The nonchromatin substructures of the nucleus: the ribonucleoprotein (RNP)-containing and RNP-depleted matrices analyzed by sequential fractionation and resinless section electron microscopy.

The nonchromatin structure or matrix of the nucleus has been studied using an improved fractionation in concert with resinless section electron microscopy. The resinless sections show the nucleus of the intact cell to be filled with a dense network or lattice composed of soluble proteins and chromatin in addition to the structural nuclear constituents. In the first fractionation step, soluble proteins are removed by extraction with Triton X-100, and the dense nuclear lattice largely disappears. Chromatin and nonchromatin nuclear fibers are now sharply imaged. Nuclear constituents are further separated into three well-defined, distinct protein fractions. Chromatin proteins are those that require intact DNA for their association with the nucleus and are released by 0.25 M ammonium sulfate after internucleosomal DNA is cut with DNAase I. The resulting structure retains most heterogeneous nuclear ribonucleoprotein (hnRNP) and is designated the RNP-containing nuclear matrix. The proteins of hnRNP are those associated with the nucleus only if RNA is intact. These are released when nuclear RNA is briefly digested with RNAase A. Ribonuclease digestion releases 97% of the hnRNA and its associated proteins. These proteins correspond to the hnRNP described by Pederson (Pederson, T., 1974, J. Mol. Biol., 83:163-184) and are distinct from the proteins that remain in the ribonucleoprotein (RNP)-depleted nuclear matrix. The RNP-depleted nuclear matrix is a core structure that retains lamins A and C, the intermediate filaments, and a unique set of nuclear matrix proteins (Fey, E. G., K. M. Wan, and S. Penman, 1984, J. Cell Biol. 98:1973-1984). This core had been previously designated the nuclear matrix-intermediate filament scaffold and its proteins are a third, distinct, and nonoverlapping subset of the nuclear nonhistone proteins. Visualizing the nuclear matrix using resinless sections shows that nuclear RNA plays an important role in matrix organization. Conventional Epon-embedded electron microscopy sections show comparatively little of the RNP-containing and RNP-depleted nuclear matrix structure. In contrast, resinless sections show matrix interior to be a three-dimensional network of thick filaments bounded by the nuclear lamina. The filaments are covered with 20-30-nm electron dense particles which may contain the hnRNA. The large electron dense bodies, enmeshed in the interior matrix fibers, have the characteristic morphology of nucleoli. Treatment of the nuclear matrix with RNAase results in the aggregation of the interior fibers and the extensive loss of the 20-30-nm particles.(ABSTRACT TRUNCATED AT 400 WORDS)

Small nuclear RNA localization during mitosis. An electron microscope study.

The localization of small nuclear ribonucleic acids (snRNAs) during mitosis in Amoeba proteus was studied by high voltage (1,000 kV) electron microscope autoradiography. By suitable micromanipulations, the snRNA's, labeled with [3H]uridine, were made to be the only radioactive molecules in the cell and thus easy to follow autoradiographically. During interphase the snRNA label, which is almost exclusively nuclear, is distributed fairly uniformly through the nucleus with a slightly higher amount of label over chromatin than over nonchromatin areas. During prophase the snRNAs, which continue to be largely nuclear, become highly concentrated in the condensing chromosomes. At metapase, almost all of the snRNAs are cytoplasmic and essentially none are associated with the maximally condensed chromatin. Beginning in early anaphase, the snRNAs resume their association with the chromosomes, with the degree of association increasing throughout anaphase. Most of the snRNAs are back in the nuclei by telophase, but the intranuclear localization is hard to determine. We conclude that snRNAs have a great affinity for the partially condensed chromosomes of prophase and anaphase, but none for the maximally condensed chromosomes of metaphase. A minor amount of snRNA localizations in association with nucleoli and the nuclear envelope are also reported. On the basis of these findings a role of snRNAs in genetic "reprogramming" or chromosome organization is proposed.

The orientation of DNA within 80-Angstrom chromatin fibers.

Squashing salivary glands of Chironomus thummi larvae, Amblystoma tigrinum erythrocytes, or Spirostromum frequently results in stretched chromatin having highly oriented DNA as determined by polarized fluorescence microscopy of acridine orange-stained preparations. The examination of such material from C. thummi in the electron microscope indicates that the individual chromatin fibers have an average thickness of 80 A as is usually found in embedded and sectioned material. It is thus concluded that the DNA lies nearly parallel to the axis of these chromatin fibers. Detailed calculations of the polarization expected from various models of DNA packing are contained in an appendix.

