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Biomedical subjects

R A Raff

Publications and source records attributed to R A Raff.

At least 73 records · Page 4Linked to original sources
PubMed1982

Regulation of tubulin gene expression during embryogenesis in Drosophila melanogaster.

Four different tubulins have been identified that are expressed during embryogenesis in Drosophila melanogaster. Two alpha-tubulin subunits (alpha 1 and alpha 2) and one beta-tubulin subunit (beta 1) are expressed throughout embryonic development. A second beta-tubulin subunit (beta 3) is expressed only for a short period in mid-embryonic development. Synthesis of beta 3-tubulin in vitro in a rabbit reticulocyte translation system is directed by RNA extracted from embryos only at the stage when the protein is expressed. Thus we conclude that the mRNA encoding beta 3-tubulin is transcribed only during the brief period of beta 3-tubulin synthesis. The expression of beta 3-tubulin is accompanied by a coordinate transient increase in the level of synthesis of the embryonic alpha-tubulins, thereby maintaining an approximately equimolar synthesis of alpha- and beta-tubulin subunits throughout embryogenesis.

Animals↗
PubMed1982

Message-specific sequestration of maternal histone mRNA in the sea urchin egg.

Nucleate and anucleate fragments of sea urchin eggs were prepared by centrifugation on sucrose step gradients. The amount of total RNA, poly(A)+ RNA, histone mRNA, actin mRNA, alpha-tubulin mRNA, and mitochondrial rRNA was determined for each fragment. Total RNA, poly(A)+ RNA, actin mRNA, and alpha-tubulin mRNA all distributed in the same ratio as the volume of the fragments. In contrast, the mitochondrial rRNA was found preferentially distributed in the anucleate fragments, coinciding with the distribution of the mitochondria. Histone mRNAs did not follow the fragment volume ratios, but rather were always found associated with the fragment containing the nucleus. To distinguish between nuclear association and possible artifacts associated with centrifugation, eggs were manually cut into nucleate and anucleate fragments and the amount of histone mRNA was determined for each set. Again only the fragments containing the nucleus had detectable amounts of histone mRNA. Although histone mRNAs were always associated with the nucleate fragment, very little histone mRNA was found associated with isolated egg nuclei prepared under gentle isotonic isolation conditions. Furthermore, embryos that have had first nuclear breakdown blocked with 6-dimethylaminopurine still initiated the recruitment of histone mRNAs into polysomes at the same time as control embryos, thus indicating that nuclear breakdown is not necessary for normal histone message utilization. These results demonstrate a message-specific sequestration of maternal histone mRNA which is physically different from that of other maternal mRNAs and which may govern the timing of maternal histone synthesis in sea urchin embryos.

Actins↗
PubMed1980

Mutation in a testis-specific beta-tubulin in Drosophila: analysis of its effects on meiosis and map location of the gene.

The structural gene for a testis-specific beta--tubulin subunit in Drosophila melanogaster was mapped genetically and cytogenetically by means of a dominant male sterile mutation, B2tD, in which a variant form of the testis beta--tubulin is expressed. The B2t locus is at 48.5 map units on the third chromosome genetic map, and in bands 85D4-7 on the salivary chromosome map. The mutation B2tD causes disruption of microtubule function in all stages of spermatogenesis, beginning with meiosis. The effects of gene dosage of B2tD on meiosis were examined in detail cytologically at the light microscope level. In testes of flies in which the variant tubulin subunit is expressed, abnormal meiotic spindle formation, improper chromosome movement and failure to undergo cytokinesis occur. The extent of these defects in microtubule function depends on the dosage of the B2tD mutation, being most severe in males homozygous for the mutation, intermediate in males heterozygous for the mutation, and least marked in males heterozygous for B2tD and a tandem duplication of the region of the genome containing the B2t locus. Chromosomal events unrelated to microtubule function, such as replication and condensation, occur normally. Results obtained during mapping of the B2t locus strongly suggest a haplo-insufficient site at or closely linked to this locus.

Animals↗
PubMed1979

Mutation in a structural gene for a beta-tubulin specific to testis in Drosophila melanogaster.

By two-dimensional gel electrophoresis of tubulins prepared from tissues of Drosophila melanogaster we have identified a beta-tubulin subunit that is present only in the testis. Furthermore, we have isolated, as a male sterile, a third chromosome dominant mutation [ms(3)KKD] in the structural gene for this beta-tubulin. Males heterozygous for this mutation produce no motile spermatozoa. Beginning with meiosis, all processes in spermatogenesis are abnormal to some extent. Many microtubules (including both cytoplasmic microtubules and doublet tubules of the axoneme) show aberrant structure in cross section, and the overall morphology of the developing spermatids is disorganized. Testes from these males were shown, by two-dimensional gel electrophoresis, to contain both the normal testis-specific beta-tubulin and an electrophoretic variant of this tubulin in equal amounts. Both wild-type and mutant testis-specific beta-tubulins were characterized by vinblastine sulfate precipitation, coassembly with purified Drosophila embryo tubulin, and peptide mapping.

Animals↗
PubMed1975

Cytoplasmic microtubule proteins of the embryo of Drosophila melanogaster.

We have been able to purify, in bulk, the cytoplasmic microtubule proteins of eggs and embryos of Drosophila melanogaster by means of in vitro self-assembly of microtubules from subunits present in a high-speed supernatant fraction of eggs or embryos. This provides the first successful application of this method to purification of microtubule protein from a source other than vertebrate brain, and the first purification of insect microtubule proteins. Our electron micrographs show that the in vitro assembled microtubules are morphologically typical and apparently are comprised of the expected 13 protofilaments. The protein we obtain from such preparations binds [3H]colchicine and has a sedimentation value of 6.4 S-6.9 S which is close to the predicted value for microtubule protein dimer. Both alpha- and beta-microtubule proteins are evident in sodium dodecyl sulfate polyacrylamide electropherograms of the isolated proteins. The apparent molecular weights of these species on dodecyl sulfate polyacrylamide gels are 54,000 and 52,000, respectively. These values as well as the amino acid composition and N-terminal methionine of the Drosophilia proteins are very closely comparable to microtubule proteins from other, unrelated organisms.

Amino Acids↗
PubMed1975

Microtubule protein pools in early development.

Microtubule protein pools have been demonstrated to exist in unfertilized eggs and the early embryonic stages of several organisms. The microtubule pool of the sea urchin embryo is constant in size (about 0.4% of the total embryo protein) throughout early development. Protein withdrawn from this pool for organelle assembly is replaced by new synthesis. Eggs and embryos of Drosophila similarly contain a pool of microtubule proteins (larger than or equal to 0.4% of the total embryo protein, congruent to 3% of the soluble protein), which is constant in size throughout early development. The Drosophila egg microtubule proteins are easily purified by self-assembly in vitro of microtubules, and are similar to microtubule proteins from other organisms in molecular weight and other properties. Synthesis of microtubule proteins in sea urchin embryos is supported by oogenetic mRNA. This appears also to be the case in molluscan (Ilyanassa) embryos. It is not known whether Drosophila embryos synthesize microtubule proteins during the early stages of development.

Animals↗