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Balbiani ring hnRNP substructure visualized by selective staining and electron spectroscopic imaging.

The Balbiani Rings (BR) in the polytene chromosomes of Chironomus salivary glands are intense sites of transcription. The nascent RNPs fold during transcription into 40-50-nm granules, containing in the mature transcript approximately 37-kb RNA. Using a new nucleic acid specific stain, osmium ammine B on Lowicryl sections, in combination with electron energy filtered imaging of sections containing BR granules, we demonstrate a RNA-rich particulate substructure (10-nm particle diameter; 10-12 particles per BR granule). Elemental imaging supports that these particles are enriched in phosphorus. The possible relationship of these RNA-rich particles to ribonucleosomes is discussed, as well as models for their arrangement in the mature BR granules.

connected via a stalk to the presumptive chromatin axis, or as mature granules within the nucleoplasm (6,36) . The packaging and arrangement of BR granules either attached to the chromatin axis, free within the nucleoplasm or traversing nuclear pores into the cytoplasm has been examined in numerous EM studies, using the methods of stereo-EM and electron microscope tomography (EMT) (19, 20, 22-26, 28, 33-35) . These methods have led to the following conclusions : (a) At least in the case of the hyperactive pilocarpinestimulated BR2 gene (23)(24)(25)(26), the nascent transcripts are close packed along the chromatin axis at -80-100 granules//Am in an imperfect helical arrangement of five to seven granules/turn . The axis DNA is estimated to be compacted about threefold (whereas, a string of nucleosomes would be compacted seven-to eightfold), implying that the chromatin is markedly unfolded during the intense transcription ; and (b) the outer diameter of the mature BR granule is -50 nm and appears to represent a coiled RNP ribbon of length, 120-nm long, 25-50-nm wide, 10-15-nm thick (33)(34)(35) . As it passes through a nuclear pore, the BR granule unfolds into the RNP ribbon . Some observations suggest that the ribbon is composed of a 10-nm diam zigzag or spiral RNP filament (20) . The BR hnRNP granules have been isolated and partially characterized (38,39) . These granules sediment at about 300S andconsistofapproximately 40% RNA and 60 protein . Treatment ofisolated 300S BR granules with RNase A revealed some unexpected results . 40S ribonucleosomal particles were never observed. Most of the granules could sustain numerous nicks in the 37-kb RNA without a change in sedimentation . The authors (39) concluded that whether hnRNP particles exist within mature BR granules can not yet be answered . To better define the structural organization of the protein and nucleic acid components of BR granules, we combined a selective nucleic acid stain with EM.
The use of selective nucleic acid staining in EM offers the potential to visualize DNA-or RNA-containing structures without the additional electron density arising from stained proteins. The most successful approach in this direction has used the Schifflike reagent, osmium ammine (OA) to stain DNA in sectioned nuclei after depurination and release of reactive aldehydes (12,16) . Because of the difficulty of reproducibly synthesizing usable preparations of OA, we (18,27) developed a new more reliable synthesis procedure and final product denoted OA-B. As yet we do not know the detailed chemical composition of OA or OA-B, or whether they share any identical chemical components which are responsible for the stain specificity. A recent study (11) has demonstrated that, under suitable conditions, OA can give high resolution staining of both RNA and DNA. These conditions include: Lowicryl K4M embedding ; no depurination, and staining with OA dissolved in 0.1 M glycine-HCI, pH 1.5, or S02-bubbled OA in water . Stain specificity was demonstrated by pretreatment of the sections with various nucleases and proteases. In the present study, we show that OA-B-treated Lowicryl sections produces a clear demonstration of an organized substructure within BR hnRNP particles .
One disadvantage with selective nucleic acid staining by OA or OA-B is the low inherent contrast of the fine osmium stain. These problems can be partially overcome by optimization of EM data collection by using a low-accelerating voltage, a small objective aperture and by photographic and digital enhancement . In this study, we optimize image contrast by collecting energy-filtered images of OA-B-stained sections . Electron spectroscopic imaging (ESI) is a relatively new method that uses an electron spectrometer situated below the specimen in a fixed beam EM, such as exists on the Zeiss EM902 microscope (Carl Zeiss, Inc., Oberkochen, Germany) (30) . Within different regions of a section, electrons are inelastically scattered depending upon the chemical elements present within that region . It then becomes convenient to image selectively for a particular element by using the spectrometer to transmit a window of inelastically scattered electrons characteristic for that element . This method has been successfully used to image phosphorus within DNA and RNA containing structures (3)(4)(5)29) . Elemental imaging requires very thin sections to minimize multiple electron scattering events. In the present study, we used the spectrometer to enhance "structure-sensitive contrast" (2,31) to minimize the contribution ofcarbon and strengthen the osmium signal in the final EM image . This was accomplished by imaging with electrons which have lost 250 eV, just before the carbon K edge (AE = 284 eV) in the energy loss spectrum . When this method was combined with selective OA-B staining, the resulting darkfieldlike images revealed a discrete RNA-containing particulate substructure in the BR granules. Phosphorus mapping was also performed on BR granules present in very thin unstained sections, providing a strong indication that the particulate substructures are highly enriched in phosphorus .
Selective Staining with OA-B OA-B prepared as described previously (18,27), now available from Polysciences, Inc. (Warrington, PA) was used as described for OA (11) . RNase and DNase pretreatment of sections followed the method described (11) . Staining was performed with 0.2 % OA-B in H2O, bubbled with S02 for 20 minjust before contact with the sections. Grids were immersed in the OA-B solution in capped Beem capsules and incubated at 37°C for 1 h. Stained grids were rinsed with water and air dried.
For EMT data collection, one microliter of 5-nm diam colloidal gold spheres (Energy Beam Sciences, Agawam, MA), diluted 1:100 in H2O was adsorbed to each surface of the stained grids. The use of gold particle positions in alignment and registration of tilt images has been described previously (24,35).

