AN ULTRASTRUCTURAL AND MICROSPECTROPHOTOMETRIC STUDY OF THE SHOOT APEX DURING THE INITIATION OF THE FIRST LEAF IN GERMINATING PINUS BANKSIANA

The morphology and anatomy of the shoot apex in germinating Pinus banksiana seeds is described by using scanning and transmission electron microscopy and microspectrophotometry, with special attention given to events preceding the appearance of the first leaf primordia at about 72 hr post-imbibition. The 2C nuclei begin DNA synthesis at about 43 hr. RNA increases until 52 hr and is followed by a reduction related to cytokinesis. Protein drops after 36 hr, apparently related to digestion of storage protein bodies, which by 48 hr are about 50% digested. The resulting protein body vacuoles do not enlarge. Starch is digested just prior to appearance of the leaves and may be mediated by α-amylase production from stacks of endoplasmic reticulum. Heterochromatin increases in the nuclei during germination and coincides with an increase in repeated nucleotide sequences. Golgi bodies increase in number after the first mitoses.     

A CYTOPHOTOMETRIC STUDY OF NUCLEIC ACIDS AND PROTEINS IN THE SHOOT APEX OF WHITE SPRUCE

During the annual three-phase growth cycle of white spruce [Picea glauca (Moench) Voss] the vegetative shoot apex changed in anatomical configuration. Relative amounts of DNA, histones, RNA, and proteins were measured in three cytohistological zones and were related to the anatomical changes during ontogeny. An extended period of DNA synthesis (S) and G₂ preceded an increase in the number of apical initial cells which were part of the mammillary apex. While DNA and histones were generally synchronous during ontogeny, the ratio of DNA to histone increased on June 20. This loss of histone and subsequent increases in RNA and cytoplasmic proteins preceded the appearance of needle primordia on next year's apex. We propose that induction of the apex to reorganize and form needle primordia occurred when the DNA was in a 2C condition, following the loss of histone on June 20.     

HISTOCHEMICAL AND AUTORADIOGRAPHIC STUDIES OF FLORAL INDUCTION IN CHENOPODIUM ALBUM

Gifford , Ernest M., Jr. , and Herbert B. Tepper . (U. California, Davis.) Histochemical and autoradiographic studies of floral induction in Chenopodium album. Amer. Jour. Bot. 49(7): 706–714. Illus. 1962.—Chenopodium album was induced to flower using short-day photoperiods. Changes in the chondriome, starch, total protein, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and histone distribution in cells of vegetative and inflorescence shoot apices were studied. The distal cells of the vegetative apex (especially the axial tunica cells) possess larger nucleoli and vacuoles, less granular mitochondria, and more differentiated plastids than do other cells of the apex; the distal cells stain lightly with dyes that indicate the presence of DNA and histone. RNA is distributed relatively uniformly in the shoot apex; the cells at sites of leaf initiation and young leaf primordia contain slightly higher concentrations of RNA than the axial cells of the shoot apex. Protein is uniformly distributed throughout the vegetative as well as the inflorescence apex. Upon induction, the chemical and morphological differences between cells in the shoot apex gradually disappear. RNA concentration of cells in the apex increases, reaching a maximum after 4 inductive cycles. Protein concentration of cells also increases, but this increase lags behind that of RNA.     

THE USE OF TRITIATED THYMIDINE IN THE STUDY OF TISSUE ACTIVATION DURING GERMINATION IN ZEA MAYS

Stein, O. L. (Montana State U., Missoula), and H. Quastler. The use of tritiated thymidinein the study of tissue activation during germination in Zea mays. Amer. Jour. Bot. 50(10): 1006-1011. Illus. 1963.—Corn embryos were exposed to H³thymidine at various times during the first 80 hr of germination. An analysis of labeled nuclei was made from autoradiographs, and the number and position of mitoses were recorded. Those tissues which approach maturity during embryogeny (root cap, coleorhiza, scutellur node) are first to resume DNA synthesis (30 hr after soaking). No mitoses were observed in these tissues. In shoot and root, mitoses usually precede DNA synthesis, indicating that the nuclei of the dormant embryo have a DNA value of 4C(twice the diploid DNA) or more. The shoot begins its activity much later than the root (50 hr). The shoot apex was the last region to boeomo active, some 70 hr after initiation of the soaking treatment.     