Presence of histones in Aspergillus nidulans.

Five major histone proteins have been extracted from chromatin isolated from purified nuclei of the fungus, Aspergillus nidulans. These proteins had chromatographic properties which were similar to reference calf thymus histones and were purified to electrophoretic homegeneity by gel chromatography of Bio-Gel P10, Bio-Gel P60, and Sephadex G-100. Electrophoresis of these proteins in three different systems (urea-starch, urea-acetic acid polyacrylamide, and discontinuous SDS polyacrylamide) showed that the A. nidulans histones H3 and H4 were nearly identical to calf thymus H3 and H4 with respect to net charge and molecular weight criteria, whereas the fungal histones H1, H2a and H2b were similar but not identical to the corresponding calf thymus histones. Amino acid analysis of A. nidulans histones H2a, H2b, and H4 showed them to be closely related to the homologous calf thymus histones. The mobility patterns of A. nidulans ribosomal basic proteins in three different electrophoretic systems were distinctly different from those of the fungal histones.

The histones associated with condensed and extended chromatin of mouse liver.

Histones were extracted from isolated mouse liver nuclei, and from mouse liver condensed and extended chromatin. Mouse liver histones were found to be very similar to those of calf thymus in their solubility properties, relative electrophoretic mobilities, and molecular weights as determined on SDS-polyacrylamide gels. Quantitative analysis by high-resolution gel electrophoresis demonstrated a remarkable similarity between the histones of condensed chromatin and those of extended chromatin. However, minor differences were found. A unique subspecies was found only in condensed chromatin histone and the relative amounts of fractions F2A1 and F2A2 differed in the two types of chromatin. The ratio of the parental to the acetylated form of F2A1 was identical in the two chromatin samples. Since DNA extracted from the condensed chromatin fraction consisted of approximately 50% satellite DNA, the general similarities between the histones of condensed and extended chromatin make it likely that even this simple, highly repetitive DNA is complexed with a number of histone subfractions.

Image analysis of chromatin in cells of reimplantation mouse embryos.

Mouse two-celled embryos and blastulae were Feulgen stained and the DNA content of their nuclei was measured with an integrating microdensitometer. The cells considered on the basis of their nuclear DNA content to be in G(1), S, and G(2) phases of the cell cycle were selected and their total chromatin area and chromatin areas at different gray levels were measured by the image analyzing computer, Quantimet. The measurements were aimed at quantitation of several features of the chromatin morphology of cells in different functional states. The total area of chromatin was found to increase, and the mean density of chromatin to decrease, from the G(1) to the G(2) phase of the cell cycle in both two-celled embryos and blastulae. The area of chromatin decreased, and the mean density of chromatin increased, as embryos developed from two-celled to blastula stage. It was concluded that nuclear morphology in preimplantation mouse embryos depends on both the phase of the cell cycle and the stage of development. The method of image analysis described was found to be useful for quantitation of changes in chromatin morphology.

Concentration of elements in mitotic chromatin as measured by x-ray microanalysis.

Unfixed frozen-dried and uncoated tissue sections of the mouse duodenum were placed on carbon planchets and analyzed in a scanning electron microscope fitted with energy dispersive X-ray equipment. Computer analysis of the X-ray spectra allowed elemental microanalysis of the nucleus, cytoplasm, and mitotic chromatin regions in the cryptal and villus enterocytes. The peak to continuum ratio of S, Cl, K, and Ca were higher in mitotic chromatin than any of the other sites measured. The redistribution of Ca at mitosis is postulated to help explain both chromosome condensation and assembly of the mitotic spindle apparatus.

Isopycnic centrifugation of chromatin in renografin solutions.

Solutions of Renografin (30-60%) can be centrifuged to form density gradients in the range from 1.0 g/cm(3) to 1.4 g/cm(3) or, alternatively, preformed gradients can be made which under appropriate conditions of centrifugation have an indefinite stability. Such solutions have a low viscosity and a relatively low ionic strength. The density of DNA in such solutions is surprisingly low ( approximately 1.14 g/cm(3)). Crude chromatin can be sedimented to an equilibrium position in such gradients, corresponding to a density of 1.2(4) g/cm(3), or slightly lower, depending on the method of preparation. The complex is shown to contain DNA, RNA, protein, and possibly some lipoprotein. Most of the RNA can be removed with RNase without any significant effect on the density of the chromatin.