General Staining with Uranyl and Lead
Salivary glands were fixed in 3 % glutaraldehyde, 50 mM Sorensen's buffer, pH 7.2, for 4 h, washed, and fixed in 1% OsO4 in the same buffer, washed, dehydrated, and embedded in Epon . Sections were stained in 2% uranyl magnesium acetate for 20 min at 48°C and for 5 min in lead citrate.

Electron Spectroscopic Imaging (ESI)
Stained specimens were imaged on a Zeiss EM902 microscope equipped with a prism-mirror-prism type electron imaging spectrometer, top-entry tilt stage and an image-intensification video camera to permit data collection at reduced dose . The microscope was operated at an accelerating voltage of 80 kV The width of the energy selecting slit aperture corresponds to 20eV. A 700 Am condenser and a 60 Am objective aperture were used . Because the tilt stage was not eucentric, the grid was rotated within the holder to bring a nucleus or BR2 onto the tilt axis, following a procedure outlined previously (28) . This procedure was performed before collecting stereo pairs or a complete tilt series . Imaging was performed at DE = 250 eV, before the carbon K edge at AE = 284eV. In some experiments the same field was photographed at selected energy losses to examine the change in contrast of the stained regions . Photographs were collected at 12, 20, 30, 50, and 85K magnification .

Phosphorus Mapping of BR Granules
Phosphorus distribution maps of BR granules were obtained using the methods described previously (3)(4)(5)29). Two energy loss images of an unstained specimen were recorded, a pre-edge image at 120 eV and a phosphorus-enhanced image at 160 eV energy loss . Regions of interest on both micrographs were digitized and normalized to account for slight changes in exposure density over regions that were judged to be devoid of phosphorus . Alignment of the two digitized regions to within one pixel was accomplished by eye, by alternating the display between a stored phosphorus enhanced image andan on-line edge image being translated and rotated .
Subtraction of the aligned and normalized pre-edge image from the phosphorus enhanced image produces a net phosphorus image. We have previously shown that the results obtained with a one-parameter (one pre-edge image) and a two-parameter(two pre-edge images) are comparable, providing the specimen thickness is not greater than -30 nm .