EARLY HISTOGENESIS AND SEMIQUANTITATIVE HISTOCHEMISTRY OF LEAF INITIATION IN TRITICUM AESTIVUM

The shoot apex of Triticum aestivum cv. Ramona 50 was investigated histologically to describe cell lineages and events during leaf initiation. During histogenesis three periclinal divisions occurred in the first apical layer, with one or two divisions in the second apical layer. This sequence of cell divisions initially occurred in one region and spread laterally in both directions to encircle the meristem. Cells of the third apical layer were not involved in leaf histogenesis. Initially, young leaf primordia were produced from daughter cells of periclinal divisions in the two outer apical layers. Nuclear contents of protein, histone, and RNA in the shoot apex were evaluated as ratios to DNA by means of semiquantitative histochemistry. Daughter cells of periclinal divisions in the outer apical layer which produced the leaf primordia had higher histone/DNA ratios than cells of the remaining meristem. However, protein/DNA and RNA/DNA ratios were similar in both regions. Leaf initial cells had a higher ³H-thymidine labeling index, a higher RNA synthesis rate, and smaller nuclear volumes than cells of the residual apical meristem.     

ONTOGENETIC AND HISTOCHEMICAL CHANGES IN THE VEGETATIVE SHOOT TIP OF CHENOPODIUM ALBUM

Gifford , Ernest M., Jr ., and Herbert B. Tepper . (U. California, Davis.) Ontogenetic and histochemical changes in the vegetative shoot tip of Chenopodium album. Amer. Jour. Bot. 49(8): 902–911. Illus. 1962.—A distinct central zone is present in apices of young, rapidly growing seedlings of Chenopodium album. This zone is clearly evident when the plane of section includes developing leaf buttresses and/or newly formed leaf primordia, but is not visible if the cells associated with these developing foliar organs are not in the plane of section. Cells in the central zone contain markedly lower concentrations of RNA than those of the peripheral zone. Differences in DNA, SH-protein, total protein, and histone concentration of cells in the apex parallel the zonal distribution of RNA, but are much less obvious. As the plant ages and the rate of leaf production decreases, the zonal distribution of the above-mentioned compounds becomes less and less obvious. Cells at sites of leaf initiation in older seedlings still have slightly higher RNA concentrations, but a central zone such as is visible in the young seedlings is no longer evident. The axial tunica cells do, however, contain noticeably lower DNA and histone concentrations than the remaining cells of the shoot apex. Changes occur in the size of nucleoli during ontogeny and the possible relationship between nucleolar size and modifying factors is discussed.     

Nuclear genetic activity in B-chromosome rye,in terms of the quantitative interrelationships between nuclear protein,nuclear RNA and histone

The nuclear basis of B-chromosome genetic activity in rye has been investigated using quantitative cytochemical techniques on isolated root tip nuclei. Nuclear DNA amount was found to be directly proportional to B-chromosome number. Relative amounts of total nuclear protein and nuclear RNA however, decreased with increasing numbers of B's but not in a strictly linear fashion. The values were disproportionately low for odd numbered B-classes of plants. Histone protein was found to increase as the number of B's went up, and in this case the values were disproportionately high for odd numbered B-classes. A negative correlation was found between histone and total nuclear protein and histone and nuclear RNA amounts.     

Macromolecular Synthesis in Saccharomyces cerevisiae in Different Growth Media

Synthesis of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein was determined in Saccharomyces cerevisiae during amino acid and pyrimidine starvation and during shift-up and shift-down conditions. During amino acid starvation, cell mass, cell number, and RNA continued to increase for varying periods. During amino acid and pyrimidine starvation, cell mass and RNA showed little increase, whereas total DNA increased 11 to 17%. After a shift from broth medium to a minimal defined medium, increase in RNA and protein remained at the preshift rate before assuming a lower rate. DNA increase remained at an intermediate rate during shift-down, and then dropped to a low rate. During shift-up from minimal to broth medium, increase in cell number, protein, and DNA showed varying lag periods before increasing to the new rate characteristic of broth medium; each of these quantities exhibited a step sometime in the first 2 hr after transfer to rich medium, suggesting a partial synchronous division. Immediately after shift-up, RNA synthesis assumed a high rate, and then dropped to a rate characteristic of growth in the rich medium after about 1 hr.     

