Noboru Sueoka

Deoxyribonucleic acid replication in meiosis of Chlamydomonas reinhardi: I. Isotopic transfer experiments with a strain producing eight zoospores☆

Abstract DNA replication in meiosis of Chlamydomonas reinhardi has been studied by 15N density-labeling experiments coupled with cytological observations. It has been shown that (1) during zygotic maturation, there is neither chromosomal DNA replication nor meiotic division; (2) during zygospore germination, meiosis occurs, and the first meiotic division is preceded by a single chromosomal DNA replication; (3) the replication of chromosomal DNA associated with meiosis is semiconservative; (4) only hybrid chromosomal DNA molecules are detected at the end of zygote germination, when eight zoospores emerge from each zygote, indicating that only one DNA replication occurs in the entire period between the mating of gametes and the germination of zygotes; (5) a DNA component (M-band DNA) replicates extensively and appears in large quantity during zygote maturation.

Transfer RNA and cell differentiation.

Publisher Summary This chapter explores that cell differentiation involves drastic changes in cell metabolism. Thus, the synthesis of many proteins is stopped and that of new ones has begun. This type of regulation could be called macroregulation in contrast to microregulation, in which the synthesis of a particular protein, or set of proteins in an operon, is specifically regulated by induction and repression. The chapter deals with the possibility of tRNA involvement as a critical factor in cell differentiation at the macroregulation level. Various mechanisms for macroregulation can be visualized. The translation of mRNA into protein involves various components, such as ribosomes, initiation and termination factors, chain elongation factors, tRNA and aminoacyl-tRNA synthetase. Among these components for code translation, tRNA is the one most likely to be involved in macroregulation because of the unique feature of being multiple for each amino acid and yet ubiquitous for the synthesis of various proteins of the cell. Controls are likely to be operating at both levels. Degeneracy of the code and degeneracy of corresponding adaptors (tRNA) have been established. On the other hand, there is generally a single aminoacyl-tRNA synthetase for each amino acid in bacteria, although, some contradictory results have been reported. In higher organisms, the mitochondria1 system seems to have its own synthetases and tRNAs in addition to cytoplasmic synthetases and tRNAs.

Modification of leucyl-sRNA after bacteriophage infection.

Abstract Aminoacyl-sRNAs of Escherichia coli B with and without bacteriophage T2 infection have been examined using methylated albumin kieselguhr column chromatography. Out of 17 aminoacyl-sRNAs examined, only one, leucyl-sRNA, was found to be clearly different before and after the infection. This alteration lies in sRNA and not in leueyl-sRNA synthetase, and is one of the earliest events after the infection. Phage T4 and T6 infections show the same modification of leueyl-sRNA, whereas infection with T1, T3, T5 and T7 and λ induction do not induce the modification. Leucyl-sRNA was examined in Shigella dysenteriae 60 and in various strains of E. coli after T2 infection, and in permissive and nonpermissive hosts after infection with various early amber mutants of T4. In all cases, the same modification of the leueyl-sRNA was observed. It is not excluded that the leucyl-sRNA change may take some role in the arrest of host-protein synthesis

Sequential replication of the Bacillus subtilis chromosome: IV. Genetic mapping by density transfer experiment☆☆☆

Abstract Genetic markers near the replication origin of the chromosome have been mapped in two strains of Bacillus subtilis , W23 and W168, which differ in replication control. A synchronization of chromosome replication during spore germination was used. Two types of density transfer experiments of spores were performed: one, the germination of deuterium oxide-grown spores in a rich aqueous medium and the other, the germination of spores of a thymine-requiring strain in a medium containing 5-bromouracil. Samples were taken at various times during germination and their lysates were centrifuged in a density gradient. The transfer of genetic markers from non-replicated, half-replicated and fully replicated DNA was followed by transformation assays. A theory of mapping applicable to replication synchronization of the chromosome and density transfer experiments (synchro-transfer analysis) has been developed and applied to the present data with special emphasis on markers close to the replication origin. This method of mapping has been extended to the whole chromosome, and the relative distances between markers was estimated by statistical analysis of the transformation data. There was no difference in the replication origin and in the marker order between the two strains.

Chromosomal location of antibiotic resistance markers in Bacillus subtilis.

Abstract Resistance loci for a number of antibiotics known to inhibit protein synthesis have been mapped on the Bacillus subtilis W168 genome by transformation, PBS1 transduction and density transfer analysis. Most of the markers are clustered near the replication origin and presumably adjacent to genes for RNA species. The map order found is ade16, nov, pac, cysA14, rfm, bry, str, neo, kan, ery-ole, spc, lin. Recombination among neomycin-resistant mutants has also been demonstrated. Novobiocin inhibits both DNA and RNA synthesis in vivo but neither DNA polymerase nor RNA polymerase in vitro. Its mode of action is unknown. The resistance of mutants to rifampicin is correlated with in vitro insensitivity of RNA polymerase to the drug. A number of resistance loci map outside the early region, including 11 spectinomycin-resistant mutants located at 0.72 on the synchro-transfer map and two neomycin-resistant mutants. The function of these loci is not known.

