Various techniques have heen adapted from protein chemistry to the cytological demonstration of proteins (e.g., ninhydrin reaction, Mazia and Jaeger, 1939; Millon reaction, Pollister and Ris, 1947; Sakaguchi reaction, Thomas, 1946;Serra, 1946) but none has found wide use for both the resolution of morphological detail in terms of protein distribution and the measurement of relative protein concentra-

Histochemical staining methods combined with specific enzymatic hydrolysis and chemical substitution procedures have permitted the cytologist to visualize, and in some instances to quantitate by microphotometric methods, many cellular constituents. Methods for histochemical detection of specific proteins or of characteristic groups in proteins are few, and some of those presently available are somewhat cumbersome for routine histological use, or result in formation of highly unstable color complexes whose quantitative relationship to the substances tested is often unknown. As far as the distinction between histones and non-histones, or, in general, between proteins of different isoelectric points, is concerned, the methods previously used have included u.v. absorption-spectroscopy,' a modification of the Millon reaction,2 and staining with acid and basic dyes at a series of different pH's.3 The last-named method, which makes use of the amphoteric nature of proteins and their resulting ability to form salts with acid and basic dye ions, has been used extensively in the past. However, up to now it has not been possible to define any set of conditions under which a particular type of protein could be visualized selectively by a single staining operation involving only one dye. The procedure described herein represents an empirical method which grew out of a series of staining experiments at controlled pH. It is believed to afford a simple, stable, and specific staining procedure for histones and, when present, protamines in cell nuclei, since other proteins with high isoelectric points (such as cytochrome C), which were found to stain under the specified conditions, do not occur in cells in sufficient concentration to affect the staining picture. Moreover, this method permits relative quantitation of the stainable material by microphotometric procedures.The method consists of the following steps: 1. Tissues are fixed for three to six hours in 10% neutral formalin, washed overnight in running water, dehydrated, and embedded in paraffin.

Addition of 0.05(y0 Os04 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.The ultrastructure of marine invertebrate tissues, especially that of eggs, has proved difficult to preserve well (Meredith Gould-Somero & Linda Holland, personal communication, and 1975 ;Harris, 1978; Parris M. Kidd, personal communication). T o study the early development of Urechis caupo (an echiuroid worm), we tried a variety of fixation methods. Two types of methods proved to be significantly better than the others.Our first success was with a method we call a 'sequential mixed fixative'. We are using the term 'mixed fixative' in the same sense as Hirsch & Fedorko (1968) to signify a freshly prepared mixture of glutaraldehyde and 0~0 4 .The eggs were first fixed for about 5 min in 1% a s 0 4 in a mixture of NaCl and sea water (1 part 0.45 M NaCl: 1 part sea water); they were then fixed for 1-3 h in 2.5% glutaraldehyde in a NaC1-sea water solution, with or without 0.5% 0~0 4 . Post-fixation was in a fresh change of the same solution used for initial fixation. The first sequential mixed fixative formula that we tried had previously been used by Diane Gowdy (personal communication) on sea urchin embryos. A similar procedure has also been used to fix the metachronal wave of cilia (Warner & Satir, 1974).Our second method, which we call the 'low osmium mixed pre-fixative technique', gave much better preservation of ultrastructure. This method consists of a brief (5-10 min) pre-fixation in a glutaraldehyde fixative to which a small amount of Os04 (0.05%) has been added immediately before use. Fixation is then continued in the same fixative without the added oso4 for 1 h. After a brief buffer rinse, a conventional Os04 post-fixation follows.Our best results so far have been achieved with a main fixative composed of 476 glutaraldehyde, 0.2 M Na cacodylate, 0.1 M NaCl (or 11 mM CaC12,56 mM MgClz), 0.35 M sucrose, pH 7.2 (or 7.8). Post-fixation was in 1 yo Os04, 0 . 3~ NaC1,0.2~ Na cacodylate, pH 7.2. The pre-fixative was prepared by adding a small measured volume of the post-fixative to a measured aliquot of the main fixative (usually 0.5 ml added to 10.0 ml of main fixative). The pre-fixative must be mixed within 5 min of the time it is used. The buffer rinse after the main fixative consisted of two changes (5-10 min each) of 0.3 NaCl in 0.2 M Na cacodylate, pH 7.2.All of the fixation methods we tried preserved the vitelline coat of Urechis eggs well, but only the sequential mixed fixative methods and the low osmium mixed pre-fixative methods preserved the cyto...