AN AUTORADIOGRAPHIC STUDY OF THE 3H-URIDINE AND 3H-THYMIDINE INCORPORATION IN THE REGENERATING MOUSE LIVER

An autoradiographic study was made of the ³H-uridine incorporation into RNA and DNA in nucleus and cytoplasm of parenchymal cells in the regenerating liver of the mouse after a pulse time of 2 hr. After a decreased uptake of precursor into the parenchymal nucleus during the first 6 hr compared with the normal value, incorporation increased and was maximal at 36 hr; normal values were restored at 72 hr. The cytoplasmic labelling, after an initial small decrease, reached a maximum at 12 hr; this changed to normal 48 hr after hepatectomy. RNase-digestion of the liver sections left a small incorporation in both nucleus and cytoplasm: presumably DNA. This incorporation is maximal at 12 hr over the nucleus and at 24 hr over the cytoplasm. After a 2 hr pulse of ³H-thymidine, there was a marked uptake of the precursor into DNA about 24 hr after hepatectomy. This was maximal at 48 hr and reached normal values at 72 hr. A small amount of incorporation of ³H-thymidine into DNA was seen immediately after the operation, and this population of weakly labelled nuclei was still rather large 72 hr later.     

THE CYTOHISTOLOGICAL AND CYTOHISTOCHEMICAL ZONATION OF THE SHOOT APEX OF BOTRYCHIUM MULTIFIDUM

Cytohistologically, the shoot apex of Botrychium multifidum is composed of three zones—a zone of surface initials in which there is usually a centrally located apical cell, a zone of subsurface initials, and a cup-shaped zone that is subdivided into a pheripheral zone and a rib meristem. The results of cytohistochemical tests for total protein, RNA, total carbohydrate, histones, and DNA localization support this concept. Thus, the cytohistological zonation of the apical meristem of the Ophioglossales is essentially identical to that of the Filicales, and furthermore, is comparable to that of the seed plants.     

NUCLEIC ACID AND PROTEIN METABOLISM DURING THE MITOTIC CYCLE IN VICIA FABA

In order to investigate some of the cytochemical processes involved in interphase growth and culminating in cell division, a combined autoradiographic and microphotometric study of nucleic acids and proteins was undertaken on statistically seriated cells of Vicia faba root meristems. Adenine-8-C¹⁴ and uridine-H³ were used as ribonucleic acid (RNA) precursors, thymidine-H³ as a deoxyribonucleic acid (DNA) precursor, and phenylalanine-3-C¹⁴ as a protein precursor. Stains used in microphotometry were Feulgen (DNA), azure B (RNA), pH 2.0 fast green (total protein), and pH 8.1 fast green (histone). The autoradiographic data (representing rate of incorporation per organelle) and the microphotometric data (representing changes in amounts of the various components) indicate that the mitotic cycle may be divided into several metabolic phases, three predominantly anabolic (net increase), and a fourth phase predominantly catabolic (net decrease). The anabolic periods are: 1. Telophase to post-telophase during which there are high rates of accumulation of cytoplasmic and nucleolar RNA and nucleolar and chromosomal total protein. 2. Post-telophase to preprophase characterized by histone synthesis and a diphasic synthesis of DNA with the peak of synthesis at mid-interphase and a minor peak just preceding prophase. The minor peak is coincident with a relatively localized DNA synthesis in several chromosomal regions. This period is also characterized by minimal accumulations of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA. 3. Preprophase to prophase in which there are again high rates of accumulation of cytoplasmic RNA, and nucleolar and chromosomal total protein and RNA. The catabolic phase is: 4. The mitotic division during which there are marked losses of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA.     