Two-dimensinal gel electrophoresis of rat liver nuclear washes, nuclear matrix, and hnRNA proteins.

The proteins of rat liver cytoplasm, nuclear washes, matrix, membrane, heterogeneous nuclear (hn)RNA proteins and chromatin were examined by two-dimensional gel electrophoresis. The inclusion in the gels of six common protein standards of carefully selected molecular weight and isoelectric point allowed us to clearly follow the distribution of specific proteins during nuclear extraction. In the nuclear washes and chromatin, we observed five classes of proteins: (a) Exclusively cytoplasmic proteins, present in the first saline-EDTA wash but rapidly disappearing from subsequent washes; (b) ubiquitous proteins of 75,000, 68,000, 57,000, and 43,000 mol wt, the latter being actin, found in the cytoplasm, all nuclear washes and the final chromatin pellet; (c) proteins of 94,000, 25,000, and 20,500 mol wt specific to the nuclear washes; (d) proteins present in the nuclear washes and final chromatin, represented by species at 62,000, 55,000, 54,000, and 48,000 mol wt, primarily derived from the nuclear matrix; and (e) two proteins of 68,000 mol wt present only in the final chromatin. The major 65,000-75,000-mol wt proteins seen by one-dimensional gel electrophoresis of nuclear matrix were very heterogeneous and contained a major acidic, an intermediate, and a basic group. A single 68,000-mol wt polypeptide constituted the majority of the membrane-lamina fraction, consistent with immunological studies indicating that a distinct subset of matrix proteins occurs, associated with heterochromatin, at the periphery of the nucleus. Actin was the second major nuclear membrane-lamina protein. Two polypeptides at 36,000 and 34,000 mol wt constituted 60% of the hnRNP. Approximately 80% of the mass of the nonhistone chromosomal proteins (NHP) from unwashed nuclei is contributed by nuclear matrix and hnRNPs, and essentially the same patterns were seen with chromatin NHP. The concept of NHP being a distinct set of DNA-bound proteins is unnecessarily limiting. Many are derived from the nuclear matrix or hnRNp particles and vary in the degree to which they share different intracellular compartments.

Fractionation of nucleosomes by salt elution from micrococcal nuclease-digested nuclei.

The solubilization of nucleosomes and histone H1 with increasing concentrations of NaCl has been investigated in rat liver nuclei that had been digested with micrococcal nuclease under conditions that did not substantially alter morphological properties with respect to differences in the extent of chromatin condensation. The pattern of nucleosome and H1 solubilization was gradual and noncoordinate and at least three different types of nucleosome packing interactions could be distinguished from the pattern. A class of nucleosomes containing 13--17% of the DNA and comprising the chromatin structures most available for micrococcal nuclease attack was eluted by 0.2 M NaCl. This fraction was solubilized with an acid-soluble protein of apparent molecular weight of 20,000 daltons and no histone H1. It differed from the nucleosomes released at higher NaCl concentrations in content of nonhistone chromosomal proteins. 40--60% of the nucleosomes were released by 0.3 M NaCl with 30% of the total nuclear histone H1 bound. The remaining nucleosomes and H1 were solublized by 0.4 M or 0.6 M NaCl. H1 was not nucleosome bound at these ionic strengths, and these fractions contained, respectively, 1.5 and 1.8 times more H1 per nucleosome than the population released by 0.3 M NaCl. These fractions contained the DNA least available for micrococcal nuclease attach. The strikingly different macromolecular composition, availability for nuclease digestion, and strength of the packing interactions of the nucleosomes released by 0.2 M NaCl suggest that this population is involved in a special function.

A model for the structure of chromatin in mammalian sperm.