BR Granule Substructure
RNA and DNA are stained specifically with OA-B in Lowicryl sections ps previously described for OA (11) . In the giant cells of the Chironomus tentans salivary gland, RNA in ribosomes and in BR granules is clearly visible . In Fig .  1, stereo pairs of a BR transcription axis are shown: the upper panel is an image of a Lowicryl section stained with OA-B; the lower panel is a section stained with uranyl and lead (22,23) . In both panels, regions within the nucleus contain a cluster of nascent BR granules along a portion of the transcription axis and individual mature BR granules located throughout the nucleoplasm . Nascent and mature BR granules exhibit obvious particulate substructure after staining with OA-B . In Lowicryl sections pretreated with RNase, the contrast of RNPs is completely obliterated (Fig. 2 E), but DNA in the banded polytene chromosome (Fig . 2 F shown) leaves the RNA staining but reduces the intensity of polytene chromosome bands only. The clarity of OA-B stained structures is striking. The stain is very fine, permitting detailed examination of specimens at high magnification .

Osmium Contrast Is Optimized Just below the Carbon K Edge
The same field of an OA-B-stained Lowicryl specimen was imaged with electrons of varying eVloss (Fig . 2) in a manner analogous to a series of images previously published (31) . bright-field image (elastically and inelastically scattered electrons) . Ribosomes in the cytoplasm, gold particles, and ImRNP are darker than the background, but the contrast is poor. Fig . 2 B is a zero-loss electron image created with only good at AE = 250 eV (Fig . 2 C). Beyond the carbon K edge, however, an image recorded at AE = 295 eV (Fig . 2 D) reveals a marked deterioration of contrast, since a high proportionof inelastically scattered electrons arise from background carbon . A more complete discussion of these contrast reversals with varying eVloss windows has been presented (30) . An electron energy-loss spectrum of an OA-B-stained specimen (Fig. 3) (Fig. 4) . The RNA distribution is obviously not homogeneous, rather, the clearly punctate pattern of stain suggests segregation of RNA-rich and of protein-rich domains . Here we note that different perspectives, determined by the angle of sectioning, suggest the coiling of a string of hnRNP particles . This interpretation of the data is most clearly demonstrated in Fig . 5 where views of BR granules from two tilt series are presented at -10' intervals . If a stereo viewer is used the rotation of the BR granules is easily observed . The top tilt series appears to rotate around the "pin-wheel" orientation of a BR granule. Due to the specificity of the stain, the presumptive RNA is easily visualized without interference from the many proteinaceous structures in the nucleoplasm, even at very high tilt angles . The bottom series resembles rotation around an "edge-on" view of a short helix . From numerous measurements of OA-B stained BR granules, we obtain the following average parameters : BR granule outer diameter, 43 .6 f 0 .6 ran (SEM), n = 89 ; particle diameter, 9 .3 t 0.1 nm (SEM), n = 166 . By contrast, the aver-487 Figure 4. High magnification stereo EM pairs of mature BR granules demonstrate the organization of the RNA substructure from various perspectives . Notice the different orientations ranging from en face "pinwheel" structure to tilted views . Bar, 100 run . age diameter of cytoplasmic ribosomes (combining together measurements of long and short axes) was: 17.7 f 0.5 run (SEM), n = 65. It is difficult to estimate accurately the number of particles per BR granule . Careful study of many stereo images suggests that up to 10-12 particles can sometimes be observed; most BR granules appear to have less. At present, we assume that the lower numbers could arise for several reasons : (a) overlap of discrete particles in the images ; (b) fusion or proximity of adjacent particles below the resolution of our methods ; and (c) removal of some particles during sectioning . There may also be more than one type of BR granule substructure ; BRl, 2, and 6 transcribe 35-40-kb mRNA (7,10) . Granules of similar outer diameter but distinctly fibrous substructure can also be readily identified within the nucleoplasm . None-the-less, the morphology of the BR granules with particulate substructure was so distinctive, and the proportion so great, that this class could be easily studied .