INTRODVCTIONRecent advances in several independent fields of biological research have tended to indicate that the desoxyribose nucleic acid (DXA) of clironiosomes exhibits a number of properties which may be coilsidered essential for the identification of a Iiypothetical genic material. These properties are :1. High specific activity, such as shoxn by the transforming principle of microorganisms (Avery, MacLeod and Mc-Carty, '44 ; Roivin, Delaunay, Vcndrely and Lehoult, '45). 2. RerriarkabIe stability during metabolic activity of nniidividing cells as evidenced by low turnover rates observed in the coursc of tracer studies (Brues, Tracy and Cohn, '44; Brown, '48; Bergstrand et al., '48; Furst, Roll and Brown, 3. A quantitative relationship correlated with the knowvn quantitative variations of chromosomal material which is the generally recognizecl carrier of the genes : gross clieniicd analyses g k e strong evidence f o r the existence of characteristic quantities of DKA in cells of different organisms (Eoivin, Vendrely and Vetidrely, '48 ; Vendreley and Vcndrcly, '48 ; Mirsky and Ris, '49) ; photometric analyses operating at the lcrel of individual cells, introduced by Cas-'50).

The characteristic morphogenetic changes which occur during sperm formation are at times accompanied by profound chemical alterations of the nuclear material. Thus it has been known since Miescher's pioneer studies during the 1870's (19) that nuclei in the immature salmon testis contain nucleohistones, while ripe salmon sperm contain nucleoprotamines. Basic proteins which are intermediate in composition between histones and protamines have been found in the sperm of some species (12, 18) while histones occur in others (11, 16); histones are generally also found in somatic nuclei of animals and higher plants (12). Both protamines and histones extracted from any type of material are not single compounds but represent mixtures of related substances (13, 23). All of those known to date form a spectrum of basic nuclear proteins among which the protamines represent the extreme in basicity and in simplicity of composition and structure.More recent studies on salmon sperm composition have generally confirmed Miescher's results except for the finding that he overestimated the non-protamine protein content of the sperm nuclei. Polfister and Mirsky (21) reported that nucleoprotamines account for 91 per cent of the nuclear mass, while other workers (15) even claimed that these nuclei consist exclusively of nucleoprotamine. Such findings are in marked contrast to those typical for somatic nuclei (e.g. reference 6) and nuclei of sea urchin sperm (8) in which an appreciable fraction--at least about 25 per cent--of non-histone protein occurs. Compared to other nuclear types, the sperm nuclei of the salmon and related fishes thus may be characterized by two separate biochemical peculiarities: (a) the extreme qualitative modification of the basic proteins to protamines consisting predominantly of arginine residues, and (b) the extreme quantitative reduction of other nuclear protein fractions. When both of these changes

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Isolated testes of the locust Schistocerca gregaria were immersed in solutions of tritiated thymidine, cytidine, uridine, or arginine for short periods to study nucleic acid and protein synthesis during spermatogenesis. DNA synthesis in this tissue is completed prior to initiation of meiosis. Protein synthesis continues throughout the whole meiotic cycle as well as during spermatid development. Meiotic cells, except those in metaphase through early telophase, and early spermatids are also actively synthesizing RNA. The heteropycnotic X-chromosome does not produce RNA at any stage of spermatogenesis. The rates of protein and particularly RNA synthesis decrease as chromosome condensation progresses. Depression of RNA synthesis, however, is not always accompanied by cytologically detectable condensation of chromatin, since very little or no RNA is synthesized in spermatids in which chromatin condensation has barely begun.Most of the earlier studies on synthetic activities in meiotic tissues were concerned with DNA synthesis. With a few exceptions (Sparrow et al.,