Max Alfert

A new fixation procedure for preserving the ultrastructure of marine invertebrate tissues

Addition of 0·05% OsO4 to a conventional glutaraldehyde fixative for the first 10 min of fixation was found to improve greatly the preservation of ultrastructure in the eggs of Urechis caupo. Several workers have since confirmed this result in other marine invertebrate tissues. Specific protocols and techniques are given. We believe that the OsO4 rapidly renders the plasma membranes of cells freely permeable to glutaraldehyde, allowing faster penetration of this fixative. This method should be applicable to a wide variety of tissues that are difficult to fix.

Composition and Structure of Giant Chromosomes

Publisher Summary This chapter discusses the composition and structure of giant chromosomes. Recent advances in chromosome chemistry, the salivary gland-type chromosome, the lampbrush chromosome, and a comparison of both types and their relation to nuclear function are also discussed. The nucleoprotein composition of the interphase nucleus in a variety of cell types is investigated by a combination of biochemical techniques and by microspectrophotometric studies in situ and on isolated cell components. Deoxyribonucleic acid (DNA) appears to be quantitatively correlated with the chromosome complement present in a nucleus and is by far the least variable of the known nuclear fractions. A characteristic quantity of DNA is associated with the haploid chromosome set of each species, and depending on their degree of ploidy, nuclei in nondividing tissues contain multiples of the basic amount. Correlated with chromosome reproduction, nuclear DNA doubles prior to division in mitotically active cells and increases in geometric steps when nuclei become polyploid.

Cytochemical studies on the protamine-type protein transition in sperm nuclei after fertilization and the early embryonic histones of Urechis caupo☆

Abstract Cytochemical staining characteristics of nuclear histones during postfertilization maturation division and various early embryonic stages in Urechis have been studied. The transition of protamine-type protein to adult histones in the sperm nucleus is accomplished by 15 min after entrance into the egg cytoplasm. Newly synthesized egg proteins migrate into enlarging male and female pronuclei after this transition, followed by pronuclear DNA synthesis and fusion. The shift from protamine-type protein to adult histones, which occurs in the absence of RNA synthesis during the postfertilization maturation division of the egg, may be one of the processes involved in the normal structural reorganization of chromosomes. Such a reorganization is likely to be a prerequisite for chromosome replication and mitosis. No qualitative differences are detected in the stainability of histones of unfertilized eggs and embryos at the cleavage and later stages of development.

The nucleolus and RNA metabolism in Urechis eggs

Abstract RNA synthesis in unfertilized and fertilized eggs of Urechis caupo has been studied by autoradiography using H 3 -uridine. The nucleolar apparatus as well as the chromosomes synthesize RNA in unfertilized eggs. RNA synthesis stops in disappearing nucleoli and in chromosomes after nuclear membrane breakdown following sperm entry and does not resume during 2- to 4-cell stages. The persisting nucleolar remnant at prometaphase-metaphase I retains most of the labeled RNA synthesized immediately before sperm entry. This suggests that the large fraction of the nucleolar substance which disappears by prometaphase-metaphase I consists of ribonucleoproteins which had been produced at an earlier time, prior to labeling of unfertilized eggs.

Cytochemical studies on the concurrent synthesis of DNA and histone in primary spermatocytes of Urechis caupo.

Abstract The results of a combined autoradiographic and cytophotometric study show that primary spermatocytes of Urechis caupo incorporate about 2 to 4 times more tritiated arginine, lysine, or phenylalanine during S-phase than during G1 and G2 stage. This increased incorporation occurs mainly in histones which do not incorporate 3 H-tryptophane and which are acid labile.

DIFFERENTIAL MICRONUCLEAR POLYTENY IN A POPULATION OF THE CILIATE TETRAHYMENA PYRIFORMIS

Summary1.A population ofT. pyriformis, variety 1, mating type I, was observed to have conspicuously larger micronuclei than other populations of types I and II.2.The increased micronuclear size is correlated with increased nuclear DNA content (in terms of Feulgen dye) and with the appearance of meiotic chromosomes which are thicker and which stain more intensely with the Feulgen reaction than those of the conjugating partner.3.Under certain culture conditions individuals with larger micronuclei exhibit a more rapid growth rate than other populations.

BINDING OF LABELED RIBONUCLEIC ACID TO BASIC PROTEINS, A MAJOR DIFFICULTY IN RIBONUCLEIC ACID-DEOXYRIBONUCLEIC ACID HYBRIDIZATION IN FIXED CELLS IN SITU

In an attempt to hybridize H3-ribonucleic acid (RNA) of onion roots to deoxyribonucleic acid of fixed cells in situ it has been found that such RNA binds only to cytoplasmic basic proteins (ribosomal?) as well as nuclear histones or protamines of diverse plant and animal materials. The bound RNA is relatively insensitive to mild RNase digestion. Most of these basic proteins cannot be removed from cells without distorting cell morphology, or without the prior removal of deoxyribonucleic acid. The binding characteristics of H3-RNA and basic proteins parallel alkaline fast green staining of histones. In the formalin-fixed cells, H3-RNA binds to the nucleus only after removal of deoxyribonucleic acid. The binding capacity is abolished after acetylation or deamination of lysine-rich histones; such treatments do not affect the binding ability of arginine-rich histones or protamines.

Chapter 14 Cytochemical Quantitation of Histones

Publisher Summary This chapter discusses the techniques for the cytochemical quantitation of histones. The chapter focuses on the alkaline fast green (AFG) staining technique and its application for quantitative measurements of nuclear histones. A few selected cytochemical methods that are used separately or in conjunction with the AFG technique are described to illustrate the usefulness of such techniques in studies on histone patterns during growth and development. The AFG staining is affected by the type of fixative used. The AFG stainability of nuclear basic proteins depends on the prior removal of DNA by hot TCA or deoxyribonuclease (DNase). Cytochemical methods are valuable tools for studying quantitative and qualitative nuclear histone changes in individual cells during growth and development. Cytochemical methods are limited to detection and analysis of the content of nuclear basic proteins and their overall composition; they do not provide information about chemical modifications and quantitative or qualitative modulations of individual histone fractions that could influence nuclear activity. Therefore, coordinated cytochemical and biochemical studies are necessary for the understanding of the role of histones in nuclear function.

Histone patterns during early embryogenesis in the echiuroid Urechis caupo

Histones were isolated from nuclei of Urechis caupo unfertilized eggs and embryos from early cleavage through gastrula. Polyacrylamide gel electrophoresis showed the presence of a slow migrating H1s fraction in the egg which was replaced by a faster migrating H1 during development. Radioactive labelling showed no synthesis of H1s from fertilization up to gastrula and a high rate of synthesis of H1 relative to the other histones during cleavage and blastula stages.