THE CULTURE OF APICAL BUDS OF XANTHIUM AND THEIR USE AS A BIOASSAY FOR FLOWERING ACTIVITY OF ECDYSTERONE

Conditions were developed for the sterile culture of shoot tips of Xanthium pensylvanicum Wallr. for use as a bioassay for flower-controlling chemicals. By using a modified Murashige-Skoog medium (minus the auxin but including kinetin) and light intensity much higher than usual for plant tissue cultures, fast growth and development of the shoot tips was achieved. Under short-day conditions (8 hr day: 16 hr night), the cultures from vegetative shoots flowered and fruited; under noninductive conditions (using a 2 hr light-break in the middle of the dark period), the shoot tips continued vegetative development. Both intact plants and cultured tips could be photoinduced in the first days after germination. Ecdysterone, a potent insect moulting hormone, was tested in the bioassay system. It was without either qualitative or quantitative effect on flowering or vegetative development on either cultured shoot tips or intact plants irrespective of whether they were under inductive or noninductive photoperiodic conditions.     

CELL ARREST IN Gl AND G2 OF THE MITOTIC CYCLE OF VICIA FABA ROOT MERISTEMS

Experiments were performed with cultured excised primary root tips of Vicia faba ‘Longpod’ to determine: (1) the proportion of meristematic cells arrested in Gl and in G2 during carbohydrate starvation, and to determine if the proportion is fixed or can be varied experimentally; (2) the effect of increased starvation on the ability of arrested cells in Gl and G2 to initiate DNA synthesis and mitosis, respectively, when exogenous sucrose was supplied; and (3) whether puromycin, cycloheximide, or actinomycin D prevented the initiation of DNA synthesis and the onset of mitosis. Microspectrophotometry of nuclear DNA and autoradiographic measurements of incorporated ³H-thymidine showed that 72 hr of starvation immediately after excision produced tissue with more than 70 % of the cells arrested in G2 and less than 30 % in Gl. If cultured for three days and then starved for 72 hr, the tissue had nearly equal numbers of cells arrested in Gl and G2. As the duration of starvation increased, the time required to initiate DNA synthesis and to divide when carbohydrate was replenished also increased. Inhibition of protein synthesis by puromycin and cycloheximide prevented the initiation of DNA synthesis and mitosis, but actinomycin D, an inhibitor of RNA synthesis, did not prevent division of cells from G2 nor DNA synthesis by cells from Gl. The experiments demonstrated that the mitotic cycle of Vicia has two major controls, one in Gl and another in G2, and that other factors determine how many cells are affected by either of these cycle controls.     

Histone gene expression and histone mRNA 3' end structure in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Background  

Histone protein synthesis is essential for cell proliferation and required for the packaging of DNA into chromatin. In animals, histone proteins are provided by the expression of multicopy replication-dependent histone genes. Histone mRNAs that are processed by a histone-specific mechanism to end after a highly conserved RNA hairpin element, and lack a poly(A) tail. In vertebrates and Drosophila, their expression is dependent on HBP/SLBP that binds to the RNA hairpin element. We showed previously that these cis and trans acting regulators of histone gene expression are conserved in C. elegans. Here we report the results of an investigation of the histone mRNA 3' end structure and of histone gene expression during C. elegans development.     

Histone gene expression during sea urchin embryogenesis: isolation and characterization of early and late messenger RNAs of Strongylocentrotus purpuratus by gene-specific hybridization and template activity