DNA in mammalian, and most vertebrate sperm, is packaged by protamines into a highly condensed, biochemically inert form of chromatin. A model is proposed for the structure of this DNA-protamine complex which describes the site and mode of protamine binding to DNA and postulates, for the first time, specific inter- and intraprotamine interactions essential for the organization of this highly specialized chromatin. In this model, the central polyarginine segment of protamine binds in the minor groove of DNA, crosslinking and neutralizing the phosphodiester backbone of DNA while the COOH- and NH2-terminal ends of protamine participate in the formation of inter- and intraprotamine hydrogen, hydrophobic, and disulfide bonds. Each protamine segment is of sufficient length to fill one turn of DNA, and adjacent protamines are locked in place around DNA by multiple disulfide bridges. Such an arrangement generates a neutral, insoluble chromatin complex, uses all protamine sulfhydryl groups for cross linking, conserves volume, and effectively renders the chromatin invulnerable to most external influences.

Selective excision of the centromere chromatin complex from Saccharomyces cerevisiae.

We have taken advantage of the known structural parameters associated with centromere DNA in vivo to construct a CEN fragment that can be selectively excised from the chromatin DNA with restriction endonucleases. CEN3 DNA is organized in chromatin such that a 220-250-bp region encompassing the elements of centromere homology is resistant to nuclease digestion. Restriction enzyme linkers encoding the Bam HI-recognition site were ligated to a 289 base pair DNA segment that spans the 220-250-bp protected core (Bloom et al., 1984). Replacement of this CEN3-Bam HI linker cassette into a chromosome or plasmid results in formation of a complete structural and functional centromeric unit. A centromere core complex that retains its protected chromatin conformation can be selectively excised from intact nuclei by restriction with the enzyme Bam HI. The centromeric protein-DNA complex is therefore not dependent upon the intact torsional constrains on linear chromosomes for its structural integrity. Isolation of this complex provides a novel approach to characterizing authentic centromeric proteins bound to DNA in their native state.

Alterations in chromatin conformation are accompanied by reorganization of nonchromatin domains that contain U-snRNP protein p28 and nuclear protein p107.

The intranuclear distribution of nuclear matrix-associated protein p107 and the 28-kD Sm antigen of U-snRNPs have been studied using double-label immunofluorescence and immunoperoxidase electron microscopy. In interphase nuclei of HeLa cells, Novikoff hepatoma cells, and rat kangaroo kidney cells, p107 was confined to discrete interchromatin domains. The domains had an irregular contour, with an average diameter of 1-1.5 micron. Each domain appeared to be composed of interconnected granules. The Sm antigen colocalized and appeared concentrated in these domains but also showed some general nucleoplasmic distribution. During mitosis, the interchromatin domains disassembled such that the Sm portion redistributed to the perichromosomal and spindle regions and the p107 component redistributed throughout the mitotic cytoplasm. During anaphase, p107 assembled into discrete clusters throughout the mitotic cytoplasm. The Sm antigen was not a component of these clusters. Double-label immunofluorescence with anti-p107 and the anti-DNA tight-binding protein, AhNa1, showed that the extranuclear p107 domains assumed an interchromatin localization only after the chromosomes had decondensed. The correlation between chromosome decondensation and the occurrence of p107 within interchromatin domains was also observed during chicken erythrocyte nuclear reactivation. We propose that the discrete interchromatin domains that contain p107 and p28 may be important for processing and splicing of RNA and that their structural assembly within nuclei is sensitive to the presence of the transcriptionally active conformation of chromatin.

Radial density distribution of chromatin: evidence that chromatin fibers have solid centers.

Fiber diameter, radial distribution of density, and radius of gyration were determined from scanning transmission electron microscopy (STEM) of unstained, frozen-dried chromatin fibers. Chromatin fibers isolated under physiological conditions (ionic strength, 124 mM) from Thyone briareus sperm (DNA linker length, n = 87 bp) and Necturus maculosus erythrocytes (n = 48 bp) were analyzed by objective image-processing techniques. The mean outer diameters were determined to be 38.0 nm (SD = 3.7 nm; SEM = 0.36 nm) and 31.2 nm (SD = 3.6 nm; SEM = 0.32 nm) for Thyone and Necturus, respectively. These data are inconsistent with the twisted-ribbon and solenoid models, which predict constant diameters of approximately 30 nm, independent of DNA linker length. Calculated radial density distributions of chromatin exhibited relatively uniform density with no central hole, although the 4-nm hole in tobacco mosaic virus (TMV) from the same micrographs was visualized clearly. The existence of density at the center of chromatin fibers is in strong disagreement with the hollow-solenoid and hollow-twisted-ribbon models, which predict central holes of 16 and 9 nm for chromatin of 38 and 31 nm diameter, respectively. The cross-sectional radii of gyration were calculated from the radial density distributions and found to be 13.6 nm for Thyone and 11.1 nm for Necturus, in good agreement with x-ray and neutron scattering. The STEM data do not support the solenoid or twisted-ribbon models for chromatin fiber structure. They do, however, support the double-helical crossed-linker models, which exhibit a strong dependence of fiber diameter upon DNA linker length and have linker DNA at the center.

