment of whole brain microtubule protein; (c) a series of cDNA "walking" experiments was done so as to obtain a non-overlapping cloned fragment corresponding to a different part of the same mRNA molecule. Upon subcloning this non-overlapping fragment into plasmid expression vectors, a fusion protein was synthesized that was immunoreactive with an anti-MAP2 specific antiserum. Thus, a single contiguous cloned mRNA molecule encodes at least two MAP2-specific epitopes; (d) the cloned cDNA probes detect an mRNA species in mouse brain that is of a size (approximately 9 kb) consistent with the coding capacity required by a 250,000-D protein. The MAP2-specific cloned cDNA probes were used in RNA blot transfer experiments to assay for the presence of MAP2 mRNA in a variety of mouse tissues. Though brain contained abundant quantities of MAP2 mRNA, no corresponding sequences were detectable in RNA prepared from liver, kidney, spleen, stomach, or thymus. We conclude that the expression of MAP2 is brain-specific. Use of the MAP2 specific cDNA probes in genomic Southern blot transfer experiments showed the presence of a single gene encoding MAP2 in mouse. The microheterogeneity of MAP2 is therefore ascribable either to alternative splicing within a single gene, or to posttranslational modification(s), or both. Under conditions of low stringency, the mouse MAP2 cDNA probe cross-hybridizes with genomic sequences from rat, human, and (weakly) chicken, but not with sequences in frog, Drosophila, or sea urchin DNA. Thus, there is significant interspecies divergence of MAP2 sequences. The implications of the above observations are discussed in relationship to the potential biological function of MAP2.

Animals↗
PubMed1986

The mammalian beta-tubulin repertoire: hematopoietic expression of a novel, heterologous beta-tubulin isotype.

We describe the structure of a novel and unusually heterologous beta-tubulin isotype (M beta 1) isolated from a mouse bone marrow cDNA library, and a second isotype (M beta 3) isolated from a mouse testis cDNA library. Comparison of M beta 1 and M beta 3 with the completed (M beta 4, M beta 5) or extended (M beta 2) sequence of three previously described beta-tubulin isotypes shows that each includes a distinctive carboxy-terminal region, in addition to multiple amino acid substitutions throughout the polypeptide chain. In every case where a mammalian interspecies comparison can be made, both the carboxy-terminal and internal amino acid substitutions that distinguish one isotype from another are absolutely conserved. We conclude that these characteristic differences are important in determining functional distinctions between different kinds of microtubule. The amino acid homologies between M beta 2, M beta 3, M beta 4, and M beta 5 are in the range of 95-97%; however the homology between M beta 1 and all the other isotypes is very much less (78%). The dramatic divergence in M beta 1 is due to multiple changes that occur throughout the polypeptide chain. The overall level of expression of M beta 1 is low, and is restricted to those tissues (bone marrow, spleen, developing liver and lung) that are active in hematopoiesis in the mouse. We predict that the M beta 1 isotype is functionally specialized for assembly into the mammalian marginal band.

Amino Acid Sequence↗
PubMed1986

Six mouse alpha-tubulin mRNAs encode five distinct isotypes: testis-specific expression of two sister genes.

Five mouse alpha-tubulin isotypes are described, each distinguished by the presence of unique amino acid substitutions within the coding region. Most, though not all of these isotype-specific amino acids, are clustered at the carboxy terminus. One of the alpha-tubulin isotypes described is expressed exclusively in testis and is encoded by two closely related genes (M alpha 3 and M alpha 7) which have homologous 3' untranslated regions but which differ at multiple third codon positions and in their 5' untranslated regions. We show that a subfamily of alpha-tubulin genes encoding the same testis-specific isotype also exists in humans. Thus, we conclude that the duplication event leading to a pair of genes encoding a testis-specific alpha-tubulin isotype predated the mammalian radiation, and both members of the duplicated sequence have been maintained since species divergence. A second alpha-tubulin gene, M alpha 6, is expressed ubiquitously at a low level, whereas a third gene, M alpha 4, is unique in that it does not encode a carboxy-terminal tyrosine residue. This gene yields two transcripts: a 1.8-kilobase (kb) mRNA that is abundant in muscle and a 2.4-kb mRNA that is abundant in testis. Whereas the 1.8-kb mRNA encodes a distinct alpha-tubulin isotype, the 2.4-kb mRNA is defective in that the methionine residue required for translational initiation is missing. Patterns of developmental expression of the various alpha-tubulin isotypes are presented. Our data support the view that individual tubulin isotypes are capable of conferring functional specificity on different kinds of microtubules.

Amino Acid Sequence↗
PubMed1985

Loss of the normal N-ras allele in a mouse thymic lymphoma induced by a chemical carcinogen.

Young mice injected with the carcinogen N-nitroso-N-methylurea develop thymic lymphomas 2-4 months later. We previously have shown that these tumors frequently contain an activated N-ras gene that can transform rodent fibroblasts in vitro. We report here the intron/exon structure of such an activated N-ras gene and the sequence of its four coding exons. A single nucleotide change is responsible for the transforming alteration, a C----A transversion in the first base of codon 61. Through the use of synthetic oligonucleotides as hybridization probes, we show that this tumor lacks the normal allele of the N-ras gene. The implications of this finding for oncogene dominance are discussed.

