Max L. Birnstiel

STUDIES ON PHYTOFERRITIN. I. IDENTIFICATION AND LOCALIZATION.

A new iron-protein complex, phytoferritin, has been identified microscopically, centrifugally, and spectrophotometrically in extracts from pea embryos. Methods for extracting 90% pure phytoferritin from pea embryonic axes, cotyledons, and young bean leaves have been developed. They include differential centrifugation and DEAE-cellulose column chromatography. The electron microscopic image, ultraviolet absorption spectrum, iron:protein ratio, and sedimentation behavior of phytoferritin closely resemble those of animal ferritin. Phytoferritin has been located electron microscopically in the proplastids of pea epicotyls and root meristems and in plastids of young light- or dark-grown bean seedlings. In addition masses of phytoferritin have been identified in the plastids of the peripheral layers of pea cotyledons.

THE NUCLEOLUS, A SOURCE OF RIBOSOMES.

Abstract The occurrence of ribosomes in sub-nuclear fractions has been investigated. It has °been found that ribonucleoprotein particles with a RNA/protein ratio of 0.5 may be extracted from nucleoli by sodium deoxycholate treatment. Several lines of evidence have been presented to show that this ribonucleoprotein does not come from cytoplasmic contamination. The nucleolar ribonucleoprotein particles are 38, 60, and 81 S with diameters of 200, 280, and 365 Angstrom. RNA of 18 and 28 S may be extracted from the nucleolus. The dissociation and association of nucleolar ribonucleoprotein particles relative to magnesium concentration resembles that of cytoplasmic ribosomes. We concude that ribosomes are present in the nucleolus.

NUCLEOLUS: A CENTER OF RNA METHYLATION.

The ubiquitously distributed complex of enzymes, the RNA methylases, the apparent function of which is the alteration of the structure of transfer RNA at the macromolecular level by the introduction of methyl groups into the component bases, are concentrated in the nucleolus, an organelle previously implicated in RNA synthesis.

Observations on fine structure in pea nucleoli in situ and isolated

The fine structure of nucleoli within the root tips of 3-day seedlings is compared to the fine structure of nucleoli isolated from the embryonic axes of the same material. Both in situ and isolated nucleoli show a fibrillar interior region and a particulate periphery. Stereoscopic photographs of in situ nucleolar particles and cytoplasmic ribosomes show that both are composed of 15–20 A fibrils. The nucleolar particles are smaller (160 A) than those of the cytoplasm (190 A). The strands, particularly of the interior, became knotted during the isolation procedure when Mg ++ is added to the suspending medium. RNase extraction of unfixed isolated nucleoli destroys the fine structure of the fibrillar components and the particles while DNase extraction appears only to loosen the knotted strands of the interior.

The nuclear synthesis of ribosomes in cell cultures

Abstract Ribonucleoprotein particles have been prepared from isolated nuclei of cultured tobacco cells. These particles possess a chemical composition similar to that of ribosomes and contain particles which possess a sedimentation coefficient of 80 S. These particles, nuclear ribosomes, are of intranuclear origin and are not the result of contamination with cytoplasmic elements. They do not, in the state in which they are present in the nucleus, support protein synthesis but are rather the products of protein synthesis within the nucleus. They appear to be unfinished precursors of the functional ribosomes of the cytoplasm.

PROTEIN SYNTHESIS BY ISOLATED NUCLEI FROM EXPONENTIALLY DIVIDING CELLS.

Abstract Nuclei have been isolated and purified from rapidly growing tobacco-cell cultures. Such nuclei incorporate as much as 1 mμmole of [ 14 C]lysine per milligram protein in 20 min. That this label is incorporated intramolecularly into peptide linkage has been demonstrated by chromatography of tryptic digests. The rate of protein synthesis is enhanced by the presence of an energy-generating system, a complete micture of amino acids, and sodium ions. Additions of RNAase (EC 2.7.7.16), Versene or chloramphenicol do not inhibit the nuclear protein synthesis.

Fractionation of isolated pea nuclei

Abstract A method for fractionation of purified and intact pea nuclei has been devised. Grinding in supersaturated sucrose in the presence of citrate and subsequent differential centrifugation yielded nucleoli,chromatin, ribosomal fraction and nuclear sap as 4 major fractions. These sub-nuclear components have been shown to be cytologically biochemically and functionally distinct.