Raymond F. Jones

Induction of Mendelian and non-Mendelian streptomycin resistant mutants during the synchronous cell cycle of Chlamydomonas reinhardtii.

SummaryIn synchronized cultures of Chlamydomonas reinhardtii N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was found to selectively mutate replicating forms of nuclear DNA. This conclusion was based on the 15- to 30-fold increase in the recovery of MNNG induced Mendelian streptomycin resistant mutants (sr-1) which was correlated with mutagenesis during the period of nuclear DNA replication. No concomitant increase in the recovery of non-Mendelian streptomycin resistant mutants (sr-2) occurred during this same period.Mutagenesis at the time of chloroplast DNA replication, however, resulted in a 1.5- to 1.6-fold increase in the recovery of both sr-1 and sr-2 induced mutants.

Periodic increases in enzyme activity in synchronized cultures of Chlamydomonas reinhardtii.

Abstract During synchronized growth of Chlamydomonas reinhardtii on a 12-h light-12-h dark cycle, net protein synthesis occurs approximately linearly in the light period but no appreciable increase is observed during the dark period of growth. Increases in activity of alanine dehydrogenase ( l -alanine:NAD oxidoreductase, EC 1.4.1.1), glutamate dehydrogenase ( l -glutamate:NAD(P) oxidoreductase, EC 1.4.1.3), phosphoenolpyruvate carboxylase (orthophosphate:oxaloacetate carboxylyase (phosphorylating), EC 4.1.1.31), aspartate carbamoyltransferase (carbamoylphosphate: l -aspartate carbamoyltransferase, EC 2.1.3.2) and ornithine carbamoyltransferase (carbamoylphosphate: l -ornithine carbamoyl transferase, EC 2.1.3.3) occur within characteristic periods of time during the life cycle of the cell. The activity of alanine dehydrogenase and aspartate carbamoyltransferase increases primarily in the light period, whereas the activity of the other three enzymes increases during the dark period of growth.

Protein turnover and macromolecular synthesis during growth and gametic differentiation in Chlamydomonas reinhardtii

Abstract Initiation of DNA replication in Chlamydomonas reinhardtii requires the synthesis of a protein and is inhibited by both actinomycin D and chloramphenicol when added a short time before the normal initiation of DNA. Protein turnover occurs in synchronized cultures of this alga during the dark period and during gametic differentiation. Protein turnover occurs in the arginine-less mutants of C. reinhardtii in the light only if NH + 4 is absent from the culture medium. C. reinhardtii possesses a relaxed control of RNA synthesis during amino acid starvation. Under these conditions both tRNA and rRNA are synthesized. After prolonged periods of starvation, tRNA may be synthesized preferentially over rRNA. However, when the alga grows in a non-synchronous culture there is a tight control of RNA synthesis during an energy “step-down”.

Isolation of stable ribosomal RNA from whole cells of Chlamydomonas reinhardtii

Abstract Sonication, rather than lysis, of cells in the presence of diethylpyrocarbonate and Mg 2+ as nuclease inhibitors has resulted in the isolation of stable high molecular weight ribosomal RNA from Chlamydomonas reinhardtii . Preparations are nuclease free and show excellent conservation of the previously thought labile 23-S rRNA species.

Changes in cytoplasmic and chloroplast ribosomal ribonucleic acid during the cell cycle of Chlyamydomonas reinhardtii

Abstract Polyacrylamide gel electrophoresis of isolated cytoplasmic and chloroplast ribosomal ribonucleic acid species during the synchronous vegetative cell cycle of the eukaryote Chlamydomonas reinhardtii suggests that a separate control of cytoplasmic and chloroplast rRNA might exist. It was found that the amount of cytoplasmic rRNA linearly increased during the entire G 1 phase of the cell cycle, whereas chloroplast rRNA accumulated only through 70% of the G 1 period. The amount of cytoplasmic rRNA per mother cell remained constant during nuclear DNA synthesis but a gradual loss of chloroplast rRNA was noted at this time. A significant decline in all four rRNA species occurred at the time of cell division.