Gene expression during outgrowth of Bacillus subtilis spores: The relationship between gene order on the chromosome and temporal sequence of enzyme synthesis

Abstract During outgrowth of Bacillus subtilis W23 spores in a defined medium, the activities of five enzymes studied—sucrase, trehalase, ornithine transcarbamylase, aspartate transcarbamylase and threonine dehydratase—appear in a sequential manner. With one exception (ornithine transcarbamylase), before DNA synthesis begins, the enzyme activities appear in the order in which the corresponding genetic loci have been located on the genetic map. Ornithine transcarbamylase activity does not appear in the first round, but in the second series of increases in enzyme activity after DNA synthesis commences. Moreover, the appearance of this enzyme activity occurs at a time that would be expected from its relative position on the genetic map.

On the formation of a homogeneous zygotic population in Chlamydomonas reinhardtii

Abstract Upon transfer into a liquid medium devoid of a nitrogen source, vegetative cells of Chlamydomonas reinhardtii undergo a gametogenic differenciation process to give rise to sexually active gametes that are capable of mating with opposite mating type gametes. Striking differences have been demonstrated in the mating efficiency (0 to 100%) of gametes induced at different vegetative growth stages in the light-dark synchronized culture. This difference in mating efficiency of gametes is independent of mating-type but directly related to the growth stage of the synchronized vegetative cultures at the time of gametogenic induction. In contrast to the synchronized culture, the mating efficiency of gametes in the continuous light, nonsynchronized vegetative culture is independent of the growth stage at the time of gametogenic induction. The mating behavior of gametes appears to be rather unstable since the mating efficiency of a given clone can be altered considerably during routine maintenance of the culture. Not all the progeny of a particular strain inherit the identical mating efficiency of their parents. The stability of the mating behavior and the transmittance of the mating behavior to the vegetative progeny have been found to vary among different clones. The high frequency at which the mating behavior alters suggests that the mating behavior is a rather complex expression of a number of different factors.

Heterogeneity of DNA in Density and Base Composition

Chromatography, on methylated-albumin columns, of DNA from calf thymus, mouse testis, and Bacillus subtilis, yielded, on elution by a sodium chloride gradient, fractions differing in density. The fractions eluted by higher sodium chloride concentrations had lower densities in a CsCl density gradient. Since DNA with higher guaninecytosine content is eluted from the column with lower concentration of sodium chloride and has higher density, the density heterogeneity of DNA is best interpreted as a result of heterogeneity of base composition. An extra band observed in calf-thymus DNA had a higher density than that of the main DNA; it was eluted at a lower concentration of NaCl, indicating a higher content of guanine and cytosine. On the other hand, an additional DNA component in the mouse-testis DNA had a lower density and also it was eluted at a lower salt concentration, possibly an indication of an unusual base component in its structure.

Characterization of a modified leucyl-tRNA of Escherichia coli after bacteriophage T2 infection.

Abstract T-even phage-specific modification of leucyl-tRNA of Escherichia coli has been established. Leucyl-tRNA F is a modified tRNA which appears one to two minutes after infection and can be detected on a methylated albumin kieselguhr column elution profile as a distinct new peak in front of all leueyl-tRNAs. Leucyl-tRNA F isolated by MAK † column fractionation can not be deacylated enzymically, nor can leucine from Leu-tRNA F be exchanged with other components of tRNA Leu . Re-acylation of Leu-tRNA F can not be achieved following deacylation. This is true when either bulk tRNA containing Leu-tRNA F or isolated Leu-tRNA F is used. The results indicate that once tRNA F Leu is charged with leucine, it can not be recognized by the synthetase. A comparison of hyperchromicity, the sedimentation coefficient and Sephadex G100 column fractionation of isolated Leu-tRNA F and bulk tRNA shows that Leu-tRNA F is approximately half the size of normal tRNA. tRNA F Leu (before aminoacylation) is an unstable molecule and the conditions which weaken the hydrogen-bonded structure destroy its chargeability. Sucrose density-gradient centrifugation of bulk tRNA containing tRNA F Leu indicates that tRNA F Leu sediments at the same rate as bulk tRNA, whereas Leu-tRNA F (after aminoacylation) sediments much slower than bulk tRNA. All the results obtained indicate that tRNA F Leu (before aminoacylation) has an intact size. However, it is broken by aminoacylation or by treatments which weaken the hydrogen-bonded structure. It is suggested that tRNA F Leu is derived from tRNA F Leu by the introduction of a nick or nicks within the molecule as the result of T-even phage infection.

POLYMER SIMILAR TO POLYDEOXYADENYLATE-THYMIDYLATE IN VARIOUS TISSUES OF A MARINE CRAB.

Several species of a genus Cancer have a DNA that has a light buoyant density and that contains mainly deoxyadenylate and thymidylate. The presence of this polymer was demonstrated previously in testes and vas deferens. By a modified procedure for isolating DNA, the muscles, liver, and eggs of Cancer borealis are also shown to contain the deoxyadenylate-thymidylate-like polymer.