BR Granule Substructure Is Retained during Pressage through Nuclear Pores
It is a simple matter to identify the nuclear envelope even though the OA-B stain does not exhibit significant binding to proteins and lipids ; the high concentration of ribosomes in the cytoplasm ends abruptly at the edge of the unstructured nucleoplasm, defining the envelope (Fig. 2 C) . A montage of stereo pairs of BR granules passing from the nucleoplasm on the left to the cytoplasm on the right is presented in Fig .  6. As visualized by OA-B staining, the unraveling BR granule The Journal of Cell Biology, Volume 117, 1992 appears cone shaped with a leading string of particles passing through the nuclear envelope, whereas the uranyl-and lead-stained stereo pair (Fig. 6, bottom) appears more like a ribbon passing through the pore.

BR Granules Exhibit Phosphorus-rich Particles
To determine whether the OA-B was identifying an RNArich particle or whether the stain is only accessible by diffusion to certain domains of the granule, we obtained phosphorus-enhanced and net phosphorus images of BR granules . The granules were imaged in unstained thin sections (N30-nm thick) of tissue . Both the phosphorusenhanced images and the net phosphorus images showed particulate structure (Fig . 7) . Since the sections are thinner than BR granule diameters (40-50 run), only portions of granules were visible . The pattern obtained with the OA-B stain and the phosphorus maps of unstained specimens suggests that the RNA is localized in discrete domains within the BR granules .