Comparison and characterization of nuclear isolation procedures as applied to chick oviduct.

A number of methods for the preparation of chick oviduct nuclei have been compared. Nuclei have been isolated in hypertonic sucrose and citric acid and the product has been characterized with respect to cleanliness, ultrastructure, RNA polymerase activity, RNA integrity, and chromatin composition. The study demonstrates that the choice of oviduct nuclear isolation procedure will depend markedly on the purpose for which the nuclei are required. Thus, nuclei prepared entirely in high-molarity sucrose retain the highest levels of RNA polymerase. Those prepared rapidly in the presence of citric acid retain nuclear RNA in an essentially undegraded state. Finally, a bulk preparation is described which, because of its adaptability and high yield of morphologically intact nuclei using large amounts of tissue, is ideal for use in preparing chromatin. Conditions are described by which isolated nuclei can be stored for up to 6 months and retain their morphology, chemical characteristics, and RNA polymerase activity.

Nuclear proteins. II. Similarity of nonhistone proteins in nuclear sap and chromatin, and essential absence of contractile proteins from mouse liver nuclei.

High resolution SDS slab gel electrophoresis has been used to examine the distribution of nonhistone proteins (NHP) in the saline-EDTA, Tris, and 0.35 M NaCl washes of isolated mouse liver nuclei. These studies led to the following conclusions: (a) all the prominent NHP which remain bound to DNA are also present in somewhat similar proportions in the saline-EDTA, Tris, and 0.35 M NaCl washes of nuclei; (b) a protein comigrating with actin is prominent in the first saline-EDTA wash of nuclei, but present as only a minor band in the subsequent washes and on washed chromatin; (c) the presence of nuclear matrix proteins in all the nuclear washes and cytosol indicates that these proteins are distributed throughout the cell; (d) a histone-binding protein (J2) analogous to the HMG1 protein of K. V. Shooter, G.H. Goodwin, and E.W. Johns (Eur J. Biochem. 47:236-270) is a prominent nucleoplasmic protein; (e) quantitation of the major NHP indicates that they are present in a range of 2.2 X 10(5)-5.2 X 10(6) copies per diploid nucleus. Most of the electrophoretically visible NHP are probably structural rather than regulatory proteins; (f) actin, myosin, tubulin, and tropomyosin, if present at all, constitute a very minor fraction of the nuclear NHP. Contractile proteins constitute a major portion of the NHP only when the chromatin is prepared from crude cell lysates instead of from purified nuclei. These studies support the conclusion that there are no clear differences between many nucleoplasmic and chromatin-bound nonhistone proteins. Except for the histones, many of the intranuclear proteins appear to be in equilibrium between DNA, HnRNA, and the nucleoplasm.

Inorganic cations in rat kidney. Localization with potassium pyroantimonate--perfusion fixation.

For localization of pyroantimonate-precipitable cations, rat kidney was fixed by perfusion with a saturated aqueous solution of potassium pyroantimonate (pH about 9.2, without addition of any conventional fixative). A remarkably good preservation of the tissue and cell morphology was obtained as well as a consistent and reproducible localization of the insoluble antimonate salts of magnesium, calcium, and sodium. All proximal and distal tubules and glomeruli were delimited by massive electron-opaque precipitates localized in the basement membrane and, to a lesser extent, in adjacent connective tissue. In the intraglomerular capillaries the antimonate precipitate was encountered in the basement membranes and also between the foot processes. In addition to a more or less uniform distribution in the cytoplasm and between the microvilli of the brush border, antimonate precipitates were found in all cell nuclei, mainly between the masses of condensed chromatin. The mitochondria usually contained a few large antimonate deposits which probably correspond to the so-called "dense granules" observed after conventional fixations.

Further observations on the occurrence of nexuses in benign and malignant human cervical epithelium.