Multiple forms of phosphoenolpyruvate carboxylase from Chlamydomonas reinhardtii

Abstract 1. 1. Two species of phosphoenolpyruvate carboxylase (orthophosphate: oxalacetate carboxylase (phosphorylating), EC 4.1.1.31), have been isolated and purified from Chlamydomonas reinhardtii by salt fractionation, ion exchange chromatography on DEAE-cellulose, and by hydroxylapatite chromatography. 2. 2. Polyacrylamide gel electrophoresis and ultracentrifugal analysis indicated that each enzyme, designated A and B, was homogeneous. The carboxylating reaction products were isolated and identified for each enzyme and sedimentation coefficients extrapolated to zero protein concentration were found to be s 20,w 0 = 6.55 for phosphoenolpyruvate carboxylase A and s 20,w 0 = 11.30 for phosphoenolpyruvate carboxylase B. These two enzymes were also found to possess different pH optima and exhibited different kinetic parameters.

An improved technique for the preparative electrophoresis and electroelution of high molecular weight ribosomal RNA

Abstract A new technique for casting flat 0.5% agarose-2.4% acrylamide preparative gels is described in this communication. The casting method presented completely eliminates mechanical problems and technical complications often encountered in producing the homogeneous loading platform critical to the entrance and proper migration of RNA species on low pore composite gels. The gels formed can be used to separate milligram quantities of RNA with excellent resolution and a method for the electroelution of a specific RNA species is also presented.

Changes in enzyme activity during differentiation in Chlamydomonas reinhardtii

The patterns of alanine dehydrogenase, glutamate dehydrogenase and malate dehydrogenase activity were studied during the normal vegetative cell cycle and during the processes of gametic differentiation and dedifferentiation in synchronized cultures of Chlamydomonas reinhardtii. During all three phases of growth and differentiation the synthesis of DNA was also measured. During gametic differentiation all three enzyme levels were suppressed compared to vegetative cells although DNA and cell number were comparable. During gametic dedifferentiation no DNA synthesis occurred during the first 24 h cycle and only a doubling during the second. It was not until the third cycle that a normal 4-fold increase in DNA was observed. Cell number followed a similar pattern. Although the levels of alanine dehydrogenase and malate dehydrogenase were uniformly low during the first cycle when glutamate dehydrogenase increased 4-fold, during the second cycle the patterns of these enzymes changed markedly. The enzymes did not attain levels characteristic of vegetative cells until the third cycle.

A rapid and sensitive method for the detection of histone peptides

Abstract Fluorescamine has been used to obtain a peptide map of a mixture of histones (H 3 , H 2 A, H 2 B, and H 4 ) prepared from oocytes of Xenopus laevis . Fluorescamine was found to be more sensitive than o -phthalaldehyde or ninhydrin-Cd for the detection of peptide fragments obtained from tryptic digestion of oocyte histones of X. laevis and the peptic digestion of the β chain of insulin. Using the β chain of insulin for a comparison, the 8 major peptide fragments could be separated by electrophoresis within 30 min and were detectable at the picomols level. Some 70 peptide spots of X. laevis oocyte histones were resolved, thus permitting the analysis of this complex mixture of polypeptides without the need for prior separation.

Molecular properties of multiple forms of phosphoenolpyruvate carboxylase from Chlamydomonas reinhardtii.

Abstract 1. 1. Using methods of gel filtration, viscosity, sedimentation velocity and sedimentation equilibrium, the molecular weights of the two isoenzymes of phosphoenolpyruvate carboxylase (orthophosphate:oxaloacetate carboxylase (phosphorylating), EC 4.1.1.31) from Chlamydomonas reinhardtii have been determined. Values ranged from 1.00·105–1.18·105 for the A conformer and 2.79·105–2.90·105 for the B conformer. 2. 2. On the basis of determination of the shape function constant, frictional coefficient and axial ratio, the A conformer appeared to possess a compact spherical type of conformation compared to a more elongated conformation for the B isoenzyme.