Discussion
The present study demonstrates that a reproducible higherorder substructure of nascent and mature BR granule RNA is observed by a combination of selective nucleic acid staining and electron spectroscopic imaging (ESI) to enhance specimen contrast . This combination of methods holds considerable promise for the study of many DNA-and RNAcontaining cellular structures . Combined with EMT (19, Oss 24-26, 28, 33, 35, 38), it should be possible to develop more precise spatial models of the position and arrangement of subunits in these higher-order structures. EMT studies on BR granules are currently in progress . Based upon extensive study of stereo images of OA-B-stained mature BR granules, we estimate that each granule contains 10-12 RNA-rich particles, with an average particle diameter of 9-10 nm . Dividing the total hnRNA length (-37 kb) by 10-12 particles, yields 3 .1-3.7-kb RNA per particle . The stereo images suggest that the particles are arranged into regular higher-order structures (e .g., "pinwheel" views are frequently observed ; see Figs . 4 and 5) . This higher-order packing appears to be disrupted when BR granules pass through nuclear pores ; during transit the BR granule appears to unravel into a string of particles .
This study raises two significant questions : (a) Is there on obvious relationship between the 10-nm RNA-rich particles observed here in BR granules and the properties of ribonucleosomes (1,8,9,13,14)? and (b) How can this particulate Olins et al . Balbiani Ring hnRNP Substructure Figure 6. The BR granule as it passes through the nuclear envelope can be observed in this montage of stereo EM pairs. The cytoplasm is at the right, the nucleoplasm at the left (compare with Fig. 2 C) . Arrowheads point the presumed direction of BR granule movement . A short string of 10-nm particles can often be observed leading the hnRNP into the cytoplasm . The bottom stereo pair was stained with uranyl and lead, contrasting lipids, proteins and nucleic acids . This image is representative of many published micrographs which demonstrate the elongation of the BR granule into a ribbon-like structure as it passes through the nuclear pore (20,33) . Bar, 100 nm .
view of the BR granule be reconciled with the coiled ribbon model (19,33,35,38)? Ribonucleosomes can be obtained as single 40S particles following extraction of RNAse-digested nuclei or RNAse treatment of reconstituted RNPs . These monomer particles consist of core proteins (30-40 kD) associated with 700 nucleotides at a protein/RNA weight ratio of 60/40, and exhibiting an overall diameter of ca . 20 tun . It is obvious that differences exist between isolated monomer ribonucleosomes and the BR granule particles ; i .e ., differences in the measured particle diameters and the estimates of associated RNA lengths . The discrepancy in particle diameters could be due to the fact that OA-B is staining RNA and not associated proteins . If we assume that the BR mRNA (37 kb) is covered with ribonucleosomes, we would estimate about 50 particles. The calculated volume of this number of ribonucleosomes would greatly exceed the volume of a BR granule . Furthermore, RNAse digestion of isolated BR granules did not yield convincing evidence for the existence of ribonucleosome subunits associated with the mRNA (39) . In Figure 7. Net phosphorus distribution maps of three BR granules are shown on the right . These were obtained by subtraction of two images of the same area of very thin unstained lowicryl sections formed at two eV loss windows : AE = 120 eV, the pre-edge image (not shown) ; AE = 160 eV, the phosphorus enhanced image (left) . The top granule gives a "pinwheel" appearance ; the middle granule resembles an "edge-on" view. The bottom granule exhibits clear 10-nn particulate structures; but, the overall diameter is <40-50 nm, perhaps because of the removal of material during sectioning. Bar, 20 run . view of the evolutionary conservation of the hnRNP core proteins (8), it is likely that these proteins are associated with mRNAs in Chironomus. It mustbe concluded, however, that current evidence does not convincingly argue that the 10-run particles are identical to ribonucleosomes . Could these BR granule substructures be spliceosomes? So far only a single 55-bp intron near the 3' end of the BR mRNA has been identified (7,33), although the sequence for at least 75% of the coding region is known . The BR genes appear to contain UI and U2 small nuclear RNP antigenic determinants within the puffs during transcription (32) . However, mature BR granules appear to be unreactive to anti-small nuclear RNP antibodies (37), arguing against an identity between the 10-nm particles and mRNA splicing complexes . At present, the basis for the particulate appearance of BR granules remains unknown . It is possible that these structures are unique to BR genes . In an effort to answer this question, we have begun an examination of other genes that synthesize very large mRNAs .
EM studies of transcribing BR genes and mature BR granules have resulted in a coiled ribbon model for the large hnRNP (19,33,35,38) . Since these sections of nuclei were stained with uranyl acetate and lead citrate, the reconstructions would not distinguish between RNA-and protein-rich regions of the BR granule. The structure proposed is a ribbon (-120-nm long x 25-50-nn wide x 10-15-nm thick) bent into a ring . The authors argue that the 5' and 3' ends of the mRNA are in close proximity in the ring, separated by a barely discernable cleft . Further, it was suggested that the ring is somewhat skewed, suggesting a left handedness. Our image data focus attention upon the particulate character of the BR granule RNA and provide very little data on their connectivity. One provocative set of images is the tilt series of an apparent "edge-on" view of a single BR granule (Fig .  5, bottom series) . This granule resembles a short left-handed Figure 8. Various EM views of BR granule substructure are modeled by a shaded surface representation . This model consists of 10 particles connected in a single left-handed helix (10-nm-diam particles ; six particles per turn; rotation angle 60°; 12/3 turns ; rise per turn 15 nm) . This figure is intended to simulate some of the images observed by EM, assuming a defined arrangement of particles, viewed at various orientations. Since the thin connections between particles are difficult to visualize, one can postulate a single helix (A-D, and F) or a zigzag connection (E) . A simulates the "edge-on" view shown in the lower panel of Fig. 5 . B and C simulate "pinwheel" views . D and E illustrate "en face" views . F simulates the top panel of our Fig . 6. More complicated models can be generated assuming more particles, and irregular spacings and connections . helix. (We mount our stereo images based on "calibration" with a specimen of known handedness.) At least two types ofmodels can be formulated in connecting the -10-run particles : (a) A single helical string 200-300-nm long, containing 10-12 particles ; or (b) more complex connections, such as a "zig-zag" pattern between particles (Fig. 8) . The nature of the arrangement ofthe 10-nm RNP particles and the order of their connections within the entire protein and RNA volume of the BR granules requires more analysis . Threedimensional reconstruction data by EMT may permit such a comparison, leading to the formulation of more definitive models .