An estimate is made of the frequency of occurrence of nexuses ("gap junctions") in a spectrum of human cervical epithelia, ranging from normal to malignant, since a deficiency of nexuses may be important in abnormal cell-to-cell communication in malignant tissues. The normal cervical epithelium has approximately ten nexuses per cell in the basal layer of proliferating cells and 200 nexuses per cell in the more differentiated intermediate zone. Nexuses are rare between invasive malignant epithelial cells (carcinoma cells). In many areas of cell proliferation near the edge of the tumor mass, fewer than one nexus per cell is present. However, up to four nexuses per cell can be found in some well differentiated regions of invasive carcinoma. Preinvasive malignant epithelia (severe dysplasia and carcinoma-in situ) have as few nexuses as invasive carcinoma. In abnormal but benign epithelia (squamous metaplasia and mild dysplasia), nexuses are abundant. The data indicate that a decrease in number of nexuses correlates with the severity of the morphological alteration in the dysplastic epithelium. Also the deficiency of nexuses in groups of carcinoma cells can occur many cell generations before the development of invasion of the malignant epithelium into the connective tissue. The diminution of nexuses before invasion suggests that a deficiency of nexuses may be one of the important factors in eventually permitting the development of the diffusely infiltrating type of invasion which is characteristic of highly malignant tumors such as squamous carcinomas.

Isolation and biochemical characterization of nuclei from chick embryo liver.

A procedure is described for the isolation of enzymatically active nuclei from chick embryo liver. It consists of the homogenization of the pooled tissue in 0.32 M sucrose-3 mM MgCl(2) followed by a slow centrifugation. The resulting nuclear pellet is then purified further in a discontinuous density gradient composed of sucrose solutions containing Mg(2+) ions, the lower portion of the gradient being 2.2 M sucrose-1 mM MgCl(2). Based on DNA recovery, the nuclear fraction isolated by the procedure described contained an average of 62% of the nuclei in the original filtered homogenate. Light and electron microscope examinations showed that 90% of the isolated nuclei were derived from hepatocytes. They appeared intact with well preserved nucleoplasmic and nucleolar components, nuclear envelope, and pores. The isolated nuclei were quite pure, having a very low level of cytoplasmic contamination as indicated by cytoplasmic enzyme marker activities and electron microscope studies. The nuclear fraction consisted of 19.9% DNA, 6.2% RNA, 74% protein, the average RNA/DNA ratio being 0.32. Biosynthetic activities of the two nuclear enzymes NAD-pyrophosphorylase and DNA-dependent RNA polymerase were preserved. The specific activities of these enzymes were: NAD-pyrophosphorylase, 0.049 micromoles nicotinamide adenine dinucleotide (NAD) synthesized/min per mg protein; Mg(2+) activated RNA polymerase, 4.3 micromicromoles UMP-2-C(14) incorporated into RNA/microg DNA per 10 min; and Mn(2+)-(NH(4))(2)SO(4) activated RNA-polymerase, 136 micromicromoles UMP-2-C(14) incorporated into RNA/microg DNA per 45 min.

Differentiation of monocytes. Origin, nature, and fate of their azurophil granules.

The origin, content, and fate of azurophil granules of blood monocytes were investigated in several species (rabbit, guinea pig, human) by electron microscopy and cytochemistry. The life cycle of monocytes consists of maturation in bone marrow, transit in blood, and migration into tissues where they function as macrophages. Cells were examined from all three phases. It was found that: azurophil granules originate in the Golgi complex of the developing monocyte of bone marrow and blood, and ultimately fuse with phagosomes during phagocytosis upon arrival of monocytes in the tissues. They contain lysosomal enzymes in all species studied and peroxidase in the guinea pig and human. These enzymes are produced by the same pathway as other secretory products (i.e., they are segregated in the rough ER and packaged into granules in the Golgi complex). The findings demonstrate that the azurophil granules of monocytes are primary lysosomes or storage granules comparable to the azurophils of polymorphonuclear leukocytes and the specific granules of eosinophils. Macrophages from peritoneal exudates (72-96 hr after endotoxin injection) contain large quantities of lysosomal enzymes throughout the secretory apparatus (rough ER and Golgi complex), in digestive vacuoles, and in numerous coated vesicles; however, they lack forming or mature azurophil granules. Hence it appears that the monocyte produces two types of primary lysosomes during different phases of its life cycle-azurophil granules made by developing monocytes in bone marrow or blood, and coated vesicles made by macrophages in tissues and body cavities.