Alleles↗
PubMed1984

Activation of a c-K-ras oncogene by somatic mutation in mouse lymphomas induced by gamma radiation.

Mouse tumors induced by gamma radiation are a useful model system for oncogenesis. DNA from such tumors contains an activated K-ras oncogene that can transform NIH 3T3 cells. This report describes the cloning of a fragment of the mouse K-ras oncogene containing the first exon from both a transformant in rat-2 cells and the brain of the same mouse that developed the tumor. Hybrid constructs containing one of the two pieces were made and only the plasmid including the first exon from the transformant gave rise to foci in NIH 3T3 cells. There was only a single base difference (G----A) in the exonic sequence, which changed glycine to aspartic acid in the transformant. By use of a synthetic oligonucleotide the presence of the mutation was demonstrated in the original tumor, ruling out modifications during DNA-mediated gene transfer and indicating that the alteration was present in the thymic lymphoma but absent from other nonmalignant tissue. The results are compatible with gamma radiation being a source of point mutations.

Animals↗
PubMed1984

Isolation, characterization, and chromosome assignment of mouse N-ras gene from carcinogen-induced thymic lymphoma.

Treatment of mice with the carcinogen N-methylnitrosourea results in the development of thymic lymphomas with frequent involvement of the N-ras oncogene. The activated mouse N-ras gene was isolated from one of these lymphomas and, by transformation in concert with restriction digestion, a map of the gene was prepared and its approximate boundaries were determined. By means of somatic cell hybrids the normal N-ras gene was found to be unlinked to other members of the ras gene family.

Animals↗
PubMed1981

Binding of microtubule protein to DNA and chromatin: possibility of simultaneous linkage of microtubule to nucleic and assembly of the microtubule structure.

Microtubule protein binds to DNA through microtubule associated polypeptides (MAPs). Among MAPs there is one high molecular weight polypeptide (MAP2) which interacts with DNA fundamentally through certain polynucleotide sequences. This interaction is not affected by the presence of histones and other chromosomal proteins. DNA can associate to assembled microtubules and when a determinate DNA/protein ratio is reached the nucleic acid behaves as a microtubule associated molecule. The nucleic acid fragments which preferentially bind to microtubules have been isolated and characterized. These fragments contain DNA regions enriched in repetitive sequences that hybridizes preferentially to the pericentromeric zone of metaphase chromosomes. These results give further support to the model of interaction microtubule-chromosome based upon the mediator function of the microtubule associated proteins.

Animals↗
PubMed1981

Factors implicated in determining the structure of zinc tubulin-sheets: lateral tubulin-tubulin interaction is promoted by the presence of zinc.

Addition of increasing amounts of zinc to a cold microtubule protein solution results in the disappearance of 30 S oligomer found in the absence of that cation and in the appearance of new tubulin oligomers, 90 S and 23 S. When a microtubule protein solution is warmed in the presence of zinc, tubulin-sheets are assembled. We have tested the influence of microtubule associated proteins and the zinc:tubulin ratio on the polymerization process. Depletion of microtubule associated proteins results in wider and longer tubulin-sheets than those polymerized in the presence of microtubule associated proteins. However by increasing zinc concentration wider but shorter tubulin-sheets were found. These results suggest that microtubule associated proteins and zinc could promote nucleation of tubulin-sheets, but zinc also promotes lateral tubulin-tubulin interaction. This interpretation was confirmed when microtubule protein was assembled at a low zinc:tubulin ratio. In such conditions composite structures of microtubules and zinc tubulin-sheets are formed. These composite structures are consequence of a lateral attachment of a zinc tubulin-sheet on a microtubule protofilament.

Animals↗
PubMed1980

Incorporation of the high-molecular-weight microtubule-associated protein 2 (MAP2) into microtubules at steady state in vitro.

Purified high-molecular-weight microtubule-associated protein 2 (MAP2) labelled with [32P]-phosphate has been obtained and used as a marker to study the incorporation of this protein into microtubules at steady state in vitro. The incorporation kinetics of protein MAP2 show two different mechanisms of addition of this protein into microtubules. A fast incorporation, which proceeds essentially independently of microtubule assembly, indicates its addition into binding positions which may be available along the structure. Once the microtubule has been saturated with the protein, its incorporation proceeds from one end of the microtubule, in a polymerization-dependent manner, at a slower rate. Loss of the protein from microtubules at steady state proceeds fundamentally at the opposite end.

Animals↗
PubMed1980

Microtubule-associated-protein MAP1 is not implicated in the polymerization of microtubules.

Addition of DNA to microtubule protein solutions results in a decrease of the amount of the high-molecular-weight microtubule-associated proteins (MAP1 and MAP2) available for polymerization. Complete removal of MAP1 from microtubules requires lower concentrations of DNA compared to MAP2. Removal of MAP1 from microtubules has little or no effect on microtubule polymerization (nucleation and propagation). In contrast, removal of MAP2 produces a marked decrease in microtubule polymerization. Incubation of microtubular protein with DNA results in complete disappearance of the 30-S ring-shaped oligomers at concentrations of DNA which are not markedly inhibitory of polymerization.

Animals↗