We have examined the molecular mechanisms responsible for the shifts in histone protein phenotype during embryogenesis in the sea urchinStrongylocentrotus purpuratus. The H1, H2A, and H2B classes of histone synthesized at the earliest stages of cleavage are heterogeneous: These proteins are replaced at late embryogenesis by a different set of histone-like polypeptides, some of which are also heterogeneous. The H3 and H4 histones appear to be homogeneous classes and remain constant. We have isolated from both early and late embryos the individual messenger RNAs coding for each of the multiple protein subtypes. The RNAs were isolated by hybridization to cloned DNA segments coding for a single histone protein or by elution from polyacrylamide gels. Each RNA was then analyzed and identified by its mobility on polyacrylamide gels and by its template activity in the wheat germ cell-free protein synthesizing system. The mRNAs for each of the five early histone protein classes are heterogeneous in size and differ from the late stage templates. The late mRNAs consist of at least 11 separable types coding for the 5 classes of histones. Each of the 11 has been separated and identified. The late stage proteins were shown to be authentic histones since many of their templates hybridize with histone coding DNA. The early and late stage mRNAs are transcribed from different sets of histone genes since (1) late stage H1 and H2A mRNAs fail to hybridize to cloned early stage histone genes under ideal conditions for detecting homologous early stage hybrids, (2) late stage H2B, H3, and H4 RNA/DNA hybrids melt at 14, 11, and 11°C lower, respectively, than do homologous RNA/DNA hybrids, and (3) purified late stage mRNAs direct the synthesis of the variant histone proteins which are synthesized only during later stages. The time course of synthesis of the late stage mRNAs suggests that they appear many hours before the late histone proteins can be detected, possibly as early as fertilization. In addition, early mRNAs are synthesized in small quantities as late as 40 hr after fertilization, during gastrulation. Thus, the major modulations of histone gene expression are neither abrupt nor an absolute on-off switch, and may represent only a gradual and relative repression of early gene expression. Two histones are detected only transiently during early cleavage. The mRNA for one of them, a subtype of H2A, can be detected in the cytoplasm for as long as 40 hr after fertilization. However, template activity for the other, a subtype of H2B, can be detected only at the blastula stage. Thus, the histone genes represent a complex multigene family that is developmentally modulated.     

RNA SYNTHESIS IN THE DIFFERENT PHASES OF CELL CYCLE OF CHICK EMBRYO CELLS

RNA synthesis was studied at different phases of the cell cycle of chick embryo fibroblasts, which were synchronized by medium replacement in the confluent phase. The synthesis of DNA started at 4 hr and continued for 8 hr. RNA synthesis increased with time after medium change. The ratio of total amount of radioactivity in nuclear RNA prepared at 0, 2 and 8 hr was 1.0:1.03:5.05. The distribution of radioactive RNA in the sedimentation pattern was similar, showing remarkable incorporation in 45S region of ribosomal precursor RNA. The base composition of newly synthesized RNA, however, varied at different time intervals after medium replacement. Even within the G₁ phase, the molar percentage of G and C was quite different. Treatment with actinomycin D at a concentration of 0.02 μg/ml for 1 hr specifically inhibited ribosomal RNA synthesis. At 2 hr after medium change, ribosomal and AU-rich RNA including larger than 28S were synthesized in about equal amounts.     

SOME MORPHOLOGIC AND KINETIC STUDIES OF THE DEVELOPING ERYTHROID CELLS OF THE COMMON GOLDFISH, CARASSIUS AURATUS

Developing erythroid cells of the goldfish Carassius auratus were obtained from kidney prints and from smears of the peripheral blood. All preparations were stained with the May-Grunwald Giemsa technique. Developing cells were divided into six different stages. The criteria used to stage the cells were degree of chromatin condensation, degree of basophilia, nuclear:cytoplasmic ratio, and cell shape. The morphology of the maturation sequence for erythroid cells in this organism was similar to that found by other workers in other non-mammalian vertebrates. Fish received intraperitoneal injections of tritiated thymidine, tritiated uridine or tritiated leucine so that the stages involved in DNA synthesis, RNA synthesis and protein synthesis could be determined by means of autoradiography. For the tritiated thymidine studies the per cent labeled cells per stage from four different series receiving 0.5, 1.0, 3.0 or 6.0 μCi/g body weight were pooled, since subjecting the average per cent labeled cells per stage at the lowest and at the highest dosages to Student's t-test showed no significant differences. In all four series the fish were killed 2 hr, 12 hr and daily, 1–8 days post-injection. The ³H-TdR studies showed that stages I-IV were engaged in DNA synthesis; they also showed that about 5 days were required for the stage V cell to become a mature erythrocyte (stage VI cell). Tritiated uridine was injected at a dosage of 5.0 μCi/g body weight and animals were killed 1/2, 1, 3 and 6 hr post-injection. Grain counts showed that stages I-IV are engaged in RNA synthesis and that the rate of this synthesis decreased as maturation proceeded. Tritiated leucine was administered at a dosage of 5.0 μCi/g body weight, and fish were killed 45 min and 3 hr post-injection. Grain counts indicate that stages I-V are engaged in the synthesis of protein (assumed to be globin). The fact that DNA and RNA synthesis ceased with stage IV cells while protein synthesis continued into stage V cells indicated that the mechanism responsible for protein synthesis in stage V cells was produced at an earlier stage and was self-sustained for about 5 days.     

