Karl M. Jakob

Poly(A)-associated RNA in plants.

The RNA associated with poly(A) sequences from Euglena gracilis and Vicia faba has been isolated by binding to millipore filters and characterized by sedimentation velocity centrifugation and electrophoretic mobility. Poly(A)-associated RNA as isolated in solution was highly aggregated. When denatured, it sedimented as a broad peak with a mean value of 16-18 S. This RNA was shown to be covalently linked to poly(A) sequences which are 150-250 nucleotides long. Our size estimates for plant poly(A) and poly(A)-associated RNA are similar to those obtained for animal cells.

CHARACTERIZATION OF THE 32,000 DALTON MEMBRANE PROTEIN—I. EARLY SYNTHESIS DURING PHOTOINDUCED PLASTID DEVELOPMENT OF SPIRODELA

The kinetics of 35S methionine incorporation into soluble and membrane proteins during the transition from steady state dark growth to greening was studied in Spirodela. A sharp increase in the rate of incorporation occurred at 3 h, which was several h before major increases in chlorophyll were apparent. Part of this enhanced incorporation was due to enhanced synthesis of a 32,000 dalton membrane protein. This synthesis was paralleled by a temporal increase in in uitro template capacity for this protein and an increase in 0.5 × 106 dalton plastid messenger RNA.

The early synthesis and possible function of a 0.5 × 106Mr RNA after transfer of dark-grown Spirodela plants to light

A 0.5 × 106Mr RNA found in plastids of the aquatic angiosperm Spirodela, is synthesized at a much higher rate than any other rapidly labeling RNA species about 3–312 h after dark-grown plants are transferred to light. The pulse labeling kinetics of the 0.5 × 106Mr RNA after transfer to light, argue against its involvement in the biogenesis of plant rRNAs. Although poly(A) RNA is found in Spirodela, poly(A) sequences are not detected in the 0.5 × 106Mr RNA; yet a sucrose gradient fraction which includes RNA of this Mr stimulates amino acid incorporation by an E. coli cell free extract more than other RNA fractions. The possible involvement of the 0.5 × 106Mr RNA as a chloroplast messenger is discussed.

Methylmethionine labeling of a higher plant tissue The incorporation into RNA and pectinic acid of nucleic acid extracts

Abstract The methylation of RNA species of actively growing tissues of higher plants by radioactive methylmethionine is difficult to study. We showed that the methyl groups of methylmethionine were incorporated much more rapidly into pectin (a methylated cell wall polysaccharide) than into RNA of the primary root meristem of germinating Vicia faba . Furthermore, the methyl-labeled pectic substances contaminated the RNA preparations and migrated as some species of RNA on polyacrylamide gel electrophoresis. This was demonstrated in phenol-extracted RNA preparations in which pectin could be detected by the hydroxylamine FeCl 3 color reaction, and in which most of the Me - 3 H radioactivity was ribonuclease and pronase resistant but sensitive to alkali, pectin (methyl) esterase and pectinase. Me - 3 H methylation of rRNA was shown qualitatively in polyacrylamide gels by the partial ribonuclease sensitivity of the Me - 3 H radioactivity of these RNA species. The Me - 3 H radioactivity of nucleotides as determined by alkaline hydrolysis and paper electrophoresis of whole RNA, was of the same low order of magnitude as the ribonuclease-removable methyl radioactivity obtained from polyacrylamide-fractionated RNA.

Removal of pectins from methylated rRNA and precursors by LiCl

The methyl group of radioactive methylmethionine is incorporated preferentially into pectin, a methylated polysaccharide, which massively contaminates RNA preparations. This makes it difficult to discern the methylated RNA species of growing tissues of higher plants. A method for the extraction of pectic substances from RNA preparations of plants is described. Ethanol precipitates containing RNA are suspended in 2m lithium chloride (LiCl) and the pectin is removed with the supernatant after centrifugation. LiCl purification of RNA from Vicia faba root meristems allows the direct identification on polyacrylamide gels of methylated RNA species labeled with [3H]methyl methionine. Presumpive precursors of rRNA which fall into the range of apparent molecular weights of 2.7–2.9, 2.2–2.4, 1.4–1.5, and 0.72–0.75 × 106 are shown to be methylated, in addition to the methylation of the fully processed rRNAs of MW 1.3 and 0.7 × 106.

Discoordination of ribosomal RNA metabolism during metabolic shifts of Spirodela plants

The effects of metabolic shifts on nucleic acid syntheses have been widely studied in prokaryotes, but not in plants because of a paucity of suitable systems. Spirodela (Duckweed) was thus used to ascertain the response of the nucleocytoplasmic (nc) and plastid ribosomal RNA metabolisms to partial and total carbon deprivation. The 0.56 X 10(6) Mr plastid rRNA is the one species of RNA most affected by metabolic shifts; unlike other species, its appearance is delayed by deprivation and it appears more rapidly than other species on transfer from dark to light. The data suggest a discoordination between the transcription and processing of plastid ribosomal precursors. Incorporation into all nc and plastid rRNAs was severely reduced and all rRNA precursors accumulated in green plants that were completely deprived of carbon by transferring to the dark, without sucrose. The amounts of nc and plastid precursors transcribed readjusted to the reduced amounts processed to mature RNA only after long periods in the dark with sucrose. This delay involved the formation of new colorless plants. Less plastid RNAs, compared to nc RNAs are found in the dark steady state.

Sex inheritance inRicinus communis L.: Evidence for a genetic change during the ontogeny of female sex reversals

Further evidence has been presented for the finding (Shifriss, 1956, 1960) that there is a higher transmission of femaleness to outcross progeny through female inflorescences of sex reversal castor bean plants, than through reverted (monoecious) inflorescences of the same plant. The change from femaleness to monoecism which occurs during the ontogeny of the plant could be transmitted through the pollen and thus represents a genetic change.

Culture And Staining of Pollen Grains of Ricinus communis

Germinating and growing pollen grains (male gametophytes) of Ricinus communis L. in liquid culture is achieved as follows: Pollen is collected over a 10-15 min period from mature anther clusters which have been removed from the male flowers and which have been kept at 25° C and 40-60% relative humidity. Samples weighing between 2.5 and 5.0 mg are brought as quickly as possible into a Desicote treated vial containing 17% sucrose and 30 ppm H3BO3 in boiled distilled water. The proportion (w/v) of pollen to culture solution should be 1:100. Shed pollen is kept in a humidity chamber whenever it is not being handled. The air in the culture vial is replaced by O2 at the pressure of 1 atmosphere plus 5 lb and the sealed vials are shaken gently for 8-10 hr while partially immersed in a waterbath kept at 30° C. The pollen is fixed by the addition to the incubation suspension of an absolute alcohol-lactic acid (4:1) fixing fluid. The proportion used is 36 parts of fixing fluid to 1 part of culture solution. The fix...

The Effect of Post-irradiation Centrifugation on Chromosome Aberrations in Vicia Faba

SummaryVicia faba primary roots were x-irradiated in air from 0–700 R and immediately centrifuged for 20 min (2500g) at room temperature. Metaphase analysis of chromosome aberrations showed that with centrifugation there are: (1) a small but consistent increase in chromosome exchanges (two-break aberrations); (2) no significant difference between the centrifugation enhancement ratios for exchanges at the various x-ray doses of the dose-response curves; (3) no increase in terminal deletions (one-break aberrations); (4) no consistent increase in interstitial deletions (ID). The results are consistent with the hypothesis that this post-irradiation treatment increased the number of sites in the interphase nucleus where chromosomes are close enough to exchange if broken by x-rays.