SPECIFIC INHIBITION OF CELL PROLIFERATION IN THE MOUSE INTESTINE BY AN AQUEOUS EXTRACT OF RABBIT SMALL INTESTINE

An aqueous extract was prepared from the mucosa of rabbit small intestine by homogenization and centrifugation at 105,000 g. After precipitation with ammonium sulfate, the 0–50 fraction (F₁) and the supernatant (F₂) were collected, dialysed against a phosphate buffer and tested on rats in vitro and mice in vivo. The F₁ fraction was found to inhibit thymidine incorporation into rat intestinal DNA in vitro, but this effect was not found to be tissue specific (liver, kidney). Two hours after a single injection of F₁ (10 mg protein content), the uptake of tritiated thymidine was decreased in jejunal and colonic DNA in mice. This effect was maximal between 2 and 4 hr and totally reversible after 7 hr; this effect was found in neither the kidney nor the testis. A slowing of cellular migration was also noticed in the jejunum and the colon. Conversely, the F₂ fraction did not inhibit the synthesis of jejunal and colonic DNA either in vitro or in vivo. Our results suggest that the F₁ fraction of the aqueous extract of rabbit small intestine contains one or more substances which may act either on intestinal DNA synthesis or on the G₁–S transition of the cellular cycle in the mouse intestine. This reversible and specific intestinal action appears to inhibit cell proliferation and presents several of the characteristics defining a chalone.     

ROLE OF NUCLEIC ACID AND PROTEIN METABOLISM IN THE INITIATION OF GROWTH AT GERMINATION

When dry decotyledonized embryos of Raphanus are supplied withwater, a brief period of water absorption (phase A) is followedby a period of no fresh weight increase (phase B) which lastsfor 8 hr at 30°. In this period, embryos become ready toadvance into the period of fresh weight increase (phase C). When embryos were exposed to various concentrations of thiouracilor actinomycin D solution from 0 hr of water supply, increasesin fresh weight and in RNA content measured at 13 hr were inhibitedin parallel with each other. Chloramphenicol and puromycin inhibitedthe fresh weight increase without affecting the RNA increase.When embryos were exposed to thiouracil or puromycin for 2,4 and 6 hr, beginning at 0 hr of water supply, the start ofphase C delayed 2, 4 and 6 hr, respectively. When these drugswere given after phase B had progressed at least for 2 hr, thedelay of the start of phase C was shorter than the period ofthe drug treatment. If given at the end of phase B, thiouraciland actinomycin D inhibited the incorporation of ¹⁴C-uracilbut not the fresh weight increase, while chloramphenicol andpuromycin inhibited the latter without inhibiting the former. During phase B, protein content per dry weight of embryo didnot increase, but the rate of ¹⁴C-leucine incorporation increasedremarkably to reach the level in phase C. Incorporation of labeledleucine was inhibited if embryos were subjected to thiouracilor actinomycin D action during phase B, but not if the drugswere given when phase B had been completed. Puromycin and chloramphenicolinhibited the incorporation whenever they were given. The increase in respiratory activity during phase B was inhibitedrelatively little by the above mentioned four drugs. In conclusion syntheses of RNA and protein seem to be essentialfor the progress of phases B and C, protein synthesis havinga more direct effect. (Received September 17, 1965; )